High-throughput sequencing techniques are used to analyse the diversity of the respiratory microbiota in health and disease. Although extensive data are available regarding bacterial respiratory microbiota, its fungal component remains poorly studied. This is partly due to the technical issues associated with fungal metagenomics analyses. In this study, we compared two DNA extraction protocols and two fungal amplification targets for combined bacterial and fungal targeted amplicon sequencing analyses of the respiratory microbiota.Creteil, France) and treated anonymously, were tested after bacterial and fungal routine culture. Two of which were spiked with Aspergillus Fumigati and Aspergillus Nigri (105 conidia/mL). After mechanical lysis, DNA was extracted using automated QIAsymphony® extraction (AQE) or manual PowerSoil® MoBio extraction (MPE). DNA yield and purity were compared. DNA extracted from spiked sputa was subjected to (i) real-time PCR for Aspergillus DNA detection and (ii) combined metagenomic analyses targeting barcoded primers for fungal ITS1 and ITS2, and bacterial V1-V2 and V3-V4 16S regions. Amplicon libraries were prepared using MiSeq Reagent V3 kit on Illumina platform. Data were analysed using PyroMIC© and SHAMAN software, and compared with culture results.Six sputa, randomly selected from routine samples in Mondor Hospital . For fungi, the relative abundances of 4/11 major taxa were impacted and AQE results were closer to culture results. The V1-V2 or V3-V4 and ITS1 or ITS2 targets assessed similarly the diversity of bacterial and fungal major taxa, but ITS2 and V3-V4 detected more minor taxa.AQE extraction provided a higher yield of DNA (AQE/MPE DNA ratio = 4.5 [1.3–11]) in a shorter time. The yield of Our results showed the importance of DNA extraction for combined bacterial and fungal targeted metagenomics of respiratory samples. The extraction protocol can affect DNA yield and the relative abundances of few bacterial but more fungal taxa. For fungal analysis, ITS2 allowed the detection of a greater number of minor taxa compared with ITS1. Streptococcus, Veillonella or Prevotella) are the most represented besides fungi, viruses, and archaea. With the development of high throughput sequencing enabling 16S/ITS targeted amplicon high-throughput sequencing and metagenomics, the bacterial component of human respiratory microbiota has become under increasing scrutiny to understand its role in health and disease ) higher than MPE , within a shorter technical time (~30 min for AQE vs. ~90 min for MPE), and an equivalent cost. Regarding the quality and purity of the DNA, we did not observe significant RNA contamination using agar electrophoresis of DNA samples. AQE method produced higher rates of single-strand DNA than MPE. However, the 260/280 nm absorbance ratio was similar for both extraction protocols .4CFU/ml of Candida krusei (teleomorph name: Pichia kudriavzevii); 7x103 CFU/ml of Candida albicans; 7x103 of Candida glabrata (of Nakaseomyces genus); 5x102 CFU/ml of Saccharomyces cerevisiae; 6x102 CFU/ml of Aspergillus section Fumigati, and 4x102 CFU/ml of Aspergillus section Nigri. Only three fungi were identified for P2: 2x104 CFU/ml of C. albicans; 2x102 CFU/ml of Aspergillus section Fumigati, and 3x102 CFU/ml Aspergillus section Nigri  revealed the presence of six fungi for P1 : 104CFU/on Nigri .Aspergillus DNA detected by 28S- and mt-qPCR was comparable (For the two spiked sputa (P1 and P2), the amount of mparable  regardlevs. 21 in P2’s samples), P1 airway microbiota was less even than P2. Of more, P1 had one vastly predominant taxon , six minor taxa , and 30 extremely low-represented taxa  , three minor taxa , and 12 extremely low-represented taxa  (Lactobacillus was identified as the only “core” taxon (> 1% abundance in every tested sample), and five major taxa were dominant in P2 . If both methods were able to detect 6/6 major and 7/9 minor taxa, we noticed that with V3-V4 a bigger number of extremely low-represented taxa were detected (15 for P1 and 9 for P2). In contrast, only 2 extremely low-represented taxa were detected with V1-V2 exclusively. We also observed that 11 genera were detected with significantly higher relative abundances using V3-V4 than V1-V2  and 94,221 reads using V3-V4  . For P2  < 0.1%) . In conte <0.1%) . AltogetParascardovia, Anaeroglobus, Actinomyces, Howardella, Staphylococcus, Alloprevotella, Pseudoramibacter, Melissococcus, Corynebacterium, Fretibacterium, Stenotrophomonas, Atopobium, Sphingomonas, Acinetobacter, Shuttleworthia, Acidaminococcus, Moraxella, Oribacterium, Delftia, Eubacterium, Lactococcus, Mogibacterium, Chelativorans, and Vagococcus.Methods used the same starting quantities of samples and the final volumes were equal. The category “Other genera” gathered 23 bacterial taxa associated with extremely low relative abundances : Aspergillus taxa, we were able to distinguish between Aspergillus Fumigati and Aspergillus Nigri sections. We detected 9/10 and 8/10 of the fungal genera in P1 and P2, respectively, and the 2 Aspergillus sections were detected in both patients , and four extremely low-represented genera . P2 had a less even mycobiota with only one vastly predominant fungal taxon , 1 minor taxon , and 4 extremely low-represented genera . We observed that 7/11 genera or sections were detected with significantly higher relative abundances using ITS2 compared with ITS1  as Candida albicans; 89.2% of Pichia reads from P1 were Candida krusei; 61.5% of Nakaseomyces reads from P1 were Candida glabrata, and 99.9% and 100% of Saccharomyces reads from P1 and P2, respectively, were Saccharomyces cerevisiae. Pooled Chao1 index was twice higher for ITS2 (15.4) than for ITS1 (7).For P1, we analysed 43,758 paired-end reads using ITS1 region , and 90,239 reads using ITS2  . For P2,patients . SettingThe bacterial community diversity analysed at genus level and estimated by Shannon and Simpson alpha-diversity indexes was comparable for P1 and P2 for the four analysed events . The pooLactobacillus, p-value = 0.008 and Rothia, p-value = 0.049) and 4 major fungal genera or sections   were higher with AQE, whereas the other half  had their abundances decreased with AQE  . The abuwith AQE .Saccharomyces, was detected in P2 samples by ITS amplicon sequencing but not by culture. All minor taxa  were detected by sequencing only. The relative abundances of the major taxa detected by culture or ITS amplicon sequencing after AQE or MPE varied significantly. For P1, culture and AQE detected similar proportions of three major yeast taxa . However, Saccharomyces and Aspergillus relative abundances were close in culture and MPE. For P2, the relative abundances of Candida and Aspergillus detected by AQE were closer to that observed in culture than MPE. But AQE detected a higher proportion of Saccharomyces compared with MPE or culture.As for fungal diversity detected by ITS targeted amplicon sequecing and culture , we obseAspergillus qPCR. In our work, the use of automated extraction enhanced the DNA yield and quality with better reliability and shorter technical time than the manual protocol. The bacterial and fungal taxonomic diversity detected in respiratory samples was not highly influenced by the extraction protocol , whereas the amplification targets had a significant influence. A higher number of low density microbial communities were detected by V3-V4 16S and ITS2 targets.In the present study, we compared, simultaneously and under the same conditions, the impact of two DNA extraction protocols  of double-strand DNA with less heterogeneous results, in shorter technical time (~30 min) and an equivalent cost compared with MPE. We did not differentiate, as most authors, the human DNA yield from the microbiota DNA yield. However, it has been demonstrated that obtaining high total DNA yields is essential for bacterial targeted amplicon sequencing, and even more for fungal sequencing due to the low load of fungal genes in human microbiota ,13,35. Cet al  comparedl  or V3-V4 16S target [light blue].Boxplot of log2 abundances of bacterial genera detected with significantly different abundances ((TIF)Click here for additional data file.S3 FigP-value < 0.05) when amplified using either ITS1 target [dark blue] or ITS2 target [light blue].Boxplot of log2 abundances of fungal genera or sections detected with significantly different abundances ((TIF)Click here for additional data file.S1 TableThe DNA was quantified (ng/ml) using Picogreen® dosage and its quality was determined using 260/280 purity ratio (Nanodrop®).(DOCX)Click here for additional data file.S2 Table5 conidia/ml of Aspergillus section Fumigati and Aspergillus section Nigri.The extraction protocols used were the Automated QIAsymphony Extraction [AQE] with DSP DNA midi kit, and the Manual PowerSoil® Extraction [MPE]. The 2 sputa were spiked with 10(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 TableAlpha diversity measurements  for bacterial (A) and fungal (B) targeted amplicon sequencing analyses performed on respiratory samples of 2 patients (P1 and P2). DNA was extracted using two extraction protocols . Amplification targets used were VI-V2 and V3-V4 for bacterial analysis and ITS1 and ITS2 for fungal analysis.(DOCX)Click here for additional data file.S7 TableAbundance Fold Change (expressed as log2 Fold Change) of bacterial (A) and fungal (B) taxa significantly different  with regard to the used extraction protocol .(DOCX)Click here for additional data file.


