Neurotransmitter release requires assembly of the SNARE complex fusion machinery, with multiple SNARE-binding proteins regulating this process to control when and where synaptic vesicle fusion occurs. Complexin (Cpx) controls spontaneous and evoked neurotransmitter release by modulating SNARE complex zippering. Although the central SNARE-binding helix is essential, post-translational modifications to Cpx’s C-terminal membrane-binding amphipathic helix modulate its activity. Here we demonstrate that RNA editing of the Cpx C-terminus regulates its ability to clamp SNARE-mediated fusion and alters presynaptic output. RNA editing of Cpx within single neurons is stochastic, generating up to eight edit variants that fine-tune neurotransmitter release by changing the subcellular localization and clamping properties of the protein. Similar editing rules for other synaptic genes were observed, indicating stochastic editing at single adenosines and across multiple mRNAs can generate unique synaptic proteomes within the same population of neurons to fine-tune presynaptic output. Single SVs also fuse spontaneously to generate mini events. Complexin (Cpx) and the Ca2+ sensor Synaptotagmin 1 (Syt1) bind and regulate SNARE complexes to control whether SVs fuse spontaneously or through the evoked pathway3. Cpx can arrest zippering of the SNARE complex at the SV/plasma membrane interface to maintain SVs in a fusion-ready state and allow Ca2+-bound Syt1 to rapidly trigger release6. Indeed, invertebrate Cpxs act as “fusion clamps” to reduce spontaneous release in the absence of Ca2+10. Changes in Cpx activity alters spontaneous release rates and regulates structural and functional synaptic plasticity14. The Cpx C-terminus has emerged as a key site for such regulatory control, as it encodes a conserved amphipathic helix that functions as a membrane curvature sensor to localize Cpx to SVs and concentrate its activity at release sites15.Neuronal communication is initiated by CaDrosophila, a single cpx gene produces two isoforms with different C-termini due to alternative splicing of exon 716. The Cpx7A isoform has a conserved membrane-tethering prenylation CAAX box, while Cpx7B lacks this motif17. Prior studies demonstrated Cpx7B is regulated by PKA phosphorylation of a serine residue (S126) within this alternatively spliced C-terminus region. PKA phosphorylation of Cpx7B reduces its clamping function at neuromuscular junctions (NMJs), leading to elevated spontaneous release that triggers activity-induced structural plasticity11. The Cpx7A isoform lacks this serine residue and instead undergoes RNA editing by ADAR (adenosine deaminase acting on RNA) to generate multiple Cpx7A proteins with unique C-terminal sequences. A-to-I editing can recode pre-spliced mRNAs by deaminating target adenosines in double-stranded RNA structures induced from exon-intron complementary pairing, causing the resulting inosine base to be read as guanosine by the translation machinery18. RNA editing of Cpx7A can trigger changes at two resides, including an isoleucine (I125) to methionine substitution. In addition, editing of two adjacent downstream adenosines can change an asparagine (N130) to a glycine (N130G), aspartate (N130D), or serine (N130S) at a site near the phosphorylated S126 residue in Cpx7B19. Given Cpx7A is expressed at higher levels and is the dominant isoform in the Drosophila nervous system16, RNA editing at this site represents an attractive mechanism for regulating neurotransmitter release and structural plasticity across a larger population of neurons.In I125M,N130S) can be phosphorylated by Casein kinase 2 (CK2). Transgenic rescue of cpx null mutants with Cpx7AI125M,N130S demonstrates RNA editing alters the protein’s subcellular localization and reduces its ability to clamp spontaneous SV fusion, leading to synaptic overgrowth. Rescue with both Cpx7AI125M,N130S and unedited Cpx7AI125,N130 indicates the N130S variant acts in a dominant fashion, consistent with a model where multiple Cpxs engage assembling SNARE complexes during SV fusion20. Such a mechanism would allow edited and unedited Cpx proteins to assert independent effects in a combinatorial fashion to control fusion dynamics. Together, these data indicate stochastic RNA editing of the Cpx C-terminus can set distinct spontaneous release rates in individual Drosophila neurons to regulate presynaptic output.Here we used CRISPR and transgenic rescue to assay the role of Cpx splicing and RNA editing in neurotransmitter release. Although Cpx7A is expressed at higher levels, the two isoforms are largely redundant in their ability to support baseline synaptic transmission. Analysis of single-cell RNAseq data from individual motoneurons reveals multiple Cpx7A RNA editing variants can be simultaneously expressed, indicating RNA editing does not act in an “all-or-none” fashion. The most prominent edit variant  to methionine (M) conserved substitution at amino acid 125 that is not predicted to alter protein function, but induces mRNA conformational changes in exon-intron base pairing that facilitates editing of two downstream adenosine residues16. At this downstream site, the unedited AAT codon encodes an asparagine (N) at amino acid 130. Editing of both residues (AAT to GGT) produces a glycine (N130G), while editing of only the 1st base (AAT to GAT) generates an aspartic acid (N130D) and editing of the 2nd base (AAT to AGT) produces a serine (N130S)  . Given Rl domain .27, helical wheel models were generated with HELIQUEST28 to examine hydrophilic and hydrophobic faces of the helix in relation to the Cpx7A N130 editing site and the Cpx7B S126 PKA phosphorylation site , spontaneous mini frequency is dramatically elevated (>50-fold), evoked release is decreased, and larval NMJ synaptic growth is enhanced7. Overexpression of either Cpx7A or Cpx7B in cpxSH1 partially rescues these phenotypes, with Cpx7A having more robust clamping properties and Cpx7B over-rescuing evoked release16. Although both variants support aspects of Cpx function when overexpressed, endogenous Cpx7A mRNA is >100-fold more abundant than Cpx7B in Drosophila adults and larvae based on quantitative RT-PCR16. As such, Cpx7A is hypothesized to play a more critical role in synaptic transmission. To directly test the endogenous function of the two splicing isoforms, CRISPR mutants