Caballeronia strains SBC1 and SBC2 from acidic permafrost suggest a new species with a facultative lifestyle via oxygen and nitrate respiration. Thus, a contribution to nitrogen cycling in cold and low-pH environments is anticipated.Annotated genomes of  Caballeronia strains SBC1 and SBC2 from acidic permafrost suggest a new species with a facultative lifestyle via oxygen and nitrate respiration. Thus, a contribution to nitrogen cycling in cold and low-pH environments is anticipated.Annotated genomes of  Single colonies were picked and purified by restreaking four times onto the same medium.Cryoturbated peat circles (PCs)  for SBC1 and 330,181 reads with an average length of 9,102 bp  for SBC2 were obtained.High-molecular-weight DNA (HWD) for Nanopore sequencing was isolated with the MasterPure complete DNA and RNA purification kit  from cells grown in liquid modified R2A medium (pH 5). HWD quality was checked on a Bioanalyzer 2100 using the DNA 12000 kit , and HWD was quantified with the Qubit double-stranded DNA (dsDNA) high-sensitivity (HS) assay kit ; 1.5 μg HWD was used for library preparation employing the ligation sequencing kit 1D (SQK-LSK109) and the native barcode expansion kit . Sequencing was performed for 72 h on the MinION Mk1B system with a SpotON flow cell R9.4.1 using MinKNOW v19.10.1, with Guppy v3.3.3 for base calling and demultiplexing. Totals of 67,997 reads with an average length of 4,873 bp   and nitric oxide (norV) reductases  was 88.lleronia , 15. SBCls v23.0 ).CP049156.1 (chromosome) and CP049157.1 to CP049160.1 (plasmids) for SBC1 and under the accession numbers CP049316.1 (chromosome) and CP049317.1 to CP049323.1 (plasmids) for SBC2. BioProject accession numbers for SBC1 and SBC2 are PRJNA604524 and PRJNA604525, and SRA accession numbers are SRP250914 and SRP250916, respectively.These whole-genome shotgun projects have been deposited in DDBJ/ENA/GenBank under the accession numbers


DNA interstrand crosslinks (ICLs) are a physical barrier to replication and therefore toxic to cell viability. An important mechanism for the removal of ICLs is the Fanconi Anemia DNA repair pathway, which is initiated by mono-ubiquitination of FANCD2 and its partner protein FANCI. Here, we show that maintenance of FANCD2 and FANCI proteins in a monoubiquitinated form is regulated by the ATR-kinase. Using recombinant proteins in biochemical reconstitution experiments we show that ATR directly phosphorylates FANCI on serine 556, 559, and 565 to stabilize its association with DNA and FANCD2. This increased association with DNA stimulates the conjugation of ubiquitin to both FANCI and FANCD2, but also inhibits ubiquitin deconjugation. Using phosphomimetic and phosphodead mutants of FANCI we show that S559 and S565 are particularly important for protecting the complex from the activity of the deubiquitinating enzyme USP1:UAF1. Our results reveal a major mechanism by which ATR kinase maintains the activation of the FA pathway, by promoting the accumulation of FANCD2 in the ubiquitinated form active in DNA repair. Many chemotherapeutic drugs kill cancer cells by inducing toxic DNA interstrand crosslinks (ICLs). ICLs prevent DNA strand separation and therefore stall DNA transcription and replication complexes . A critiFANCD2 monoubiquitination is temporally and spatially controlled at stalled forks by the FA core complex (a RING E3 ligase), USP1:UAF1 (a deubiquitinating enzyme) and ATR kinase . FANCI iA crystal structure of FANCI:FANCD2 complex revealed that the lysine residues targeted for monoubiquitination in FANCI (K522 in mouse corresponding to K523 in human) and FANCD2 (K559 in mouse corresponding to K561 in human) are embedded in the dimer interface . This rain vitro monoubiquitination and de-ubiquitination of FANCI:FANCD2 using purified proteins. Maximal monoubiquitination required the FANCB-FANCL-FAAP100 (BL100 enzyme module) and FANCC-FANCE-FANCF (CEF substrate adaptor module) components of the FA core complex  FANCI:FANCD2, human FANCB:FANCL:FAAP100, FANCC:FANCE:FANCF and UBE2T were expressed and purified as described in Human FANCI:FANCD2 complex and Avi-ubiquitin was purified as described in Xenopus laevis StrepII-FANCD2, Flag-FANCI and human Flag-FANCI were cloned into pFastBac1 plasmid (Thermo Fisher). Expression plasmids for StrepII-FANCD2, Flag-FANCI, FANCI phosphomimic mutant (S6D) and phosphodead mutant (S6A) were previously described (Xenopus FANCI with six codons encoding for serine (S) residues S557, S560, S566, S597, S618, and S630  mutated to encode either for aspartic acid (D) residues (FANCI6S→ D) or alanine residues (FANCI6→ A) were kindly provided by Alexandra Sobeck lab (Trichoplusia ni (Hi5) insect cells were co-infected with Xenopus FANCI and FANCD2 viruses or infected only with human or Xenopus FANCI (Flag-tagged) or FANCD2 (StrepII-tagged) (MOI = 2) and harvested after 72 h. Cell pellets were washed in 1X PBS and resuspended in 9 mL Flag Lysis buffer , 10% glycerol) or Strep Lysis buffer , 10% glycerol, 20 μg/mL avidin, 1 mM DTT). Lysates were briefly sonicated and cleared by centrifugation for 45 min at 16,000 × g, 4°C. 1 mL M2 Flag resin (Sigma-Aldrich) or 1 mL StrepTactin Sepharose resin  was washed with 5 CVs each of water, followed by 0.1M glycine pH 3.5 or 0.5M NaOH, and equilibrated with 10 CVs buffer A . Lysate was added to the Flag or Streptactin resin, and incubated with gentle mixing at 4°C for 2 h. Resin was washed with 10 CVs of buffer A and eluted in 1 CV buffer A with 5 μg/mL Flag peptide (Assay Matrix) or 100 μg/mL D-desthiobiotin (Sigma-Aldrich). Flag fractions containing FANCI:FANCD2 complex were pooled, and loaded onto a MiniQ anion purification column (GE) and equilibrated with buffer A. Using a gradient between buffer A and buffer B , FANCI:FANCD2 complex were eluted between 250 and 350 mM NaCl. The peak fractions were pooled, and assessed by SDS-PAGE. Protein concentration was determined by Nanodrop .escribed . Xenopusbeck lab . Differebeck lab . Trichop2 and incubated for 30 min at 30°C prior to establishment of ubiquitination reaction.Ten μg of recombinant FANCI:FANCD2, FANCI, or FANCD2 were incubated in 60 μL 20 mM Tris-HCl pH 7.4, 10 mM MgAc, 0.5 mM DTT, 0.05% Tween-20, 100 mM KCl and 0.2 mM ATP in the presence of 0.1 μg of ATR:ATRIP for 30 min at 30°C. For phosphatase experiments, 600 units of lambda phosphatase was added to reactions together with 1 mM MnCl2, 150 mM NaCl, 0.01% Triton X-100). The dsDNA substrates were generated using oligonucleotide 1 (5′-ACGC TGCCGAATTCTACCAGTGCCTTGCTAGGACATCTTTGCC CACCTGCAGGTTCACCC-3′) and oligonucleotide 2 (5′-GG GTGAACCTGCAGGTGGGCAAAGATGTCCTAGCAAGGCA CTGGTAGAATTCGGCAGCGT-3′). 20 μL reactions were set up on ice and incubated at 25°C for 90 min. To perform deubiquitination assays, FANCI:FANCD2 monoubiquitination was arrested using apyrase (NEB) and 100 nM recombinant USP1:UAF1 (Boston Biochem) were added for 30 min at room temperature. Reactions were stopped by adding 10 μL NuPage LDS sample buffer and heated at 80°C for 5 min. Reactions were loaded onto 4–12% SDS PAGE and run using NuPAGE® MOPS buffer and assessed by western blot analysis using Flag (Jomar Life Research) or StrepII (Abcam) antibody.Standard ubiquitination reactions contained 10 μM recombinant human AviTag-biotin-ubiquitin, 50 nM human recombinant UBE1, 100 nM UBE2T, 100 nM pUC19 plasmid or dsDNA oligonucleotide substrate, 2 mM ATP, 100 nM FANCI:FANCD2 complex wild type (WT) or ubiquitination-deficient (KR), in reaction buffer  at 55°C for 1 h and alkylated with 50 μL 55 mM iodoacetamide in 25 mM NH4HCO3 at room temperature for 45 min in the dark. Samples were incubated overnight with 20 μL trypsin . The resulted proteolytic peptides were subjected to sonication with 50 μL of 50% ACN, 5% formic acid and analyzed by mass spectrometry after concentration under vacuum to a 10–15 μL final volume. Mass spectra of digested protein gel bands were obtained on ESI-quadropole-time-of-flight mass spectrometer coupled to reverse-phase HPLC-MS/MS. The analysis program MASCOT was used to identify phosphorylation sites on FANCI.Proteins were separated by SDS-PAGE, and the band excised for manual digestion to maximize sensitivity and efficiency. Protein bands were destained and dehydrated with 500 μL acetonitrile (ACN). Subsequently, proteins were reduced with 500 μL 10 mM dithiothreitol (DTT) in 25 mM ammonium bicarbonate . 100 μg/ml FANCD2 or FANCI was diluted in buffer F and further diluted three times with a dilution factor of two. To measure the interaction between FANCD2 and FANCI, the association and dissociation times were 180 and 300 s, respectively, for every analyte concentration. In total, four Streptavidin sensors (ForteBio) were used to measure four different analyte concentrations in parallel, while one sensor was used to measure the buffer reference. All steps were performed at 25°C with an agitation speed of 1000 rpm. Sensorgrams were measured on a ForteBio BLItz instrument and referenced against the buffer reference signal using the Data Analysis software 7.1.0.36 (ForteBio). The sensorgrams obtained with the concentrations: 334.7, 167.3, 83.7, and 41.8 nM were fitted with the BiaEvaluation software 4.1 from Biacore using a 1:1 binding model.Sixty bp fluorescently labeled dsDNA substrates were prepared as described in Two μg of purified Flag-FANCI proteins were incubated with 2 μg of His-USP1:UAF1 (Boston Biochem) for 30 min at 25°C in 40 μL of pull-down buffer . 10 μL of TALON metal affinity resin (Takara Bio) were added to the reaction mixtures and were gently mixed for 30 min at 25°C. The resin were then washed three times with 1 mL pull-down buffer. The proteins bound to the resin were separated by SDS-PAGE and stained with Coomassie blue. The band intensities of FANCI were quantitated and visualized by Licor Odyssey system.We determined that a percentage of recombinant FANCI purified from baculovirus infected insect cells is phosphorylated within a previously described S/TQ cluster domain . Tandem in vitro ubiquitination reactions using recombinant FA core complex. In both human  showed faster monoubiquitination rates than for WT xFANCI:FANCD2 complex. In particular, the FANCI-ADD and -DDD variants stimulated the highest rate of xFANCD2 monoubiquitination. Conversely, two of the FANCI-3SQ mutants (AAD and AAA) and both of the FANCI-6SQ mutants (S6D and S6A) showed lower xFANCD2 monoubiquitination than WT Ub. Overall, the rate of FANCD2 deubiquitination when bound to FANCI-ADD or WT-FANCI is fourfold slower than for FANCI-AAA  mutated in this variant may cause structural malformation of FANCI, given their location within α-helices of the solenoid structure of the protein. As such, FANCI-S6D only binds weakly to FANCD2 and does not stimulate its mono-ubiquitination. This finding supports the in vivo cell-based studies of We found that three sites on the surface of FANCI, serines 557, 560 and 565, are the main targets of recombinant ATR kinase in vitro or in cells. For example, Casein Kinase 2 phosphorylation of FANCD2 at a cluster of serines between residues 882–898 inhibits its DNA association and subsequent monoubiquitination  it prevents the retention of FANCD2 at spurious, non-repair sites in the nucleus and (2) it allows completion of DNA repair . A major in vivo . Togethe in vivo . Our in FANCD2 monoubiquitination by FA core complex and deubiquitination by USP1:UAF occurs at steady state levels associated with ongoing replication . We propGiven the critical importance of FANCI as an ATR target, the phosphorylation state of the FANCI-3SQ cluster may be an appropriate biomarker of the effectiveness of several classes of ATR kinase inhibitor currently in clinical trial for the treatment of cancer . Our worThe datasets generated for this study are available on request to the corresponding author.WT performed protein purification, biolayer inferometry, ubiquitination, deubiquitination, and DNA binding assays. ST performed deubiquitination assays. VM generated FANCI phosphomutants and assisted with protein purifications. WT and AD wrote the manuscript and designed experiments for this article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Here, we found that RNA demethylase ALKBH5 expression was significantly upregulated in endometrial cancer, ALKBH5 was then identified to positively regulate proliferation and invasion of endometrial cancer. Mechanistically, the m6A eraser ALKBH5 demethylated target transcripts IGF1R and enhanced IGF1R mRNA stability, consequently promoting IGF1R translation and activating IGF1R signaling pathway. Thus, we demonstrated that ALKBH5 promoted proliferation and invasion of endometrial cancer via erasing IGF1R m6A-modifications, which suggests a potential therapeutic target for endometrial cancer.N6-methyladenosine (m Endometrial cancer is the most prevalent malignancy of the female genital tract in many countries. Women have a 2-3% lifetime risk of developing this malignancy 6N-methyladenosine (m6A) is the most prevalent messenger RNA modification in eukaryotes. The biological effects of this reversible modification are mostly through methylase, demethylase and association recognize proteins 6A modification is reported to participated in regulating RNA structure 6A-dependent mRNA regulation affects diverse biological processes in mammals  Alkbh5 knockout mice have shown compromised spermatogenesis 6A carried mRNA regulates the differentiation of stem cells by affecting mRNA stability and regulating transcriptome switching during embryonic development 6A mRNA methylation also affects cancer initiation and progression in a variety of cancers 6A mRNA methylation as an oncogenic mechanism in endometrial cancer and identify m6A methylation as a regulator of AKT signaling “Insulin like growth factor (IGF) axis” plays a key role in regulating cell growth and survival and affecting virtually every organ in the body in vitro. Together, our results may add insights to the m6A demethylase ALKBH5 as an important regulator of the insulin-like signaling pathway in promoting proliferation and invasion of endometrial cancer, providing potential targets to prevent the development of endometrial cancer.Herein, we found that RNA demethylase ALKBH5 expression was significantly upregulated in endometrial cancer clinical samples. ALKBH5 knockdown decreased the expression of key molecular IGF1R and inhibited the activation of insulin-like growth factor signaling pathway and inhibited proliferation and invasion of endometrial cancer All samples were obtained with informed consent under a protocol approved by the ethics committee of Shanghai First Maternity and Infant Hospital, China. The study is compliant with all relevant ethical regulations regarding research involving human participants. All Endometrial cancer samples were diagnosed and assessed in accordance with the International Federation of Gynecology Oncology (FIGO) criteria (2009). All the Endometrial cancer samples must have been diagnosed as endometrial adenocarcinoma and underwent initial surgery. All patients had no history of malignancies. Clinical and pathological data of all patients were retrieved from their records at our department. Patient's characteristics are listed in Table 6A (202003) was from Synaptic Systems.Antibodies used for experiment were as follows: β-Actin , and HRP-coupled secondary antibodies (sc-2749) were from Santa Cruz Biotechnology . ALKBH5 (ab174124) antibody was from Abcam . IGF1R (#9750) antibodies were from CST company. Antibody specific to mHEC-1-A cell lines were obtained from American Type Culture Collection  and cultured as grown in McCoy's 5A medium  supplemented with 10% fetal bovine serum (FBS) (Gibco), and 1% penicillin-streptomycin (Gibco). The RL95-2 cells were purchased from ATCC and grown in DMEM: F-12 medium supplemented with 10% FBS, 0.005 mg/mL insulin, and 1% penicillin-streptomycin. The T-HESCs were purchased from ATCC and grown in DMEM: F-12 medium supplemented with 10% FBS, 1% ITS-premix, 1 mM pyruvate, 0.5 µg/mL puromycin and 1% penicillin-streptomycin. Cells were transfected with siRNAs  and plasmids with Lipofectamine™ 2000  according to the manufacturer's instructions. All siRNAs were obtained from GenePharma shown below: IGF1R sense #1: GCGGAGAGAUGUCAUGCAATT, antisense #1: UUGCAUGACAUCUCUCCGCTT, sense #2: GCUUCACCGUUUACUACAATT, antisense #2: UUGUAGUAAACGGUGAAGCTT; Negative control, Sense: UUCUCCGAACGUGUCACGUTT, Antisense: ACGUGACACGUUCGGAGAATT.To establish stably transfected HEC-1-A cells, G418 was added (1000 μg/mL) 48 h after transfection and maintained at 800 μg/mL for 3 weeks for positive selection. For stably transfected cells, ALKBH5 and mutant H204A expression were confirmed by Western blot. Lentiviruses harboring human ALKBH5 shRNAs were purchased from OBIO technology . HEC-1-A cells were infected with the lentivirus (MOI: 10:1). After HEC-1-A cells were transfected for 72 h, 5 μg/mL puromycin was added for 72 h and maintained at 1 μg/mL for 3 weeks for positive selection of stably knockdown ALKBH5. The sequences encoding the short-hairpin RNA are shown below: Human Alkbh5, #1: TCGTGTCCGTGTCCTTCTT, #2: GACTGTGCTCAGTGGATAT; Human Mettl3, CGTCAGTATCTTGGGCAAGTT; Negative control, TTCTCCGAACGTGTCACGT.HEC-1-A cells were transfected with indicated shRNA, siRNAs or plasmids. 48 h post-transfection, total RNA was extracted by TRIZOL reagent (Invitrogen). RNA was reversed-transcribed using the Reverse Transcription System from Toyobo . The reverse transcription products from different samples were amplified by real-time PCR and analyzed as described previously For invasion assays, cell-culture inserts  were pre-coated with Matrigel  in serum-free medium for 30 mins at 37 °C. For migration assays, inserts were not pre-coated. 40,000 cells per insert were seeded in the upper chamber of the insert and cultured in 300 μL RPMI-1640 medium supplemented with 2% FBS. Complete RPMI -1640 medium supplemented with 10% FBS was used in the lower chamber. Following 16-24 h of migration or invasion, fluorescent stain  was added to each lower chamber and incubated for 30 mins. Images were collected with a Nikon Eclipse Ti2 with NIS Elements imaging software (version 5.02) and images analyzed with ImageJ (version 1.51i).500 cells were seeded per well in six-well culture dishes. After 7 to 10 days, the culture medium was removed and the cells were washed twice with PBS, fixed with 4% paraformaldehyde for 20 min, stained with 0.1% crystal violet (in 25% methanol) for 20 min, washed with water and dried. Colonies were counted manually.5000 cells were seeded per well in a 96-well plate. The cell proliferation was assessed by assaying the cells at various time points using the Cell counting kit-8  following the manufacturer's protocols. For each cell line tested, the signal was normalized to the value observed about 24 h after seeding.6A-qRT-PCR, total RNAs were firstly subjected to mRNA purification by Poly(A) selection , but not randomly fragmented to facilitate RT with oligo dT and PCR amplification. Then it was performed as described in m6A-seq assay but without generating cDNA library. For comparing m6A abundance changes, relative enrichment was first normalized with inputs, and then analyzed by comparing the data from m6A-immunoprecipitated sample. All samples were analyzed in triplicate qPCR.The procedure was adapted from the previous report HEC-1-A cells were seeded in 24 well plates at 60% confluency. After 24 h, actinomycin D  was added to 5 mg/mL 6 h, 4 h, 2 h and 0 h before collection. The total RNA was purified using an RNeasy kit with an additional DNase-I digestion step on the column. RNA quantities were determined by qRT-PCR. The HPRT1 gene was used as a reference gene when carrying out qPCR.Alkbh5 Forward: CCGGAATTCATGGCGGCCGCCAGCGGCTAC,Reverse: GCCGGTACCTCAGTGCCGCCGCATCTTCACCTTT;Alkbh5 (H204A) Forward: GGCTGCATCGTGTCCGCCGTGGACCCCATCCA,Reverse: GCCGGGCTGGTAGTCGTTGATGACGGCGC;IGF1R Forward: CCGGAATTCATGAAGTCTGGCTCCGGAGGAGG,Reverse: CGCGGATCCTCAGCAGGTCGAAGACTGGGGC.Related genes were obtained from human endometrial cell by RT-PCR and subsequently cloned into pcDNA vectors. Each construct was confirmed by sequencing. The corresponding primers used in this study are listed below:Cells were lysed using Cell Lysis Buffer  supplemented with cocktail protease inhibitor . Protein concentrations of the extracts were measured using BCA assays  and equalized with the extraction reagent. Equivalent amounts of extract were loaded to SDS-PAGE, transferred onto PVDF membranes, and then blotted as described previously t-test. Data were analyzed with GraphPad Prism Software. Statistical values achieving p<0.05 were considered to be statistically significant.All experiments were independently repeated at least three times. Comparisons between two groups were performed using Student's 6A mRNA methylation as an oncogenic mechanism in endometrial cancer and identify m6A methylation as a regulator of AKT signaling 6A modification associated enzymes maybe the key regulators of the endometrial cancer. Intriguingly, we identified that the mRNA levels of RNA m6A demethylase ALKBH5 were significantly upregulated in endometrial cancer compared to adjacent normal endometrium  and the matrix metallopeptidase 9 (MMP9) expressions in HEC-1-A Figure L. These To further confirm that our results extend beyond the HEC-1-A endometrial cancer cell line, we tested the effects of knockdown and overexpression of ALKBH5 in a second endometrial cancer cell line (RL95-2), as well as human endometrial stromal cells (T-HESCs), a normal non-transformed cell line, and found similar effects on the activity of endometrial cell Figure , these r6A-seq results showing decreased m6A methylation were enriched in KEGG terms related to focal adhesion (IGF) signaling pathway 6A methylation modifications according to m6A-seq data 6A antibody followed by RT-qPCR, and found that, in addition to negative control HPRT, IGF1R and TNS3 (positive control) mRNAs were enriched in the m6A antibody-bound fraction, confirming the presence of m6A sites on the IGF1R transcripts  (Figure 6A modification of its substrates IGF1R.As ALKBH5 is an m6A modification. We found that knockdown of ALKBH5 decreased the mRNA and protein levels of the IGF1R transcripts and IGF1R transcripts showed increased RNA decay rates upon knockdown of ALKBH5 (Figure IGF1R transcripts and IGF1R transcripts showed decreased RNA decay rates upon ALKBH5 overexpression (Figure We next explored that whether the expression of IGF1R were affected after erasing mWe then investigated the potential roles of IGF1R in regulating the activity of endometrial cell. Firstly, we confirmed that IGF1R was efficiently knocked down by two different siRNAs Figure A,B. We fTo investigate whether the expression of IGF1R protein in endometrial cancer adjacent and endometrial cancer were consistent with the ALKBH5, we found that IGF1R were indeed upregulated in endometrial cancer Figure J. Taken 6A modifications of IGF1R transcripts, and promotes IGF1R expression leading to the activation of the IGF signaling pathway, which consequently induces COL1A1 and MMP9 expression, subsequently increases the invasion and migration of endometrial cells.On the basis of our findings, we propose the following working model to explain how ALKBH5 promotes the activity of endometrial cell. ALKBH5 expression significantly upregulates in endometrial cancer, and ALKBH5 demethylate the min vitro. Mechanically, ALKBH5 promotes the expression of IGF1R via demethylating m6A modification and increase the expression of COL1A1 and MMP9, which can promote the invasion of endometrial cell. Together, our results indicate that the m6A demethylase ALKBH5 maybe a potential target to prevent the tumorigenicity of endometrial cancer.We find that the RNA demethylase ALKBH5 expression upregulated in endometrial cancer. Downregulation of ALKBH5 can inhibit endometrial cell proliferation and invasion 6A methylation when compared with matched normal endometrium, which were probably caused by either mutation of METTL14 or reduced expression of the METTL3 methyltransferase 6A methylation, this is consistent with previous observations that ALKBH5 was significantly upregulated in many cancer tissues It is reported that about 70% of endometrial tumours exhibit reduced m6A mRNA levels and enhance proliferation of endometrial cancer cells in vitro, m6A-seq of endometrial cancer patient tumors and cell lines revealed that reduced m6A mRNA methylation could promote cell proliferation by altering the expression of key enzymes that affect the AKT signaling pathway Either METTL14 mutation or METTL3 downregulation could reduce m6A methylated mRNA could have effects on cellular physiology, especially if the key transcripts are affected 6A demethylase; here we found that the key transcripts IGF1R in IGF signaling pathway were demethylated upon ALKBH5 overexpression, and accordingly the decay of IGF1R mRNAs were inhibited after demethylation. These results indicated that the m6A methylation as an important factor promoting cancer progression, reveal that reduced m6A mRNA methylation on the key transcript is most likely an oncogenic mechanism underlying mostly endometrial cancers and identify m6A methylation as a regulator of the cell growth.The dynamically mChanges in IGF1 expression and signaling play key roles in the regulation of normal uterine physiology