disrupting 7A (cpxΔ7A) or 7B (cpxΔ7B) were generated by introducing an early stop codon at the beginning of exon 7A or 7B, respectively. Western analysis of adult head lysates with a Cpx antibody that recognizes both variants showed an 87% reduction in overall Cpx expression in cpxΔ7A (p < 0.0001) and a milder 10% reduction in cpxΔ7B (p = 0.9398), consistent with Cpx7A being the predominant isoform  and no detectable decrease in cpxΔ7B mutants (p = 0.9567).Before characterizing the impact of RNA editing on Cpx7A function, we first examined the role of Cpx alternative splicing to gain greater insight into the endogenous roles of Cpx7A and Cpx7B. In  isoform . Immunosr effect , C, within vivo, adult and larval motor behavior were examined in cpxΔ7A and cpxΔ7B and compared to control and cpxSH1 nulls. Complete loss of Cpx in cpxSH1 severely disrupts behavior and reduces viability, with the few escaper adults that emerge from the pupal case displaying a profound loss of motor control and an inability to walk in a coordinated manner. Loss of the predominant Cpx7A isoform also strongly disrupted motor behavior, though not as severely as cpxSH1. In contrast to cpxSH1 where homozygous adults are rarely observed, cpxΔ7A could be maintained as a homozygous stock, indicating the remaining endogenous Cpx7B can improve viability and fertility compared to animals lacking both isoforms. However, cpxΔ7A adults showed a complete inability to climb in a negative geotaxis assay . To examine larval locomotion, crawling velocity of 3rd instar larvae was assayed in cpxΔ7A and cpxΔ7B mutants . In contrast, cpxΔ7B mutants showed only a mild decrease in velocity (p = 0.1155). Together, these data indicate Cpx7A has a more prominent role in supporting larval and adult motor behavior.To assay functional requirements for the splice variants is assay . In cont mutants . SimilarcpxΔ7A and cpxΔ7B mutants, the well-characterized 3rd instar larval glutamatergic NMJ preparation was used29. Synaptic bouton and active zone (AZ) number were quantified by immunostaining for neuronal membranes (anti-HRP) and the AZ protein Bruchpilot (Brp) (p = 0.0012) and AZ  number in cpxSH1, synaptic growth was largely unaffected in cpxΔ7A  and cpxΔ7B  mutants. Despite the differences in overall levels of the two splice isoforms at NMJs (cpxΔ7A mutants had similar NMJ morphology to cpxΔ7B (p = 0.9999). We conclude that Cpx7A and Cpx7B are functionally redundant in their ability to regulate synaptic growth, with Cpx7B having a more important role than expected based on its lower expression level.To examine synaptic morphology and neurotransmitter release in ot (Brp) , F, G. I at NMJs , cpxΔ7A rd instar larval muscle 6 in abdominal segment A3. In contrast to the dramatic increase in spontaneous release rate observed in cpxSH1 (>55-fold), endogenously expressed Cpx7A or Cpx7B was able to substantially lower mini frequency (cpxΔ7B mutant returned spontaneous release rates to control levels (p = 0.2641). The residual Cpx7B in cpxΔ7A mutants was not able to fully clamp spontaneous fusion, with a 2-fold increase compared to controls (p < 0.0001). In contrast to spontaneous release, the presence of only one of the two splice isoforms was not as effective in driving normal levels of evoked fusion (cpxSH1 compared to control (p < 0.0001). Although less severe, cpxΔ7A displayed a 46% reduction (p < 0.0001) and cpxΔ7B a 25% reduction (p = 0.0269) compared to controls. These data indicate Cpx7A and Cpx7B are both required for wildtype evoked responses, with the higher levels of Cpx7A in cpxΔ7B mutants supporting more evoked fusion than the lower levels of Cpx7B in cpxΔ7A.To assay synaptic function, two-electrode voltage-clamp (TEVC) was used to measure evoked and spontaneous neurotransmitter release at 3requency , B. The d fusion , D. A 714. While peak eEJC amplitude primarily captures synchronous release, eEJC charge, half-width, and the time course of cumulative release provide kinetic insights into synchronous and asynchronous fusion. These evoked variables were compared across control, cpxΔ7A, cpxΔ7B and cpxSH1 NMJs  Cpx7A edit variants were differentially expressed between the two neuronal populations; and (2) editing of Cpx7A altered its function. To determine the abundance and diversity of Cpx7A edit variants , single-cell RNAseq datasets we previously generated from ~200 larval Ib and Is motoneurons were analyzed33. For these experiments, whole cell electrodes were used to collect cytosolic and nuclear content from individual Ib or Is neurons that expressed GFP using Gal4 drivers specific to each cell type32. High-resolution paired-end deep single-cell RNA sequencing was performed on RNA extracted from each individual motoneuron, generating ~4 million reads per cell and allowing identification of RNA editing diversity at single neuron resolution. Prior studies using mRNA obtained from pooled neuronal populations proposed RNA editing occurs in an “all-or-none” fashion at individual edit sites within a cell. For example, more than 99% of mammalian AMPA GluA2 receptor subunit transcripts undergo RNA editing at specific times during brain development39. Given our method provides individual neuron resolution of RNA editing, we could directly test the “all-or-none” model. Strikingly, individual larval motoneurons showed highly stochastic RNA editing of the three adenosine bases  that are subject to possible editing in Cpx exon 7A. Editing rates for these adenosines ranged from 0 to 100% across the ~200 neurons, with 98% of Ib cells and 94% of Is cells showing some level of exon 7A editing (n = 95 cells) and 30.3 ± 2.4% in Is (n = 86 cells). Only 3% of Ib and 1% of Is neurons fully edited all Cpx mRNA to I125M. For all Cpx7A mRNA edit variants, the average Ib motoneuron expressed 53% unedited Cpx, 32% CpxI125M, 0.0% CpxN130D, 1.5% CpxN130S, 0.3% CpxN130G, 0.2% CpxI125M,N130D, 9.5% CpxI125M,N130S and 3.7% CpxI125M,N130G . A similar ratio was observed in Is neurons , no correlation of editing percentage at this site and expression levels of adar mRNA in that cell was observed for Ib or Is motoneurons 30 alone . Ib neur neurons , B. In ta neuron . In cont neurons , B. For oneurons , D. We cn = 11 Is cells), while Syx1A M244V was edited in the same neurons at a rate of only 5.4 ± 3.8% per cell (n = 11 Is cells). Similar to Cpx, no