CSN2, CSN1S1, CSN1S2, CSN3 were 11P, 13P, 17P and 6P, respectively in 105 casein phosphopeptides. Whey proteins BLG and LALBA showed 19 and 4 phosphosites respectively. Post translational modification was observed in 45 low abundant non-casein milk proteins mainly associated with signal transduction, immune system, developmental biology and metabolism pathways. Pasp is reported for the first time in 47 sites. The rare conserved peptide sequence of (SSSEE) was observed in αS1 and αS2 casein. The functional roles of identified phosphopeptides included anti-microbial, DPP-IV inhibitory, anti-inflammatory and ACE inhibitory. This is first report from tropics, investigating post translational modifications in casein and non-casein goat milk proteins and studies their interactions.Goat milk is a source of nutrition in difficult areas and has lesser allerginicity than cow milk. It is leading in the area for nutraceutical formulation and drug development using goat mammary gland as a bioreactor. Post translational modifications of a protein regulate protein function, biological activity, stabilization and interactions. The protein variants of goat milk from 10 breeds were studied for the post translational modifications by combining highly sensitive 2DE and Q-Exactive LC-MS/MS. Here we observed high levels of post translational modifications in 201 peptides of 120 goat milk proteins. The phosphosites observed for  Milk protein composition exhibited high heterogeneity due to numerous genetic variants, and isoforms with different degrees of posttranslational modifications such as phosphorylation and glycosylation in caseins3. Milk proteins exhibit conformational structure due to post translational modifications and constitutive levels of proteolytic activity produce a range of significant peptides. The posttranslational modifications of the polypeptide chain occur in the Golgi apparatus of the mammary epithelial cells4. Casein phosphorylation at amino acid serine or threonine is catalyzed by kinase enzymes5. Phosphorylation is affected by different factors such as protein sequence, efficacy of kinase enzymes, gene expression, substrate availability and access to phosphorylation site which is responsible for the specific protein conformation6.Milk is the primary source of nutrition for mammals and serves as a major vehicle of maternal immunity transfer, thus, plays a vital role in inclusive development of the neonates. The milk proteome is extremely complex due to abundant post-translational modifications and various proteolytic processes7. Phosphorylation stabilizes calcium phosphate nano clusters in casein micelles8. The micellar structure of casein enables milk to carry calcium and phosphate to the neonate by channelizing the risk of mammary gland bio-calcification10. Phosphorylation state of caseins varies widely from 1P to 3P on CSN3, 4P to 5P on CSN2, 8P to 9P on CSN1S1, 10P to 13P on CSN1S211. In bovine milk, CSN1S1 accounts for about 35% of the total casein and has 2 common phosphorylation isoforms: CSN1S1-8P and CSN1S1-9P. Similarly, CSN1S2 accounts for about 10% of the total casein and is present with isoforms from 10 to 14P and occasionally with 9P or 15P12.Protein functions such as binding, stabilization, biological activity, interactions with proteins and other biomolecules are regulated by phosphorylation-dephosphorylation of protein14. However, advances in proteomics have largely enhanced the quotient of protein identification. Phosphorylation of milk proteins has been studied in llama15, camel16 and goat milk fat globule membrane proteins17 using MS/MS proteomic approach. A total of 8 phosphopeptides corresponding to 18 phosphorylation sites were identified in CSN1S1, CSN1S2 and CSN2 of goat milk using nLC-MS/MS18. Casein phosphopeptides in goat milk have been studied by Olumee-shabon and Boehemer18 and phosphoproteome of goat milk fat globule membrane have been reported by Henry and others17. Phosphorylation have been reported for bovine caseins using inductively coupled plasma mass spectrometry (ICP-MS)19, equine CSN1S1 and and CSN2 by nESI-MS/MS21, donkey CSN2 by MALDI-TOF and nESI-MS/MS22 and for CSN1S1 and CSN2 of water buffalo by MS23.The identification and analysis of phosphopeptides has been challenging because of the relatively low stoichiometry, inherent lower ionization efficiency and variation of phosphorylation sites24. These peptides show various biological activities and are released by proteolytic digestion of caseins and milk proteins in gut or during fermentation. This study was designed to gain an insight into molecular diversity of goat milk proteins and to identify various degree of PTM in casein and non-casein proteins. The characterization of high and low abundant milk proteins by gel based proteomic approach i.e. 2DE and nLC-MS/MS. The present study analysed post-translational modification such as phosphorylation, oxidation, and carbamidomethylation on casein and non-casein proteins in goat milk. As post-translational modifications (PTMs) are acting as the major means of intracellular communication, therefore, the interactions of various proteins involved in post translational modifications were analysed.Bovine milk is extensively used, due to its high biological value and plasticity as it can be transformed to cheese and several other dairy products. The functional knowledge of casein and whey proteins has been identified for the presence of bioactive peptidesThe present study has analyzed the post translational modifications in low and high abundant proteins in goat milk of genetically diverse goat breeds/genotypes reared in varied ecological and grazing condition in India. The present study generated a comprehensive profile of PTM in goat milk and identified PTM sites in relation to sample variations.The milk samples from 10 Indian goat breeds were investigated regarding their genetic variation, proteome composition and post translational modifications. The selected populations were mapped in their home tract and reared in semi-intensive system and the animals were apparently healthy. Altogether 1240 milk samples were analyzed by means of 14% SDS-urea-PAGE and different allelic combinations of caseins were resolved to identify the protein variants. Further, the selected variants were analyzed by combining highly sensitive 2DE and Q-Exactive nLC-MS/MS using Thermo Fisher in-house reference database. Distinctive protein spots (n=144) selected from 21 variants in 2DE gels were processed by nLC-MS/MS. The MS data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD013593. The .raw files generated from nLC-MS/MS of goat milk proteins were analysed for Sequest search using Proteome Discoverer (v2.2). By comparing the databases, a milk proteome of 578 peptides for 348 genes (800 Uniprot accessions) were identified using reference database. The post translational modifications were identified in 201 peptide sequences for 86 proteins (120 Uniprot KB accessions).The annotations of peptides observing modifications including cysteine carbamidomethylation, oxidation, acetylation and phosphorylation in each protein are presented in Table CSN2) showed a maximum of 17 phosphosites, 4D, 5T, 6S and 2Y ; α-S2 casein (CSN1S2) showed 13 phosphosites  ; α-S1 casein (CSN1S1) showed 11 phosphosites  ; and κ-casein (CSN3) showed 6 phosphosites , respectively. Whey proteins β-lactoglobulin (BLG) (P02756) showed 19 phosphosites  and α-lactalbumin (LALBA) showed 4 phosphosites .The observed phosphosites for the high abundance proteins in goat milk are presented in Table CSN1S1 showed 5P at S78-S82 and peptide 58–73 showed 1P or 3P resulting in isoforms 10P or 12P. Similarly, peptides from CSN1S2 showed variations leading to different isoforms. Peptides 19–40, 19–37 showed 3 variations in site of phosphorylation such as 1P S24 or 2P S24, S25 or 3P S23, S24, S25. Peptide 142–153 showed 1P S145 or 1P S147 or 3P S145, T146, S147 and peptides 154–165/166 showed 1P S159 or 2P S159, T160 resulting in 8P isoform. β-casein peptides showed variations 1P or 6P in peptide 129–147, 2P or 3P in peptide 148–184 and may result in 10P and 11P isoforms. The identified fragment of κ-casein showed 4P to 5P variation by the level of phosphorylation of peptides 23–40 at 2P or 3P and 52–65/66 at 1P.The phosphorylated peptides generated from these proteins showed different isoforms and are presented in Table LALBA peptide 78–89 showed 2P and 1P variation at D82, D83; peptide 113–127, 114–127 showed 1P at D116 and peptide 118–127 showed 1P at Y122 resulting in one more possible isoform up to 4P. In BLG the highest level of variation in phosphorylation was observed in peptide 33–58 with 3P, 4P, 5P and 6P at sites T36, Y38, S39, S45, D46 and S48. Furthermore, the peptides 59–78, 143–153/156 showed 1P and 2P variations resulting in 13P isoform of BLG. These findings indicated the different levels of isoforms in phosphorylation of the caprine milk proteins.Similarly, H2 (2P), olfactory receptor OR51D1 (3P), GYCAM1 (1P), putative transcription factor (2P), Proteoglycan 4 (8P), and other proteins with varying level of phosphorylation were identified. Antiviral interferon tau BB4 found only in goat milk was also detected with 7P level of phosphorylation.The sensitivity for the identification of phosphorylation sites in low abundance proteins was enhanced by combining 2DE and nLC-MS/MS approach. Phosphorylation was identified in 45 low abundant proteins and presented in Table CSN1S1 (Q8MIH4)9P  was identified in samples of Himalayan local goats, whereas, D100, S138 phosphosites were observed in Osmanabadi goat. Similarly, CSN1S2 (P33049) 10P  was observed in Jamunapari goat. Phosphorylation at  and T164 was observed in Himalayan local goat and Osmanabadi goat, respectively. CSN2 (Q95L76) showed 13P  in Jakhrana goats. CSN3 (Q7YRX4) 5P  was observed in Attapady Black goats. α-lactalbumin (P00712) exhibited 4P  in Himalayan local goat and β-lactoglobulin (P02756) showed presence of 16P  in Osmanabadi, 15P in Attapady Black, 13P in Gaddi goats and sites S48, D51 and S54 were also noted.The identified 123 common UniprotKB ID proteins with varying level of phosphorylation across the 10 breeds were observed by comparing databases. The phosphosites and phosphoproteins observed in each breed are presented in Table PIK3C2A, CEP152 observed 7P each and SLC1A6 1P in Barbari. Interferon tau BB4 (7P), CREBBP (5P), Cationic trypsin (8P) isoforms were observed in Jamunapari. PDZD9 and SMC5 observed exhibited 5P and 7P in Osmanabadi goats. Emilin-2 (3P), FAM222B (4P), MnSOD and OVOL1 each with 2P isoforms were observed in Sirohi goats.Similarly, the low abundant proteins were identified in different breeds with varying sites of phosphorylation. Proteins Proteoglycan 4 (PRG4) showed 8P isoform in Barbari, LALBA and BLG showed both oxidation and carboxymethylation other than phosphorylation. β-casein showed carboxymethylation on 7 sites  whereas; α-S2 casein displayed 3 oxidation sites M42, M157, M206. PTM in proteins α-lactalbumin were observed as C80, C110, C130, C139 and C84, C178, M42, M163 on β-lactoglobulin. Low abundant proteins also exhibited oxidation and carboxymethylation such as interferon tau BB4 , laminin subunit LAMC2 , olfactory receptor OR2K2 , ND5 , proteoglycan , keratin KRT1  and histone showed M60, M63 and/or C111 modifications.Other post translational modification such as oxidation and carbamidomethylation were also observed and presented in Table P value 3.63 E−08), cellular component organization , response to stress , anatomical structure development , defense process , response to hypoxia . The proteins mostly localised in membrane bound organelle , and extracellular region  and extracellular exosome . The molecular functions (MF) largely involved binding activity such as protein binding, DNA binding, nucleosome binding, anion binding, antioxidant activity  and structural molecular activities  were also observed. The UP_Keywords comprised antimicrobials, phosphoprotein, disease mutation and methylation.The identified proteome was classified into functional categories such as biological process (BP) (87.8% genes), cellular component (CC) (97.6% genes) and molecular function (MF) (92.7% genes) using DAVID 6.8. The detailed annotations including UP-Keywords from DAVID software are available in Table The interaction of proteins was analysed by STRING by MCL clustering of the identified proteome categorised the proteins in 16 clusters at highest confidence (>90%) score as depicted in Figure P value 0.02069), response to stress , response to drug , biogenesis , structural molecular activity , protein dimerization activity  and extracellular space .The GO functional annotations for PTM genes subset were enriched using DAVID 6.8 for classification into BP (87.5% genes), CC (94.4% genes) and MF (87.5% genes). The details of annotations are presented in Table H2BC12, H2BC21 and KRT6A with antimicrobial humoral immune response mediated by antimicrobial peptide. Proteins BMPR2, NPPC, ZBTB16 found in defense response to gram-positive bacteria, CAD, CSN2, CSN3 in lactation and neurofilament in structural constituent of cytoskeleton. Estrogen signaling pathway was detected and proteins H3C1, H3C13, H3C15 were associated with pathways such as alcoholism, histone modifications, systemic lupus erythematosus ; CSN2, CSN1S1, CSN3 and LALBA were identified in single cluster and histone proteins interacted closely with CREB-binding protein.The PTM gene subset exhibited a network comprising of 8 clusters in STRING Fig. c The kerp < 0.05  were analysed in Reactome database, where 61 identifiers were found in 332 pathways hit by at least one of them. The majority of proteins were associated with signal transduction, immune system, developmental biology and metabolism pathways. Pathways such as signaling by nuclear receptors, WNT, Notch and RHO GTPase were significant with The 201 peptide sequences with identified PTM sites were grouped as long (>25 amino acids), medium (7–25 amino acids) and small (<7 amino acids). The identified phosphopeptides were categorized as long (62 peptides), medium (145 peptides), and small (1 peptide). The functions of these peptides were determined from MBPDB for 80% alignment with the known peptides are presented in Table 26. Post-translational modifications (PTMs) of milk proteins contributed to their biological functions and their compositional complexity27. It has been commonly observed that phosphorylation of casein occurs at S or T amino acid residues in tripeptide sequences S/T-X-A, where X represents any AA residue and A is an acidic residue5. In the present study, we have identified phosphorylation occurring in S, T, Y, D amino acids and other post translational modifications as carboxymethylation, oxidation and acetylation. The phosphosites on Asp residues have been reported for the first time in goat milk. Post translational modification of proteins plays an important role to regulate the cellular processes, protein function, protein localization, and formation of protein complex. In eukaryotic cells, protein phosphorylation is among the most frequent post translational modifications28. Post-translational modifications in proteins are crucial for the activity state, localization and protein-protein interactions29. Therefore, molecular diversity of goat milk proteins needs to be explored to identify post translational modifications for studying potential biological role and protein–protein interaction.Non-bovine milk is attracting the researcher’s attention due to its nutrition and therapeutic applications. Goat milk, is leading in the area for nutraceutical formulation and drug development using goat mammary gland as a bioreactor. Goat milk has unique chemical, biochemical, physical and nutritional characteristics and has higher digestibility and lower allergenicity over cow milk32. Electrospray ionization (ESI) mass spectrometry (MS) is suitable for studying PTM, including phosphorylation and glycosylation, since the technique provides molecular mass determination of native proteins. In this study, we analysed the goat milk proteins to identify both casein and non-casein PTM sites using nLC-MS/MS. We processed distinct protein spots by mass spectrometry for identification of phosphorylation, oxidation, acetylation and caramidomethylation. A peptidome of 201 peptide sequences with post translational modifications identified 86 proteins/120 UniprotKB accessions  to 13P for others (αS2 CN)40. Bijl and others41 demonstrated that high αS1-CN-8P concentration in bovine milk is a great benefit for the production of uncooked curd cheese because αS1-CN-8P is hydrolyzed more efficiently by chymosin during ripening. Bovine proteome analysis showed more than 30 phosphorylated proteins which included 5 CSN2, 15 CSN1S1, 10 CSN1S2 and 4 CSN3 casein components42. Similarly, donkey milk showed 11 CSN3, 6 CSN1S1 and 3 CSN1S2 casein components43.Phosphorylation has been well characterized in the bovine caseins49. The present study resulted in 105 phosphopeptides from casein and non-casein proteins from all the analysed samples. The conserved peptide sequence of (SSSEE) in casein was also observed in CSN1S1 and CSN1S2 at 1P, 2P and 3P. The phosphosites were identified in casein and whey proteins and on 45 other low abundance proteins. The total number of phosphosites observed in the major milk proteins associated with S, T, Y and D residues were 32, 18, 11 and 21, respectively. The phosphorylation sites observed for CSN2, CSN1S1, CSN1S2, and CSN3 were 11P, 13P, 17P and 6P, respectively  and LALBA showed 4 phosphosites . The identified phosphopeptides resulted in 12P, 8P, 11P, 5P, 13P and 4P isoforms of CSN1S1, CSN1S2, CSN2, CSN3, BLG and LALBA, respectively. Beta casein showed highest degree of variation in phosphorylation with identification of 17P sites and other PTM such as oxidation in the identified peptides. A higher number of casein phosphopeptides and phosphorylation sites are reported in the present study in comparison to previous study18.Goat milk proteins have been analysed using MS/MS50. The phosphorylation degree of αS-casein is a prime factor affecting the technological properties of milk. Therefore, “signature peptides” and “caseome” analysis are being used to investigate adulteration in milk of different species51. The identification of cheese from different species has been authenticated by proteolytic peptides53. Therefore, proteome analysis of fermented milk products should be carried out due to their nutritional and health economic importance.Casein and whey proteins are post translationally modified by proteolysis by the milk enzymes, formation of disulphide bond by oxidation of cysteine, differential phosphorylation levels of serine and threonine, and glycosylation of threonine residuesThe present study reported 45 non casein phospho proteins assigned to various metabolic pathways Table . The ide54. Peptides with carbamidomethylation are mainly used in peptide mass fingerprinting for identification and characterization of proteins55.The other post translational modifications such as acetylation, oxidation, and carbamidomethylation have also been reported Table . The posPRSS1) and histone proteins interacted with CREB binding protein (CREBBP). The identified phosphoproteins were associated with lactation, response to stress, histone modification, cornification and signaling pathways  in casein was observed in casein phosphopeptides . Phosphorylation regulates protein functions, such as biological activity, interaction and stabilization by causing conformational changes in the protein. Therefore, the identification of the post-translational modifications of the milk proteome has become necessary and may provide newer insight to extend the milk proteome and its potential biological role.The work flow of the present study has been depicted in Fig. Goat milk samples were collected from 1240 animals belonging to 10 goat breeds/ genotypes during the postpartum days 30–65. Milk samples were collected from natural habitats of the breeds belonging to different geographical and agro-climatic regions of India. Samples were collected between 7:30 and 9:00 hrs. by hand milking. After disinfecting the udder, 30–40 ml of milk was collected directly to the collection tubes, transported to the laboratory at 4ºC within 24 hrs. and stored at -20ºC. Milk subsamples for protein analysis were stored at -40ºC until further analysis. All sample collection was conducted in accordance with institutional practice and the study was approved by Institutional animal ethics committee (IAEC).The breeds analysed in the study belonged to arid, semi-arid, humid, coastal and mountain regions with different grazing conditions Fig. . The det44. The skimmed milk samples were centrifuged at 12000g, 4ºC, 15 min to obtain clear transparent aqueous layer. This layer was separated, quantified and reduced in Laemmli’s sample buffer for analysis by SDS-PAGE. Milk proteins were resolved on 14% SDS and urea PAGE using 8 µg in each lane. The gels were stained with commasie brilliant blue (R250) and subsequently scanned in the Gel documentation system . Milk protein variants were identified by comparing the allelic variation with reference samples (confirmed by sequencing) by determining the molecular weight.The milk protein variants were analysed by SDS-PAGE and details have been described elsewhereThe protein variants (n=21) identified in the 10 breeds using SDS-PAGE were selected for analysis by 2DE. Individual protein samples were subjected to in–gel rehydration of IPG strips (SERVA IPG Bluestrip 3–10 NL/7cm). Rehydration was carried out at room temperature for 16 hrs. with protein sample diluted in rehydration buffer . For first dimensional electrophoresis, IPG strips were transferred to Hoefer IPG phor II. Iso electro focusing (IEF) was then performed at 20°C by a series of steps as follows: constant 250 V, 1:00 hrs.; constant 500 V, 1:00 hrs.; gradient 1000 V, 1:00 hrs.; gradient 3000 V, 2:00 hrs.; constant 3000 V, 2:00 hrs. The strips were then equilibrated in SDS equilibration buffer  for 20 min. and loaded onto a 14% acrylamide gel for second dimension resolution. Second dimension run was carried out with mini gel SE 260 at 4V/cm. The 2DE protein spots were visualised by staining with 0.2% silver nitrate solution.44 and used with a minor modifications for PTM analysis.Protein digestionThe details of nLC-MS/MS analysis has been described elsewhereb.Liquid chromatography mass spectrometry analysisThe silver stained protein spots in gels were excised carefully with help of a sterilized spatula and transferred into separate vials. Destaining and acetone precipitation was carried out. In-gel digestion was performed; samples were reduced with 5 mM TCEP (Tris 2-Carboxyethyl Phosphine) at 55°C for 1 hour and further alkylated with 50 mM iodoacetamide for 30 min at room temperature in the dark. The gel spots were shrinked with acetonitrile and air-dried for few minutes at room temperature followed by digestion with trypsin  for 16 hours at 37°C. Digests were dried using speed vac for 1 hour and pellet was dissolved in buffer A . Digests were cleaned by Sep-Pak. Titanium method was performed for phospho binding. Digests were cleaned up again using C18 silica cartridge  following manufacturer’s protocol and dried using speed vac. The desalted dried pellet was reconstituted in buffer A .c.Protein identification and PTM analysisAll the experiments were performed using EASY-nLC 1000 system (Thermo Fisher Scientific) coupled to Q Exactive mass spectrometer  equipped with nano electrospray ion source. Peptide mixture (1.0 µg) was loaded on a precolumn and was resolved using 5 cm PicoFrit column  filled with 1.9 µm of C18-resin . The peptides were loaded with buffer A and eluted with a 0–40% gradient of buffer B  at a flow rate of 500 nl/min for 10 min. The QExactive was operated using the Top10 HCD data-dependent acquisition mode with a full scan resolution of 70,000 at m/z 400. MS/MS scans were acquired at a resolution of 17500 at m/z 400. Lock mass option was enabled for polydimethylcyclosiloxane (PCM) ions (m/z = 445.120025) for internal recalibration during the run. MS/MS data was acquired using a data-dependent top 10 method dynamically choosing the most abundant precursor ions from the survey scan.Capra hircus, Ovis aries, Homo sapiens and Bos taurus). Due to scarcity of available protein annotations for goat, the analysis was carried out with goat, human, sheep and cow database with 7056, 20117, 27666, and 23869 entries respectively. For Sequest search, the precursor and fragment mass tolerances were set at 10–15 ppm and 0.5 Da, respectively. The protease used to generate peptides, that is the enzyme specificity was set for trypsin/P (cleavage at the C terminus of “K/R”: unless followed by “P”) along with maximum missed cleavages value of 2. Carbamidomethyl on cysteine as fixed modification and oxidation of methionine and N-terminal acetylation and phosphorylation  were considered as variable modifications for database search. Peptide spectrum match and protein false discovery rate (FDR) were set to 0.01.The .raw files generated were analyzed using Proteome Discoverer (v2.2) against the in-house Uniprot reference proteome database (http://www.uniprot.org/). Protein classification of the identified proteome and PTM genes subset were performed based on their functional annotations using Gene Ontology (GO) for biological process, subcellular localization and molecular function using the Database for annotation, visualization and integrated discovery (DAVID) version 6.8. The enrichment was performed with Homo sapiens database in background for the identified gene names and measured by fisher exact test in DAVID system.All the identified milk proteins were assigned their gene symbol via the Uniprot knowledgebase . STRING networks were calculated at highest confidence score of 0.900 for the entire set of milk proteins and for PTM protein subsets and interactions were clustered using MCL algorithm. The gene search for network was performed in 69 at threshold of 80% identity match for their known functions.The peptides showing modifications were grouped based on the length as small (<7AA), medium (7–25 AA) and long (>25AA). The functions of these peptides were determined by Milk bioactive peptide database (MBPDB)Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.