significant difference in editing percent of Syn or Syx1A was observed between Ib and Is neurons  within Synapsin (Syn) and one site (M244V) in Syntaxin 1A (Syx1A) were compared in the same motoneurons that edited Cpx7A to I125M . Both Sy neurons . We concI125M,N130S is the most abundant editing variant on the hydrophilic face of the amphipathic C-terminal helix and resides near the Cpx7B S126 phosphorylation site, the functional significance of RNA editing to N130S or the potential phospho-mimetic version N130D was examined. Unlike Cpx7B, PKA did not phosphorylate unedited Cpx7AI125,N130 or Cpx7AI125M,N130S in in vitro phosphorylation assays (data not shown). As such, N130S might represent a target for different kinases or instead alter structural properties of the amphipathic helix independent of phosphorylation. Computational analysis of candidate phosphorylation motifs in exon 7A predicted a Casein Kinase 2 (CK2) consensus sequence of S/T E/D at the N130S site that is found in some CK2 targets40. To determine if Cpx7AI125M,N130S can be phosphorylated by CK2, in vitro phosphorylation assays were performed. Indeed, Cpx7AI125M,N130S was phosphorylated by CK2 (p = 0.0003), while unedited Cpx7AI125,N130 was not (p = 0.9554)  , G, pinpI125M,N130S, Cpx7AI125M,N130D or unedited Cpx7AI125,N130 were generated and expressed pan-neuronally using elavC155-Gal4 in the cpxSH1 null that lacks both Cpx7A and Cpx7B. All three Cpx7A transgenic proteins were overexpressed at similar levels when assayed by western analysis of adult brain lysates (p < 0.0001 to control) or anti-Cpx immunostaining at larval muscle 4 NMJs (p = 0.0001 to control). Given N130S and N130D alters the hydrophilic face of the C-terminal amphipathic helix that controls SV membrane binding and Cpx localization ), cpx nulls  and the three rescue strains: (1) elavC155;; cpxSH1, UAS-Cpx7AI125,N130 (unedited Cpx7A); (2) elavC155;; cpxSH1, UAS-Cpx7AI125M,N130S; (3) elavC155;; cpxSH1, UAS-Cpx7AI125M,N130D. Endogenous Cpx accumulates along the periphery of presynaptic boutons  at control NMJs . Expression of Cpx7AI125M,N130S or Cpx7AI125M,N130D in the null background had the opposite effect, with a greater fraction of Cpx in non-synaptic regions of the axon  and ~57% compared to unedited Cpx7AI125,N130 (p < 0.0001). Cpx7AI125M,N130S had a more striking subcellular change, with a nearly one-to-one ratio of its abundance at synapses and along axons . As such, unedited Cpx7AI125,N130 is more strongly enriched at synapses, while the predominant splice variant altering the C-terminal amphipathic helix  distributes equally between axons and synapses. Taken together, these data indicate Cpx distribution at control NMJs likely represents a combinatorial expression of both unedited and edited Cpx proteins.To examine if RNA editing alters Cpx function at synapses, transgenic UAS rescue lines expressing Cpx7Alization , Cpx sub boutons , co-locarol NMJs , F. Exprthe axon , F. Cpx7in vivo, morphological and electrophysiological assays were performed in rescue lines. Quantification of synaptic bouton and AZ number revealed significant differences in the ability of different Cpx7A proteins to rescue synaptic overgrowth in cpxSH1 null larvae , slightly less than the doubling of AZ and bouton number observed in cpxSH1 . Electrophysiological analysis of spontaneous fusion rates revealed a similar pattern of rescue as observed for synaptic growth . In contrast, Cpx7AI125M,N130S expression failed to properly clamp spontaneous release, similar to its inability to rescue synaptic overgrowth. Compared to the 102 Hz mini frequency at cpxSH1 null NMJs, Cpx7AI125M,N130S expression was able to reduce spontaneous fusion by only ~50% to 47 Hz (p < 0.0001 to control). Rescue with unedited Cpx7AI125,N130 created a more effective fusion clamp, decreasing mini frequency to 13 Hz (p = 0.0034 to control), although this rate was still ~3-fold higher than controls that also contain endogenous Cpx7B or the Cpx7AI125M,N130D rescue. RNA editing of Cpx7A also impacted evoked release, though not as severely as spontaneous release. Both unedited and edited Cpx7A improved eEJC amplitude and release kinetics compared to null mutants , while Cpx7AI125M,N130S and Cpx7AI125M,N130 displayed intermediate rescues. We conclude that RNA editing of Cpx7A regulates its functional properties, with N130D and N130S displaying several opposing effects compared to unedited Cpx7A. The N130D edit improved Cpx’s ability to clamp spontaneous release, in contrast to the N130S edit which reduced clamping and failed to prevent synaptic overgrowth. Based on the distinct phenotypes of N130S and N130D, it seems unlikely the primary effect of N130S is downstream of phosphorylation, but instead reflects a change to the functional properties of the C-terminal amphipathic helix. Alternatively, the N130D change does not act as a phospho-mimetic and instead alters the Cpx7A C-terminal helix in a distinct manner.To directly test the functional impacts of RNA editing of Cpx l larvae , H. Cpx7n cpxSH1 , H. Exprc growth , B, cons mutants –F. SimilI125M,N130S representing the most abundant edit to the N130 residue. To determine if co-expression of unedited Cpx7AI125,N130 with Cpx7AI125M,N130S, as would be observed in vivo, results in competition for Cpx interactions with the SNARE fusion machinery, the two proteins were co-expressed in the cpxSH1 null background. If unedited Cpx7A restored mini frequency to baseline rates, RNA editing would likely have effects on synaptic transmission only when neurons predominantly express edited Cpxs. Alternatively, if co-expression of Cpx7AI125M,N130S increased mini frequency beyond unedited Cpx7AI125,N130 rescues alone, a combinatorial role for unique Cpx edit variants to modulate presynaptic output from a neuron would be more likely. An intermediate effect on spontaneous fusion was observed when unedited Cpx7AI125,N130 and Cpx7AI125M,N130S were co-expressed in cpxSH1. Co-expression of the two proteins resulted in a spontaneous release rate of 23 Hz . Co-expression of unedited Cpx7AI125,N130 and Cpx7AI125M,N130S also displayed a larger evoked response than unedited Cpx7AI125,N130 alone, suggesting the N130S acts in a dominant fashion for promoting evoked release  containing a stop codon at the end of exon 616.In the current study, we examined the functional consequences