K700E mutation induces DNA damage via generation of genotoxic R-loops and stalled replication forks, defective homologous recombination, and increased replication fork degradation, which can be targeted with PARP inhibitors.The cancer-associated SF3B1 SF3B1 have been identified across several cancer types. This key spliceosome component promotes the efficient mRNA splicing of thousands of genes including those with crucial roles in the cellular response to DNA damage. Here, we demonstrate that depletion of SF3B1 specifically compromises homologous recombination (HR) and is epistatic with loss of BRCA1. More importantly, the most prevalent cancer-associated mutation in SF3B1, K700E, also affects HR efficiency and as a consequence, increases the cellular sensitivity to ionizing radiation and a variety of chemotherapeutic agents, including PARP inhibitors. In addition, the SF3B1 K700E mutation induced unscheduled R-loop formation, replication fork stalling, increased fork degradation, and defective replication fork restart. Taken together, these data suggest that tumor-associated mutations in SF3B1 induce a BRCA-like cellular phenotype that confers synthetic lethality to DNA-damaging agents and PARP inhibitors, which can be exploited therapeutically.Mutations in K700E mutation induces DNA damage via generation of genotoxic R-loops and stalled replication forks, defective homologous recombination, and increased replication fork degradation, which can be targeted with PARP inhibitors.The cancer-associated SF3B1  The DNA damage response (DDR) is a highly complex network of pathways that play an essential role in maintaining genome stability and protecting cells from malignant transformation. It is becoming clear that the DDR does not solely focus on regulating cellular pathways linked with DNA metabolism, such as DNA replication and repair, but has also been implicated in controlling a variety of different RNA-regulatory processes, such as transcription, mRNA splicing, RNA export, and nonsense-mediated decay. In relation to this, we previously found that a key DDR protein, BRCA1, forms a DNA damage–inducible complex with several key splicing factors including BCLAF1 and SF3B1, to stimulate the pre-mRNA splicing of a specific set of target genes, which function to facilitate DNA damage and DNA repair (SF3B1 is the most commonly mutated in human cancers. Mutations in this gene are recurrently observed in myelodysplastic syndromes (MDS) and other myeloid/lymphoid neoplasms, with the heterozygous missense substitution K700E being the most prevalent  at 3′ splice sites, thereby promoting efficient mRNA splicing . As a reMycoplasma free.293T  and U2OS (RRID:CVCL_0042) cells were obtained from the Cancer Research UK Cell Repository . 293T and U2OS cell lines were maintained in DMEM (Sigma-Aldrich) containing 10% FCS. U2OS-DR-GFP cells were a kind gift from Prof. Jeremy Stark . K-562 (RRID:CVCL_0004) cell lines were obtained from DSMZ and maintained in RPMI1640 supplemented with 10% FCS. Polyclonal, stable inducible GFP-RNaseH1 K562 cells were generated via lentiviral infection with pCLX-pTF-R1-DEST-R2-EBR65-RNH1-GFP lentiviral particles and selection was done with 10 μg/mL blasticidin. H2595 (RRID:CVCL_A545), H2591 (RRID:CVCL_A543), REN (RRID:CVCL_M202), and H28 (RRID:CVCL_1555) were cultured in RPMI1640 media supplemented with 10% FCS plus nonessential amino acids for the REN cell line. H2595 has the SF3B1-K700E mutation and the remaining cell lines are wild-type for SF3B1. All cell lines stocks have been authenticated by short tandem repeat profiling and were regularly verified as Irradiations (IR) were carried out using an X-RAD 225 kV X-ray generator (Precision X-ray Inc.) at a dose rate of 0.591 Gy/minute.P < 0.05; **, P < 0.01; ***P < 0.001; ****, P < 0.0001.293T and U2OS cells were seeded as single cells at various densities following transfection with siRNAs and 62 hours incubation. Ten hours later, cells were exposed to IR. After 7–14 days, cells were fixed, stained with crystal violet, and colonies counted and the surviving fractions calculated. Clonogenic survival assays performed on the K-562 suspension cell line were done using Methylcellulose base media . Cells were seeded at a density of 10,000 cells per mL, and the compound being tested was added at the desired concentrations and vortexed briefly before plating. After 10 days, Methylcellulose was stained for 16 hours at 37°C with 8 mg/mL of iodonitrotetrazolium chloride in ethanol. Experiments were carried out a minimum of three times, with significant differences in survival assessed by two-way ANOVA, with Tukey multiple comparisons test. Significant differences are indicated by *, siRNAs were obtained from QIAGEN. The sequences are listed as follows: SF3B1_5 – TACGAGTTTGCTTGGTCAGAA; SF3B1_7 – GACCGGGAAGATGAATACAAA and siSCR – AGCAGCACGACTTCTTCAAGT. siBRCA1 - ACCATACAGCTTCATAAATAABglII + NotI and cloning into the BamH1 NotI sites of pENTR1A (RRID:SCR_021263). The RNH1-GFP cassette was then transferred to the pCLX-pTF-R1-DEST-R2-EBR65 lentiviral plasmid, a gift from Patrick Salmon (RRID:Addgene_45952) via an LR (recombination reaction between attL and attR sites) recombination reaction, creating a single vector that expresses the Tet-repressor and blasticidin resistance gene under the control of a constitutive promoter and containing the RNHA1-GFP cassette under the control of a Tet-repressor regulated promoter. SF3B1-K700E and RNAseH1 D210N mutations were created by site-directed mutagenesis.pCMV6-AN-Myc-DDK  and pCMV6-AN-mRFP  were purchased from Origene. Inducible GFP-RNAseH1 plasmid was generated via restriction cloning an RNH1-GFP cassette from pEGFP‐RNASEH1, a gift from Andrew Jackson and Martin Reijns (RRID: Addgene_108699) using Whole-cell extracts (WCE) were prepared by lysing cells in two volumes of Lysis buffer . Thirty to 60 μg of WCE was resolved on 4%–12% Bolt Gels (Thermo Fisher Scientific). Protein was transferred to nitrocellulose membrane (Thermo Fisher Scientific) and blotted for SF3B1 ), Flag M2 , mRFP  and γ-tubulin , ATM , BRCA2 , ATR , Mre11 , BRCA1 , RAD51 , γH2AX , GAPDH , vinculin , β-actin , and GFP .t tests, with post hoc Holm–Šidák multiple comparisons tests. Significant differences are indicated by *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.Following treatment and appropriate recovery as indicated, cells were fixed in 4% PFA/PBS and then permeabilized in 0.4% Triton X-100/PBS followed by blocking in 3% BSA/PBS. Cells were stained with γH2AX , 53BP1 , or RAD51  primary antibodies and anti-mouse AlexaFluor 488/568 (Thermo Fisher Scientific) secondary antibodies. Cells were counterstained with DAPI to identify cellular nuclei. Representative images were acquired using a Nikon Eclipse Ti-S fluorescence microscope, with 60× objective or using an ArrayScan XTI High-content Imaging system with 40× objective (Thermo Fisher Scientific) and using the spot detection algorithm for foci/cell scoring. Cells were scored as positive or negative for unrepaired DNA damage as indicated in figures, with a minimum of 100 cells per condition scored with each condition per experiment carried out a minimum of three times independently. Significant differences between quantified data were assessed using multiple two-tailed D210 was induced for 24 hours with 1 μg/mL doxycycline, after which, nonbound GFP-RNH1D210 was extracted with extraction buffer  at 4°C for 2 minutes before fixation with 4% PFA. Fixed cells were then stained with DAPI and nuclear GFP intensity quantified via high-content imaging using a Thermo ArrayScan XTI. Significant differences between quantified data were assessed using multiple two-tailed t tests, with post hoc Holm–Šidák multiple comparisons tests. Significant differences are indicated by *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.Cells were fixed in 4% PFA/PBS followed by permeabilization with 0.4% Triton-X100 and blocking with 3% BSA/PBS. Cells were stained with S9.6 , and anti-nucleolin  primary antibodies and AlexaFluor 488/568 (Thermo Fisher Scientific) secondary antibodies. Cells were counterstained with DAPI before being imaged using a Nikon Eclipse Ti-S fluorescence microscope with 60× objective with quantification of nuclear S9.6 staining carried out by measuring mean fluorescence intensity within DAPI stained regions using imageJ (RRID:SCR_003070). Regions of nucleolin staining were subtracted from final quantification. Alternatively, GFP-RNH1U2OS-DR-GFP cells were a kind gift from Prof Jeremy Stark  and DR-GFP assays carried out as described previously .http://n2t.net/addgene:44026; RRID:Addgene_44026) was transfected into SF3B1WT and SF3B1K700E K-562 cells using the Amaxa 4D-Nucleofector as per the recommended protocol for K562 cells, after which, stably transfected cells were selected with puromycin (1 μg/mL). Cells were then infected with the I-SceI–carrying adenovirus AdNGUS24i  at an estimated multiplicity of infection of 10. NHEJ was assessed using flow cytometry to quantify GFP-positive cells 96 hours after AdNGUS24i infection. GFP positivity was assessed using flow cytometry with a minimum of 10,000 cells per condition with each condition/experiment carried out a minimum of three times. Significant differences between quantified data were assessed using multiple two-tailed t tests, with post hoc Holm–Šidák multiple comparisons tests. Significant differences are indicated by *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.The pimEJ5GFP non-homologous end joining (NHEJ) reporter plasmid, a kind gift from Jeremy Stark , the cells were spun at 1,400 rpm for 4 minutes, the supernatant was discarded. The cell pellet was washed in 20 mL HU-containing media and pelleted as before. The supernatant was discarded and cells resuspended in 2 mL of HU-containing media and incubated at 37°C for 2 hours. Cells were then pulse labeled with 250 μmol/L IdU and harvested for DNA fiber analysis as above. For fork degradation, 8.0 × 105 cells were grown for 24 hours and pulsed labeled with 25 μmol/L CldU for 20 minutes, followed by 250 μmol/L IdU for 20 minutes as above, after which, cells were washed and treated with 4 mmol/L HU for 5 hours. DNA fiber analysis was carried out as described previously .SF3B1eviously . For ongWT and SF3B1K700E K-562 cells were maintained at an exponential growth phase. Cell viability was determined using Trypan blue exclusion assay (minimum of 98% viability) prior to preparation for injection. Each mouse  received a single subcutaneous injection into the flank of a hind leg containing 1 × 106 cells per 100 μL of Matrigel suspension (100 μL/injection). Tumors were measured using a digital caliper until an average size of 100 mm3 was reached. Animals were then randomized into appropriate group sizes of average tumor size and treated with either vehicle (n = 6) for 5 days per week, 50 mg/kg olaparib (n = 6) for 5 days/week, or 20 mg/kg etoposide (n = 6) 1 day per week. Animal weight was assessed prior to each treatment and tumor volume was assessed three times weekly. Animals were humanely euthanized when maximum tumor size was reached . Fold change from initiation of treatment was calculated for each tumor per mouse, with mean tumor volumes per group reported. Significant differences in tumor volume were assessed at day 28 using multiple two-tailed t tests, with post hoc Holm–Šidák multiple comparisons tests. Multiple comparisons Padj values have been reported.All animal studies within this manuscript were approved by the Queen's University Belfast Animal Welfare and Ethics Review Board (AWERB) and the Northern Ireland Department of Health. SF3B15 cells were seeded and grown for 24 hours before 10 μmol/L BrdU was added for 2 passages (48 hours). Cells were then treated with 2 Gy IR and allowed to recover for 36 hours, after which, 0.2 μg/mL colcemid (KaryoMax) was added and cells incubated for 12 hours. Cells were then pelleted and resuspended gently in 10 mL prewarmed 0.075 mol/L KCl incubated at 37°C for 20 minutes, following which, 2 mL of Carnoy's fixative (3:1 methanol/acetic acid) was mixed in and the cells pelleted. Cells were resuspended in 10 mL Carnoy solution and cells left overnight at 4°C. Cells were then pelleted and resuspended in 2 mL Carnoy solution and then dropped onto wet slides prewarmed at 45°C. Slides were then incubated in 10 μg/mL Hoescht for 20 minutes in the dark, followed by incubation in 20xSSC buffer for 5 minutes. 