of alternative splicing and RNA editing to the C-terminus of 55 provides a candidate mechanism for Cpx expression to differentially impact these two release pathways. For spontaneous release, a smaller number of Cpx proteins could block enough SNARE zippering events to prevent reaching the minimum required for fusion. For rapid Ca2+-triggered release, excess Cpxs would be needed to bind more SNARE complexes in the radial assembly at the SV-plasma membrane interface to control Syt1 activity and SNARE zippering.Given Cpx7B clamped spontaneous fusion and promoted evoked release at only ~15% of the Cpx7A expression level, the two splice variants likely have intrinsic differences in their activity. Like mammalian CPX1 and CPX2 that are present at most central nervous system (CNS) synapses, Cpx7B lacks the C-terminal CAAX motif, potentially endowing these non-prenylated Cpx proteins with greater mobility to interface with Syt1 and SNAREs and enhance release kinetics. Indeed, the onset of evoked release was slightly faster in larvae expressing only Cpx7B. While Cpx7B was able to reduce elevated mini frequency 28-fold compared to nulls, it was only able to rescue ~50% of evoked release, suggesting Cpx abundance is more critical for promoting evoked fusion than clamping minis. The estimated requirements for zippering of 3 to 11 SNARE complexes in a radial assembly for a single SV to undergo action potential-triggered fusion11. PKA phosphorylation of S126 also controls fusion pore dilation and cargo release from Drosophila dense core vesicles50, indicating changes to the C-terminal amphipathic domain of Cpx modulate its function in membrane fusion. Although Cpx7A lacks this PKA phosphorylation site, it undergoes RNA editing to generate up to eight unique C-terminal sequence variants . Single-cell RNAseq revealed multiple Cpx7A editing variants are simultaneously expressed in Ib and Is motoneurons. As such, ADAR-mediated RNA editing does not act in an “all-or-none” fashion in Drosophila motoneurons, but instead stochastically deaminates A-to-I residues with distinct efficiencies. Generally, unedited Cpx7A was the dominant form of Cpx in motoneurons, with editing variants represented at lower levels. However, in some cases, a single edit variant was the only Cpx mRNA expressed in that neuron  prevents CPX1 from stimulating liposomal fusion46. In Drosophila, presynaptic CK2 has been demonstrated to control synaptic stability by regulating Ankyrin2 function40, preventing a detailed analysis of its role in neurotransmitter release due to synapse loss. Although CK2 can phosphorylate Cpx7AI125M,N130Sin vitro, it remains unclear if this is relevant to Cpx function in vivo. In particular, the putative phospho-mimetic variant Cpx7AI125M,N130D was able to fully clamp spontaneous release and support normal synaptic growth, outperforming even unedited Cpx7A. As such, Cpx7AI125M,N130S may instead alter the structure or binding properties of the amphipathic helix in unique ways to prevent normal interactions with SNAREs or other protein/lipid targets.To determine if Cpx7A edit variants within a single neuron alter presynaptic output, transgenic rescues were used to assay their function. The most prominent variant Cpx7AI125M,N130S with unedited Cpx7AI125,N130 could allow distinct Cpx proteins to independently alter release output. Indeed, the N130S isoform acted in a partially dominant manner, preventing unedited Cpx7A from fully clamping spontaneous fusion, while supporting higher levels of evoked release. These data support a model where each Cpx variant has independent access to assembling SNAREs, allowing each to fine-tune presynaptic output in unique ways. Indeed, disruptions of the amphipathic helix in C. elegans Cpx alter SV release43, further highlighting a key role for this domain.Given the stochastic expression of Cpx edited proteins within single motoneurons, and the requirement for multiple SNARE complexes to drive fusion, we assayed if co-expression of Cpx7ADrosophila motoneurons. As such, stochastic RNA editing across multiple mRNAs is likely to generate unique synaptic proteomes within the same neuronal population and contribute to heterogenous properties of individual cells with similar transcriptomes. Such a mechanism would be a robust way to alter multiple features of neuronal output given ADAR editing alters the function of proteins that contribute to both synaptic release and membrane excitability64.Beyond stochastic RNA editing of Cpx7A, we observed similar heterogeneity in the percent of RNA editing in Synapsin and Syx1A mRNAs across rd instar larvae were used for imaging and electrophysiological experiments. Western blots were performed on adult brain extracts. Males were used for experiments unless otherwise noted. Experiments were performed in a w1118 (Bloomington Drosophila Stock Center #3605) genetic background unless otherwise noted.Flies were cultured on standard medium and maintained at 25 °C. Late 3Cpx edit variants that were subcloned into modified pValum construct, as previously described11. The resulting constructs were injected into a yv;;attP 3rd chromosome docking strain by BestGene Inc. . UAS lines were recombined into the cpxSH1 null mutant background and elavC155-GAL4 (BDSC #8765) was used for pan-neuronal expression of transgenes.QuikChange Lightning (Agilent) was used for site-directed mutagenesis on unedited Cpx7A to generate specified Cpx truncation lines were generated (cpxΔ7A and cpxΔ7B) using a CRISPR genome engineering approach. Four guide RNAs (gRNAs) flanking the splice acceptor site of exon 7A or 7B were selected using the CRISPR Optimal Target Finder65. gRNAs were cloned into the pCFD5 expression vector (Addgene #73914)66 and donor constructs were generated to encode a floxed P3>DsRed reporter cassette (Addgene #51434) in the reverse orientation flanked with one kb homology arms upstream and downstream of the splice acceptor site of either exon 7A or 7B by Gibson assembly protocol using NEBuilder HighFidelity DNA Assembly Cloning Kit (E5520). An early stop codon was inserted between homology arms for each respective exon construct, with several amino acid coding sequences maintained to preserve proper exon splicing. gRNA binding sites of donor template were mutated using silent mutations that did not alter amino acid sequence. Template