1 mL of 20xSSC was added to each slide and a coverslip placed on top before slides were placed under a UV lamp for 1.5 hours. Following this, slides were placed in 20xSSC buffer for 1 hour at 60°C. The slides were washed in water before being stained with Giemsa (1:20 dilution in water) for 15 minutes, washed again in water for 3 minutes, and left to dry overnight. Coverslips were mounted with Cytoseal and imaged and exchanges counted using a Nikon Eclipse Ti-S microscope. Quantification of sister chromatid exchanges was carried out from three independent experiments with ≥50 spreads were scored/experiment. Significant differences in the number of SCEs were assessed using two-tailed t tests. ***, P < 0.001; ****, P < 0.0001.8.0 × 10Total, nuclear, and cytoplasmic RNA preparations, RNA sequencing (RNA-seq), and downstream data analysis, including differential splicing analysis using dSpliceType was carried out as described previously . IngenuiRNA-seq data is available in the ArrayExpress (EMBL-EBI) repository, under accession E-MTAB-7192.We have previously shown that the BRCA1 mRNA-splicing complex, which includes SF3B1, facilitates DNA repair, in part through its ability to promote the efficient splicing of genes involved in the DNA damage response . Intriguingly, while we did not observe any appreciable changes in the expression of key HR proteins such as BRCA1, Rad51, ATM, ATR, Mre11, and BRCA2 in SF3B1-mutant cells , as outlined above, we did observe A3SS usage and SEs in a large number of genes involved in HR, suggesting that SF3B1 mutation may have a polygenic effect on HR. Taken together with the role for SF3B1 in DSB repair highlighted above, we next tested the impact of the cancer associated SF3B1K700E mutation on DSB repair. Indeed, the SF3B1K700E cells were unable to resolve 53BP1 or γH2AX marked DSBs at late times postirradiation compared with their SF3B1WT counterpart (K700E in SF3B1 wild-type cells and a mesothelioma-derived cell line harboring the SF3B1K700E mutation (H2595) compared with a matched SF3B1 wild-type control mesothelioma-derived line . In addition, using the DR-GFP HR reporter, HR-directed repair was impaired in U2OS cells transfected with ectopic SF3B1K700E compared with those transfected with SF3B1WT . Intriguingly, we also observed increased 53BP1 foci in SF3B1K700E cells compared with SF3B1WT cells, even in the absence of DNA damage, suggesting increased basal damage in these cells .To assess the biological impact of this deregulated splicing and export, we performed IPA on genes with 3ASS and SE in SF3B1se cells  and B. BK700E mutation on HR, we assessed the formation and resolution of Rad51 foci, a key marker of HR function, in our isogenic models following IR-induced DNA damage. Intriguingly, unlike cells lacking BRCA1, SF3B1K700E cells exhibited a normal ability to recruit Rad51 to sites of DSBs, indicating a functional BRCA1/Rad51 pathway. Interestingly, however, Rad51 foci persisted in the SF3B1K700E cells at the 24 hours timepoint postirradiation, indicative of a defect in the later stages of HR following Rad51 loading and nucleofilament formation . In keeping with this, both the increased 53BP1 foci in unperturbed SF3B1K700E cells, as well as unresolved 53BP1 foci 24 hours following IR, predominantly occurred in EdU-positive cells, suggesting that these DSBs arise and/or fail to be repaired in S-phase . To examine the impact of SF3B1 mutation on HR further, we assessed sister chromatid exchanges (SCE), an end product of a proportion of HR-mediated repair events. Indeed, in both unperturbed cells, and following IR, we observed significantly decreased SCEs in SF3B1K700E cells, compared with wild-type cells .Importantly, HR defects consistently induce sensitivity to DNA-damaging chemotherapeutics, such as etoposide, and to PARP inhibition, through synthetic lethality. Therefore, we assessed the sensitivity of SF3B1nterpart  and H. IWT and SF3B1K700E cells using a fluorescent NHEJ reporter that measures NHEJ mediated joining of two tandem endonuclease cut sites, resulting in the juxtaposition of an active promoter upstream of a functional GFP gene .To ensure that the increased sensitivity of SF3B1-mutant cells to etoposide was specifically caused by a defect in HR, we also assessed NHEJ function in SF3B1K700E cells. Importantly, a number of cancer-associated mutations in mRNA splicing factors that disrupt splicing efficiency and/or splice-site recognition have been shown to induce R-loops, which can themselves lead to DNA damage, particularly when encountered by an advancing replication fork, resulting in replication fork stalling .Our data clearly suggests a role for SF3B1 in the DDR, particularly in HR-mediated DNA repair following DNA damage. However, we have also observed increased DSBs and reduced HR in unperturbed SF3B1stalling . Given tnterpart . We also R-loops . This re R-loops . ImportaK700E-mutant expressing cell line affects DNA replication, we examined replication fork dynamics using the DNA fiber assay .To test whether the increased R-loops observed in SF3B10E cells . CombineK700E cells displayed an increased sensitivity to etoposide and olaparib, we utilized the DNA fiber assay to determine whether this requirement for SF3B1-dependent repair and restart of damaged replication forks could underlie the hypersensitivity of the mutant cells to etoposide and olaparib. In keeping with this notion, the mutant SF3B1 cells displayed a striking reduction in replication fork progression following exposure to either etoposide or olaparib, when compared with their SF3B1WT counterpart .Because we had previously shown that SF3B1K700E cells display a BRCA-like phenotype, we investigated whether mutations in SF3B1 could also compromise replication fork stability. This was carried out by dual labeling of newly replicated DNA with equal sequential short pulses of CldU and ldU, followed by a prolonged HU-induced replication fork arrest . Taken together, our data demonstrate that the common tumor-associated mutation in SF3B1, K700E, recapitulates many of the cellular deficits that are characteristic of loss of BRCA1/2.Finally, it is known that following replication arrest, several proteins, including BRCA1 and BRCA2, are required for preventing uncontrolled nucleolytic degradation of damaged forks and loss of this function contributes to the hypersensitivity of HR-deficient tumors to replication stalling genotoxic agents. Given that we have shown that SF3B1k arrest . Interesterparts . In keepK700E mutation gives rise to an HR defect, we hypothesized that this could be exploited therapeutically to treat tumors that have acquired this mutation. To test this hypothesis, we engrafted the SF3B1WT and SF3B1K700E cell lines subcutaneously into immunocompromised mice, allowed the tumors to grow, treated the mice with etoposide, olaparib, or a vehicle control and monitored tumor growth for 28 days . These observations provide the first evidence that tumor cells harboring the SF3B1K700E mutation can be selectively targeted in vivo using a synthetic lethality approach.Because our data indicate that the SF3B1 28 days . In keepT tumors . In addiSF3B1, with SF3B1K700E occurring in a wide range of cancer types at varying frequencies. Despite the high frequency of SF3B1 mutations, the functional impact of these on tumor development remains unknown. Moreover, the impact of SF3B1 mutations on overall survival is disease dependent, for example, SF3B1 mutations in uveal melanoma are associated with improved overall survival, while associated with poor overall survival in acute myeloid leukemia . Notably, both genes have been previously shown to be important for HR downstream of Rad51 loading (K700E arise directly from aberrant splicing of either of these genes.Whilst a number of studies have linked loss of mRNA splicing machinery components with aberrant DNA damage response (DDR) activation and/or defective DNA repair , it is cSPR/Cas9 . Importaor cells . More imechanism . Interes loading  althoughK700E cells displaying increased levels of spontaneously stalled forks, which can be reduced following the expression of RNaseH1. Despite this, the inability of SF3B1K700E cells to protect and restart replication forks stalled with HU, or to efficiently replicate in the presence of DNA-damaging agents, known to induce replication fork reversal, cannot be adequately explained simply by an increased presence of R-loops. In line with this, the induction of RNAse-H1, which degrades R-loops, did not alter the sensitivity of SF3B1K700E cells to etoposide or olaparib. In this respect, it is more plausible that these phenotypes can be explained by missplicing or reduced mRNA export of a gene(s) involved in HR. Intriguingly, unlike the other Rad51 paralogs, XRCC3 has been recently shown to be important for the restart of stalled replication forks , whom are often not deemed fit for treatment with DNA-damaging therapies such as etoposide, which can be relatively toxic. Therefore, these patients would likely benefit from a less toxic, targeted approach, such as that offered by PARP inhibitors.Importantly, irrespective of the underlying mechanism, we demonstrated that the HR and replication defects arising as a consequence of the SF3B1K700E mutation gives rise to a BRCA-like cellular phenotype and as such, indicates that the presence of this mutation and perhaps other tumor-associated spliceosome mutations could form the basis of a clinical trial to explore the efficacy of synthetically lethal inhibitors as a treatment option to specifically target these tumor cells for killing.Taken together, our data demonstrate that the SF3B1K.I. Mills reports grants from Queen's University Belfast during the conduct of the study. D. Harkin reports grants from Medical Research Council and grants from Cancer Research UK during the conduct of the study; other support from Almac Diagnostic Services outside the submitted work. G.S. Stewart reports grants from Cancer Research UK and grants from Great Ormond Street Hospital Charity and SPARKS during the conduct of the study. K.I. Savage reports grants from Cancer Research UK and grants from Medical Research Council UK during the conduct of the study. No disclosures were reported by the other authors.Supplementary DataClick here for additional data file.Supplementary TableClick here for additional data file.Supplementary TableClick here for additional data file.