and gRNA plasmids were co-injected into vasa-Cas9 embryos (BDSC #56552) by BestGene Inc  and Ds>Red positive transformants were selected by BestGene Inc . The modified locus with stop codons inserted into exon 7A or 7B were confirmed by sequencing.Two endogenous 67. Briefly, eight cohorts of ten adult males  per genotype were separated after eclosion and allowed to recover from CO2 for 24 hours on standard fly medium. After 24 hours, each cohort was moved to a chamber made from two clear plastic vials taped together, with a line drawn around the lower vial 8 cm from the bottom. Each cohort was allowed to acclimate in the chamber for five minutes before assays began. Negative geotaxis was measured as percent of the cohort that crossed the 8 cm line within ten seconds after being tapped to the bottom of the chamber. Each cohort was subjected to ten rounds of negative geotaxis assay, with a one-minute rest period between each. The percent of flies that crossed the 8 cm line after each round was averaged to produce a pass rate per cohort. Larval crawling was assayed in 3rd instar larvae of both sexes, as previously described69. Larvae were briefly washed in room-temperature water before placing onto the center of a 5 cm petri dish containing 2% agarose, with five animals from a single genotype placed together (n = 10 larvae per genotype). The petri dish was placed over a grid and velocity was measured as average distance (in mm) traveled during the first 30 seconds following placement.Adult geotaxis was measured in adult male flies aged 2–3 days as previously described2, 10 NaHCO3, 5 trehalose, 115 sucrose, 5 HEPES, pH 7.2) and fixed in 4% paraformaldehyde for 18 minutes. Larvae were washed three times for five minutes with PBST (PBS containing 0.1% Triton X-100), followed by a thirty-minute incubation in block solution (5% NGS in PBST). Fresh block solution and primary antibodies were then added. Samples were incubated overnight at 4°C and washed with two short washes and three extended 20 minutes washed in PBST. PBST was replaced with block solution and fluorophore-conjugated secondary antibodies were added. Samples were incubated at room temperature for two hours. Finally, larvae were rewashed with PBST and mounted in Vectashield . Antibodies used for this study include: mouse anti-Brp, 1:500 , Iowa City, IA)); rabbit anti-Cpx, 1:50007; goat anti-rabbit Alexa Fluor 488, 1:500 ; goat anti-mouse Alexa Fluor 546, 1:500 ; DyLight 649 conjugated anti-HRP, 1:500 .Larvae were dissected in hemolymph-like HL3.1 solution  and quantify synaptic bouton number and NMJ area. Brp puncta quantification was used to measure AZ number. Muscle 4 area was used to normalize quantifications for muscle surface area. For Cpx fluorescence quantification, the HRP-positive area was used to outline NMJs and axons. Total Cpx fluorescent intensity was measured in the outlined area, with background fluorescence of mean pixel intensity of non-HRP areas subtracted. For NMJ/axon ratios, background subtracted mean NMJ Cpx fluorescence was compared to background subtracted mean axon Cpx fluorescence within the same image.Imaging was performed on a Zeiss Pascal confocal microscope  using a 63X 1.3 NA oil-immersion objective (Carl Zeiss Microscopy). Images were processed with the Zen (Zeiss) software. A 3D image stack was acquired for each NMJ imaged  and merged into a single plane for 2D analysis using FIJI image analysis softwarerd instar muscle 6 at segment A3 using two-electrode voltage clamp with a −80 mV holding potential. Experiments were performed in room temperature HL3.1 saline solution . Final [Ca2+] was adjusted to 2 mM unless otherwise noted. Motor axon bundles were cut and suctioned into a glass electrode and action potentials were stimulated at 0.5 Hz (unless indicated) using a programmable stimulator . Data acquisition and analysis was performed using Axoscope 10.0 and Clampfit 10.0 software  and inward currents were labeled on a reverse axis for clarity.Postsynaptic currents were recorded from 3Western blotting of adult head lysates (three heads per sample with one head loaded per lane) was performed using standard laboratory procedures with mouse anti-Tubulin  at 1:10000 (UAS rescue experiments) or 1:1000000 (CRISPR experiments) and rabbit anti-Cpx at 1:5000. IRDye 680LT-conjugated goat anti-mouse, 1:5000  and IRDye 800CW conjugated goat anti-rabbit, 1:5000  were used as secondary antibodies. Blocking was performed in a solution containing four parts TBS  to one part Blocking Buffer (Rockland) for one hour. Antibody incubations were performed in a solution containing four parts TBST (1X TBS with 1% Tween-20) to one part Blocking Buffer. A LI-COR Odyssey Imaging System  was used for visualization and analysis was performed using FIJI image analysis software. Relative Cpx expression was calculated by normalizing to Tubulin intensity.I125M,N130S (termed N130S). Recombinant Cpx fused with GST was expressed in E. coli (BL21) and purified using glutathione sepharose 4B (Fisher Scientific). Peak fractions were concentrated and further purified by gel filtration as previously described11. In vitro kinase assays were performed using purified recombinant Cpx proteins and the catalytic subunit of CK2 . Briefly, 10 mg of purified GST-fusion protein  was used per reaction and incubated with 2,500 units of recombinant kinase and [32P]ATP (Perkin Elmer). Reaction products were separated by SDS-PAGE and gels were stained with Bio-Safe Coomassie Blue (Bio-Rad), dried, and exposed to autoradiography film at room temperature. Mean integrated density of each band was quantified using FIJI and relative density of phospho-Cpx (pCpx) was calculated by normalizing to input band intensity determined by Coomassie staining.QuikChange Lightning (Agilent) was used for site-directed mutagenesis of unedited Cpx7A to generate Cpx7Ard instar larval motoneurons obtained using isoform Patchseq protocols33 were analyzed using the Integrative Genomics Viewer (IGV)71. To create single-cell Cpx RNA editing expression profiles, single RNA reads were analyzed for Cpx and included in the analysis if all three C-terminal Cpx7A edited bases were represented on a continuous single read. The percent of each Cpx7A edit variant was determined by the number of edited variant reads divided by total RNA reads for each cell, creating an RNA editing profile for each neuron. To compare single base editing across different genes, the edit percent at each base of interest was analyzed and compared to known edits in other genes within the same neurons. Neurons were excluded if each base of interest did not contain ten or more reads for all edits of interest.RNAseq data from 105 single MN1-Ib and 101 single MNISN-Is 3p-values: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001, with ns = not significant.Statistical analysis and plot generation was performed using GraphPad Prism software version 9.5.1 . Appropriate sample size was determined using a normality test. Statistical significance for comparisons of two groups was determined by a two-tailed Student’s t-test. For comparisons of three or more groups of data, a one-way ANOVA followed by Tukey’s Multiple Comparisons test was used to determine significance when the largest sample standard deviation was no more than twice as large as the smallest sample standard deviation. When the largest sample standard deviation was more than twice as large as the smallest sample standard deviation, a Brown-Forsythe and Welch’s one-way ANOVA followed by Dunnett’s T3 Multiple Comparisons test was used to determine significance. For comparisons of two factors with three or more groups of data, as described in


We conducted an analysis across multiple PD-L1 combined positive score (CPS) indications to establish concordance of a 22C3 antibody–based laboratory-developed test (LDT) on the Ventana BenchMark XT or BenchMark ULTRA platform and the regulatory-approved PD-L1 IHC 22C3 pharmDx in cervical cancer (CC), esophageal squamous cell carcinoma (ESCC), head and neck squamous cell carcinoma (HNSCC), triple-negative breast cancer (TNBC), and urothelial carcinoma (UC). Tumor specimens from each tumor type were stained with 22C3 antibody and scored using the 22C3 antibody–based LDT, and scores were compared with those using PD-L1 IHC 22C3 pharmDx. PD-L1 status was measured by the pathologist using CPS as a continuous score and using clinically relevant cutoffs . The agreement between the BenchMark platforms and PD-L1 IHC 22C3 pharmDx was assessed by intraclass correlation coefficient (ICC) and a contingency table for clinical interpretation. A total of 522 samples were evaluated for the pan-tumor analysis . Most clinical interpretations of PD-L1 status were concordant between the BenchMark XT and PD-L1 IHC 22C3 pharmDx for all five tumor types with regard to negative percentage agreement , positive percentage agreement , and overall percentage agreement ; the ICC by tumor type was high (≥0.88). Importantly, the pan-tumor ICC was 0.95 (95% CI 0.94–0.96). Thirty additional TNBC samples were evaluated using the BenchMark ULTRA and PD-L1 IHC 22C3 pharmDx; the NPA, PPA, and OPA were 100%. The 22C3 antibody–based LDT on Ventana BenchMark XT and BenchMark ULTRA platforms demonstrated high concordance with the regulatory-approved PD-L1 IHC 22C3 pharmDx across multiple tumor types. These findings suggest the comparability of PD-L1 IHC 22C3 pharmDx with an LDT based on the 22C3 antibody. Pembrolizumab is a highly selective humanized monoclonal antibody that blocks the interaction between programmed death 1 (PD-1) and its ligands, PD-L1 and PD-L2, which helps restore T-cell responses against tumor cells . The antPD-L1 IHC 22C3 pharmDx is a qualitative immunohistochemical (IHC) assay using the monoclonal anti–PD-L1 clone 22C3 to detect PD-L1 protein in formalin-fixed, paraffin-embedded (FFPE) tumor tissues and is designed for use on the Dako Autostainer Link 48 (Agilent) [An analytical harmonization study, which fits the 22C3 monoclonal antibody to the Ventana BenchMark XT platform, has been published . High coThe aim of the current study was to establish the concordance between the 22C3 antibody–based LDT and the gold standard PD-L1 IHC 22C3 pharmDx using CPS in five tumor types separately  and together in a pan-tumor analysis.Archival FFPE tumor blocks from patients with CC, ESCC, HNSCC, TNBC, and UC were sourced from various suppliers Precision for Medicine, BioIVT, and MTgroup). Samples considered eligible for this study were <7 years old per technical specifications and had preserved tumor morphology, at least 1.5 × 1.5 cm of tumor, necrosis <20%, tumor cellularity >50%, and sufficient thickness for three separate cutting sessions. IHC testing was performed between 2019 and 2021 at the Hadassah Hebrew University Medical Center in Jerusalem, Israel. This analytical study used fully anonymized human tissue samples sourced from various suppliers; patient informed consent and approval by an institutional review board or an independent ethics committee were not required.Archival FFPE tissue blocks were sectioned at a 4-μm thickness and attached to positively charged glass slides; slides were stored at room temperature. Staining was performed using consecutive serial sections ≤14 days after sectioning. PD-L1 IHC 22C3 pharmDx was performed on the Autostainer Link 48 platform following the manufacturer’s specifications [For the LDT, samples were stained with the 22C3 antibody (Agilent) diluted 1:33 in antibody diluent with casein using the BenchMark XT platform (Ventana). The LDT used for this analysis has been previously described . CharactTo minimize interobserver bias, all samples were reviewed, analyzed, and scored for PD-L1 CPS  by one trained pathologist (GWV) according to the same CPS clinical algorithm for PD-L1 IHC 22C3 pharmDx. First, the slides (per each indication) were stained using the Autostainer Link 48 platform and evaluated. Each slide was evaluated twice, with a washout period of 1 week. The final score was the average of both readings. Then, after a mandatory washout period of ≥3 weeks, the process was repeated for the LDT-stained slides, as before. The final BenchMark XT or BenchMark ULTRA platform–based LDT score was the average of both readings. Only after the reading was complete was the final score that was obtained using the LDT compared with that obtained using the gold standard (PD-L1 IHC 22C3 pharmDx).