Genetic differences between C57BL/6 substrains lead to different cardiovascular traits; a null mutation in Mylk3 likely causes cardiomyopathy in C57BL/6N mice, whereas C57BL/6J Nnt-null mice do not develop cardiomyopathy. Nnt) null mutation in the C57BL/6J substrain, which has been linked to cardiovascular traits in mice and cardiomyopathy in humans. To assess whether Nnt loss alone causes a cardiovascular phenotype, we investigated the C57BL/6N, C57BL/6J mice and a C57BL/6J-BAC transgenic rescuing NNT expression, at 3, 12, and 18 mo. We identified a modest dilated cardiomyopathy in the C57BL/6N mice, absent in the two B6J substrains. Immunofluorescent staining of cardiomyocytes revealed eccentric hypertrophy in these mice, with defects in sarcomere organisation. RNAseq analysis identified differential expression of a number of cardiac remodelling genes commonly associated with cardiac disease segregating with the phenotype. Variant calling from RNAseq data identified a myosin light chain kinase 3 (Mylk3) mutation in C57BL/6N mice, which abolishes MYLK3 protein expression. These results indicate the C57BL/6J Nnt-null mice do not develop cardiomyopathy; however, we identified a null mutation in Mylk3 as a credible cause of the cardiomyopathy phenotype in the C57BL/6N.The C57BL/6J and C57BL/6N mice have well-documented phenotypic and genotypic differences, including the infamous nicotinamide nucleotide transhydrogenase ( Many genetic differences are known to exist between these two closely related substrains with perhaps the most commonly studied the nicotinamide nucleotide transhydrogenase (Nnt) mutations in the B6J.Differences in background genetics can produce many different baseline phenotypes in the ever-growing library of laboratory mouse strains, and these can have profound influences on the expression of particular trait(s) , 3, 4, 5NNT was identified as the causative gene in 15 families with familial glucocorticoid deficiency, a disorder of adrenal insufficiency without heart involvement   . We also6J-Nnt]) , 15, 17.Mylk3), which is only present in B6N substrains. MYLK3 phosphorylates myosin light chain 2 (MYL2), which is itself essential for the assembly of actin fibres in the heart and mutated in many human cases of CM , and there were no significantly enriched pathways between the B6N and B6J-Nnt, nor the B6J and B6J-Nnt (data not shown). We subsequently filtered the expression data for genes which were regulated in a strain-dependent manner , myosin light chain 4 (Myl4), myosin light chain 7 (Myl7), troponin T1, skeletal type (Tnnt1), natriuretic peptide precursor A (Nppa), and natriuretic peptide precursor B (Nppb). These genes are markers of hypertrophy and are often altered in the heart in disease states as the tissue reverts to a more “foetal” expression pattern Myh7 and Tnnt1), whereas the others showed a strain-dependent trend. No sequence variants were found in these genes to explain their differential expressions (Table S5).Within the cluster of genes associated with muscle contraction, we found many genes typically associated with cardiac remodelling up-regulated in the B6N: myosin heavy chain 7 , 43 variants amongst the B6N and B6J substrains were identified, 38 in the B6N and 5 in the B6J, potentially affecting 63 genes (Table S5). A variant in Mylk3, Chr8:85365179A>T, c-5T>A, SNP ID rs245783224, was considered a good candidate because it is almost exclusively expressed in the heart, with ablation resulting in cardiac dysfunction, which is modest in the germ line knockout , immediately upstream of the luciferase start codon. Luciferase signal was normalised by co-transfecting with a GFP vector and normalising luminescence to fluorescence  or variant (B6N) 5′ UTRs of rescence . Lucifernslation . This wapression  and no opression .Mylk and Mylk2 expression levels were very low in the heart; however, Mylk4 expression level was similar to Mylk3. Interestingly, Mylk4 expression was higher in the B6N mice and was identified as a strain-dependent gene by RNAseq (Table S2). MYLK4 could phosphorylate key targets in the absence of MYLK3 in the B6N, and this may allow the mice to survive to adulthood. RT-qPCR revealed Mylk4 was ∼1.8-fold higher in the B6N group at both 3 and 12 mo; however, there was no difference in protein expression and is, therefore, unlikely to be able to compensate for loss of MYLK3 (data not shown).We analysed the other three myosin light chain kinase genes, suspecting one of these may compensate for the absence of MYLK3 in the B6N mouse. RNAseq data revealed Nnt mutations in B6J mice, many groups have used this mouse in conjunction with the B6N to study the role of Nnt, as one might with a traditional knockout and corresponding wild-type model  was previously described and, although that study found no evidence of DCM at an early time point (16 wk), it did show the B6N heart papillary muscles contracted with less force than those from B6J hearts  and angiotensin II treatment observed in other studies comparing C57BL/6 mouse substrains , 17. Thefter TAC . Interesfter TAC . We beliMyh7 than hearts from the two B6J substrains. Myh7 is one of two main cardiac myosin heavy chain isoforms, the other being myosin heavy chain 6 (Myh6)  . In huma6 (Myh6) , 55. In 6 (Myh6) . Althoug6 (Myh6) . IsolateMyh7 in B6N reflects a stress adaptation of the tissue due to the dilation, particularly as the RNAseq was performed on 18-mo heart tissues. Under conditions of stress, transcription of foetal genes such as Myh7 are often increased and this is thought to be due to a de-repression of their expression by microRNAs , and the National Institutes of Health Knockout Mouse (KOMP) have moved to the C57BL/6N as a background strain because of greater ES cell efficiency for homologous recombination , 59.Diabetes, 63% of all published articles between 2010 and 2014 that used a variant of the C57BL/6 strain and made no mention of the exact substrain used , temperature (21°C ± 2°C), and humidity (55% ± 10%) conditions. They had free access to water (9–13 ppm chlorine) and were fed ad libitum on a commercial diet . The mouse strains used were C57BL/6NTac originally from Taconic (Taconic Biosciences), C57BL/6J distributed by Charles River (Charles River UK), and C57BL/6J mice carrying a bacterial artificial chromosome transgene to restore murine Nnt TG1Rdc/H). All mice used were no more than 10 generations of mating removed from restock.All mice were bred, housed, and culled at Medical Research Council Harwell, and therefore, the husbandry was identical for all three substrains. Only male mice were used in this study. Mice were kept and studied in accordance with the UK Home Office legislation and local ethical guidelines issued by the Medical Research Council . Mice were kept under controlled light  according to the IMPC phenotyping protocol. More details of this procedure can be found on g in a refrigerated centrifuge set to 8°C. Plasma samples were analysed for sodium, potassium, chloride, glucose, triglycerides, free fatty acids, and total-, high-density lipoprotein–, and low-density lipoprotein cholesterol on board a Beckman Coulter AU680 clinical chemistry analyser using reagents and settings recommended by the manufacturer and as per the IMPC phenotyping pipeline (http://www.mousephenotype.org/impress/procedures/7).Free-fed mice were terminally anaesthetised at 3, 12, or 18 mo with isoflurane, and blood was collected from the retro-orbital sinus using uncoated glass capillary tubes  into either lithium heparin or EDTA-coated paediatric tubes . Lithium heparin samples were kept on wet ice and centrifuged within 1 h of collection for 10 min at 5,000After bleeding, mice were dissected under a laminar flow tissue culture hood. Tissues were removed and immediately snap frozen in liquid nitrogen or immersed in 4% PFA. All animals were culled between 1,000 and 1,130 h. All animal protocols in this study were approved by the United Kingdom Home Office.Frozen isolated heart tissues were weighed to an accuracy of 0.1 mg. Tibia lengths of C57BL/6N and C57BL/6J mice were measured using X-ray at 13 wk.Nnt status using previously published primers (Genomic DNA was extracted from the mouse tail tissue using a DNeasy tissue kit (QIAGEN). Mice were genotyped for  primers .Five 18-mo-old mice of the three different substrains were used per group. RNA was isolated from intact hearts using the RNeasy Mini Kit (QIAGEN). RNA samples were processed by INVIEW Transcriptome Discover service (GATC Biotech) and resultant FASTQ files processed as follows.http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/), a wrapper tool around Cutadapt (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/), to filter reads with quality Phred score cutoff less than 20 and trim Illumina TruSeq adapters in FASTQ files.We used Trim Galore (Cutadapt  and FastM. musculus reference genome (GRCm38)  , discard(GRCm38)  to calcuhttps://software.broadinstitute.org/gatk/documentation/article.php?id=3891). We used the STAR 2-pass method from STAR aligner . We used SplitNCigarReads from GATK (Preprint) for variant calling. Variants were validated by visualising alignments in the Integrative Genomics Viewer browser , 79 variants amongst the B6N and B6J substrains were identified, potentially affecting 98 genes. Visual inspection of these variants in Integrative Genomics Viewer browser allowed us to eliminate 35 variants on the basis (mostly) of misalignment of short-read sequencing in repetitive regions, leaving 43 variants which differed from the B6J reference sequence with the potential to affect 63 genes. 38 of these varied in the B6N substrain with the remaining five in B6J, all were biallelic with the exception of one variant, in cue domain-containing protein 1 (Cuedc1) which was monoallelic in B6J. This apparent heterozygosity is likely due to alignment of short sequencing reads to more than one locus.Compared with the Mylk3, Chr8:85365179A>T, c-5T>A, SNP ID rs245783224, was considered a likely candidate because MYLK3 is almost exclusively expressed in the heart, with ablation resulting in cardiac dysfunction, which is modest in the germ line knockout  and synthesised by Sigma-Aldrich (Merck). Primer sequences are listed in the following table.g for 15 min, and the resultant supernatants were normalised for total protein concentration with a bicinchoninic acid assay. Protein lysates were added to an equal volume of Laemmli buffer. Lysates were heated at 95°C for 5 min and then loaded into 4–12% SDS gels. Immunoblots for NNT were performed with unheated samples. Proteins were then separated based on size using electrophoresis and transferred to a nitrocellulose membrane (Sigma-Aldrich) by semi-dry transfer blot (Bio-Rad) at 15 V for 1 h. Membranes were probed with a target antibody and GAPDH. Antibodies used are as follows: mouse anti-NNT , mouse anti-TNNT2 , mouse anti-Sarcomeric Alpha Actinin , rabbit anti-MYLK3 , rabbit anti-GADPH , and rabbit anti-MYL2 .Protein lysates were obtained by homogenising and lysing tissues in radio immunoprecipitation assay buffer for 2 h at 4°C. The lysates were sonicated at 20 kHz twice for 10 s, then centrifuged at 16,000Freshly isolated hearts were immersed in 4% PFA on ice overnight. Tissues were then dehydrated and embedded in paraffin wax. We cut 7-μm sagittal sections through the tissues, making note of serial section number. Sections obtained at similar planes were then used for haematoxylin and eosin staining using a Leica Autostainer We stained paraffin-embedded heart sections as described in reference We used two detection methods for protein localisation, one using chromogenic diaminobenzidine oxidation assays and the other epifluorescence coupled with conjugate fluorescent dyes. Chromogenic diaminobenzidine staining of MYLK3 was performed by a Ventana automated staining station using the OmniMap Rabbit HRP (Roche). We imaged stained sections using a Panoramic 250 (3D Histech) slide scanner with CaseViewer software.For epifluorescence staining, paraffin tissue sections were cleared and dehydrated followed by boiling in a citrate buffer to expose protein epitopes. Specimens were then blocked in a BSA–serum mixture, followed by primary antibody incubation overnight. Samples were washed and incubated with secondary antibody, washed and finally mounted in an aqueous mountant. For immunofluorescent sections, tissues were counterstained with DAPI and wheat germ agglutinin Alexa Fluor 555 conjugate (Thermo Fisher Scientific) before background removal with TrueView Autofluorescence quenching kit (Vector Labs) followed by mounting in VectaShield mounting medium. Fluorescent slides were imaged on a Zeiss LSM 880 with Airyscan and quantified by MyofibrilJ package in ImageJ. DAB-stained slides were imaged using a Panoramic 250 (3D Histech) slide scanner with CaseViewer software. For sarcomeric morphometrics, longitudinal and transverse heart sections were used to measure the long axis  and CSA (transverse section) of individual cardiomyocytes delineated by WGA, alpha-actinin , and MYL2  staining. Confocal microscopy was performed with an LSM880 Microscope (Zeiss) on a 63× objective.Mylk3 transcript variant 1 from B6N or B6J mice was cloned immediately upstream of the luciferase ORF in PGL3 (Promega) by Gibson cloning (The 5′ UTR of  cloning  from a gA MYLK3 ORF clone generated by GenScript (Clone ID: OMu23765) in the pcDNA3.1(+) vector was used as the backbone for subsequent 5′UTR cloning. 5′ UTR of B6N and B6J MYLK3 was cloned immediately upstream of the canonical ATG and immediately downstream of the T7 promoter by Gibson Assembly  and confStatistical tests are described on a per experiment basis in the figure legends describing the experiment in question.PRJNA531833, http://www.ncbi.nlm.nih.gov/bioproject/531833.The dataset supporting the conclusions of this article is available in the NCBI Bioproject repository: Bioproject ID