ρ) were used to analyze the concordance of CPS by the 22C3 antibody–based LDT on the BenchMark XT platform versus PD-L1 IHC 22C3 pharmDx. Analyses were performed by each individual tumor type and in a pan-tumor analysis. For an additional 30 TNBC samples, ICC and Spearman’s ρ were used to analyze the concordance of CPS by the 22C3 antibody–based LDT on the BenchMark ULTRA platform versus PD-L1 IHC 22C3 pharmDx.Scatterplots of CPS as a continuous variable were generated using GraphPad Prism 6 . The intraclass correlation coefficient (ICC) and Spearman’s correlation coefficient , CPS ≥1 (positive), and CPS ≥10 (positive) for CC samples; CPS <10 (negative) and CPS ≥10 (positive) for ESCC, TNBC, and UC samples; CPS <1 (negative), CPS ≥1 (positive), and CPS ≥20 (positive) for HNSCC samples , 10. AftpharmDx-positive control slides from Agilent were first used as analytic controls for both IHC platforms . Becauseρ = 0.93)  . All CC  = 0.93) .ρ = 0.89)  . Of 41 E = 0.89) .ρ = 0.96)  . Almost  = 0.96) .ρ = 0.83) (ρ = 0.88)  . Almost  = 0.83) . An addi = 0.88) . All sam = 0.88) .ρ = 0.92)  . Almost  = 0.92) .ρ = 0.93)  .As with the analytic control, morphologic evaluation of stained tissue samples showed high similarity of PD-L1 staining patterns and intensities between PD-L1 IHC 22C3 pharmDx and the 22C3 antibody–based LDT on the BenchMark XT platform in CC, ESCC, HNSCC, TNBC, and UC samples .Identifying patients who are most likely to benefit from a given therapy based on biomarkers is increasingly important in creating a personalized treatment plan in oncology. Although anti–PD-1/PD-L1 immunotherapy has improved the prognosis substantially across a spectrum of cancers, some patients do not respond to therapy . AlthougPrevious studies comparing validated PD-L1 IHC assays and PD-L1 LDTs based on different anti–PD-L1 antibodies have reported inconsistent and divergent results –16. ThesUsing both a 22C3 antibody–based LDT on the Ventana BenchMark XT platform previously validated for NSCLC and the PD-L1 CPS algorithm, we evaluated PD-L1 expression in tumor samples from patients with CC, ESCC, HNSCC, TNBC, and UC and compared it with assessments using the gold standard, the regulatory-approved PD-L1 IHC 22C3 pharmDx. In our study, CPS as a continuous variable demonstrated a high correlation between the 22C3 antibody–based LDT on the BenchMark XT platform and PD-L1 IHC 22C3 pharmDx across all assessed tumor types. A high correlation between the 22C3 antibody–based LDT on the BenchMark ULTRA platform and PD-L1 IHC 22C3 pharmDx was observed in a small set of TNBC samples. Furthermore, high concordance between the 22C3 antibody–based LDT on the BenchMark XT or BenchMark ULTRA platform and PD-L1 IHC 22C3 pharmDx was also reported for the clinical interpretations of PD-L1 status by tumor type. These findings suggest that this 22C3 antibody–based LDT has the potential to standardize PD-L1 scoring using the Ventana BenchMark XT or Benchmark ULTRA platforms, thereby serving an unmet need in pathology laboratories for PD-L1 testing.Perhaps the best evidence of the equivalence of the two assays is the excellent correlation for CPS as a continuous variable Figs  and 3. APublished literature shows that a few other 22C3 antibody–based LDT protocols have been evaluated in NSCLC (using TPS) –20, gastPotential limitations of this study include the relatively small sample set sizes for CC (n = 77) and ESCC (n = 80) compared with those for TNBC , UC (n = 121), and HNSCC (n = 126). However, the analytic comparability of the LDT used in this study was already successfully assessed in NSCLC (using TPS) and the analytic comparability in this study was successfully assessed in TNBC, UC, and HNSCC (using CPS). For this reason, we determined that an assessment of the smaller sample set sizes for CC and ESCC was sufficient because the goal was to ensure that the protocol did not fall outside of a reasonable confidence level. We did not report on the NPA for CC data because of the high prevalence of CPS ≥1 (positive) in the CC samples, which dictated that a much larger cohort would be needed for the NPA to be calculated. This study also did not consider interobserver variability, given that all samples were scored by a single experienced pathologist.In conclusion, our 22C3 antibody–based LDT on the BenchMark XT or BenchMark ULTRA platforms demonstrates high concordance with PD-L1 IHC 22C3 pharmDx for the assessment of PD-L1 CPS across multiple tumor types, including CC, ESCC, HNSCC, TNBC, and UC. These findings suggest that the 22C3 antibody–based LDT on the BenchMark XT or BenchMark ULTRA platforms performs comparably with the gold-standard PD-L1 IHC 22C3 pharmDx for the assessment of PD-L1 status and can therefore be used to identify patients who are indicated for pembrolizumab therapy. Lastly, findings from our study will help laboratories who do not have access to the approved companion diagnostic assay for pembrolizumab (PD-L1 IHC 22C3 pharmDx and/or the DAKO Autostainer platform) to use the validated protocol for using 22C3 antibody on a Ventana BenchMark XT platform (and Ventana BenchMark ULTRA platform for TNBC) and enable them to use existing platforms without requiring the purchase of new equipment.


IL7R) in MS-risk. Based on evolutionary and functional arguments, we postulated that DDX39B enhances immune tolerance thereby decreasing MS risk. Consistent with such a role we show that DDX39B controls the expression of many MS susceptibility genes and important immune-related genes. Among these we identified Forkhead Box P3 (FOXP3), which codes for the master transcriptional factor in CD4+/CD25+ T regulatory cells. DDX39B knockdown led to loss of immune-regulatory and gain of immune-effector expression signatures. Splicing of FOXP3 introns, which belong to a previously unrecognized type of introns with C-rich polypyrimidine tracts, was exquisitely sensitive to DDX39B levels. Given the importance of FOXP3 in autoimmunity, this work cements DDX39B as an important guardian of immune tolerance.Genes associated with increased susceptibility to multiple sclerosis (MS) have been identified, but their functions are incompletely understood. One of these genes codes for the RNA helicase DExD/H-Box Polypeptide 39B (DDX39B), which shows genetic and functional epistasis with interleukin-7 receptor-α gene ( IL7R) is mediated by alternative splicing of IL7R transcripts , an autoimmune disease of the central nervous system, have been identified, but their mechanisms of action are incompletely understood . We estanscripts . The disnscripts . Elevatenscripts . Given tnscripts .DDX39B shows genetic and functional epistasis with IL7R in enhancing MS risk (DDX39B is conserved and located in the major histocompatibility complex (MHC) class III region in all vertebrates , plays roles in RNA splicing and nucleocytoplasmic transport , but was MS risk . Based o MS risk . The alttebrates , suggest+/CD25+ regulatory Tcells , a master regulator of the development, maintenance and function of CD4s Tregs; , and a rs Tregs; . Splicin+ T cells from six healthy human donors (Donors 1–6) and shown that DDX39B depletion led to increased skipping of IL7R exon 6 (+ T cells from two of these donors (donors 1 and 4). More transcript level changes were detected with Sh3 treatment than with Sh5 treatment  in primary human CD4R exon 6 . To idenreatment , which wreatment  and DDX3reatment . Therefo+ T cells from both donors 1 and 4   , we iden T cells . Among 6 T cells . To conf T cells . CMap is T cells . The GSE+ T cells and primary Tregs ) were significantly downregulated in DDX39B-depleted CD4ry Tregs . Other Din Tregs –3. TheseH1 cells, but increased expression of GATA3, a transcriptional regulator of TH2 cells -poor and cytosine (C)-rich py tracts, which on the average are more likely to have a C at each position, except positions –19 and –20 (measured relative to the guanine (G) in the 3’ splice site AG marked as position –1)  . This cotion –1)  and prevtion –1) , where tFOXP3 introns are conserved in mammals from both monotreme and theriiformes subclasses, which diverged over 200 million years ago  trend towards the C-rich py tracts of mammals, while the other (FOXP3b) has U-rich tracts  and U2AF2 (U2AF65)  composed of U2AF1 (U2AF(U2AF65) . U2AF2 b(U2AF65)  and this(U2AF65) . U2AF2 b(U2AF65) . We conj(U2AF65) . While e(U2AF65) , did not(U2AF65) . On the (U2AF65)  rescued (U2AF65) . Of note(U2AF65) . These rDDX39B is genetically associated with MS, an autoimmune disease  was proposed to be an anti-inflammatory factor  and we h disease . Previou disease , which e disease . Complem disease . While tOur work highlights the importance of post-trancriptional mechanisms in the regulation of FOXP3. These have not been noted before and indeed CRISPR screens to discover regulators of FOXP3 did not discover DDX39B , but thiH2-like self-reactive effector cells  syndrome  and the or cells . The DDXDDX39B resides in the class III region of the MHC and many of its neighbors code for proteins involved in RNA transactions (e.g. DXO and SKIV2L)  larger than 1200 nucleotides . There i SKIV2L) . This ovDDX39B, which is also known as UAP56, was characterized as a factor required for formation of splicing complexes . Here weMT-2 cells were kindly provided by Bryan R. Cullen (Duke University) and grown at 37 °C in Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher Scientific) with 10% (v/v) fetal bovine serum (FBS) (Genesee Scientific) and 1% (v/v) penicillin-streptomycin (Thermo Fisher Scientific). Human embryonic kidney 293T/17  and HeLa (ATCC CCL-2) cell lines were obtained from the Duke University Cell Culture Facility. The HeLa Flp-In T-Rex cell line (HeLa-Flp-In) was kindly provided by Dr. E. Dobrikova (Duke University). HEK293T/17, HeLa, and HeLa-Flp-In cells were cultured at 37 °C in Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Fisher Scientific) with 10% (v/v) FBS, 1% (v/v) penicillin-streptomycin and 2.5 μg/ml Plasmocin (InvivoGen). HeLa_Flp-In cell lines expressing wild-type or mutant versions of DDX39B  were grown as described above except media was supplemented with 2.5 μg/ml blasticidin (InvivoGen) and 200 μg/ml Hygromycin B (Thermo Fisher Scientific). All cells were free of Mycoplasma contamination as confirmed by routine testing using the MycoAlert Mycoplasma Detection Kit (Lonza).+ T cells were isolated and cultured as described previously (+ T cells were further isolated using CD4+ T cell isolation kit (Miltenyi Biotec). The isolated cells were cultured in RPMI medium supplemented with 20% (v/v) FBS and 100 ng/mL human recombinant IL-2 (Peprotech). Two days prior to transduction, CD4+ T cells or MT-2 cells were activated with media containing 50 ng/mL anti-CD3 (eBioscience) and 100 ng/mL anti-CD28 (BD Biosciences) antibodies.Primary human CD4eviously . In brie+ CD25+ CD127dim/- Regulatory T Cell Isolation Kit (Miltenyi Biotec). The isolated Treg cells were expanded in culture for 14 days with TexMACS medium (Miltenyi Biotec) supplemented with 5% (v/v) Human AB Serum (Sigma), 500 IU/mL recombinant human IL2 (PeproTech) and Treg Expansion kit (Miltenyi Biotec) which contains beads pre-loaded with anti-CD3 and anti-CD28 antibodies. Induced T regulatory cells (iTregs) were purchased from IQ Biosciences (IQB-Hu1-iTr-1) and were expanded in culture for 6 days using the same media and Treg Expansion kit as the primary human Tregs.Primary human T regulatory cells (Tregs) were isolated from PBMCs using CD4DDX39B into the pLCE vector. Splicing reporter plasmids  were constructed by inserting introns 7, 9 or 11 of FOXP3 (NC_000023), or intron 2 of HGB1 (NC_000011) within the open reading frame of Renilla Luciferase (Rluc) at nucleotide positions 599, 601, 408, or 253, respectively, in the pcDNA3.1 vector (pcDNA3.1-Rluc). Splice sites (SS) mutations were introduced in the corresponding parental plasmid using In-Fusion HD Cloning kit (Takara Bio) to generate pcDNA3.1-Rluc_FOXP3 Intron 7 5’-SS G-U, pcDNA3.1-Rluc_FOXP3 Intron 7 3’-SS PolyU, pcDNA3.1-Rluc_FOXP3 Intron 11 5’-SS G-U, and pcDNA3.1-Rluc_FOXP3 Intron 11 3’-SS PolyU. To construct pcDNA3.1-Rluc_FOXP1 Intron 19 WT and 3’ C-rich py tract mutant, synthesized DNA fragments with the FOXP1 intron sequences (NG_028243.1) were purchased from Integrated DNA Technologies and were subcloned into the pcDNA3.1-Rluc plasmid. Point mutants DDX39B K95A, E197A and D199A were introduced in the parental pcDNA5/FRT/TO-DDX39B plasmid (shRNA pLKO.1 plasmids expressing non-targeting control shRNA (NTC: SHC002) or anti-DDX39B shRNAs  were purchased from Millipore Sigma. The construct pLCE-DDX39B was generated by cloning the coding sequence of  plasmid  using QuGeneration of the inducible HeLa cell line expressing wild-type DDX39B or DDX39B mutant D199A was described previously . HeLa ce7 293 T/17 cells were seeded in 15 cm dishes in DMEM media and cultured overnight (three 15 cm dishes per construct). Cells were co-transfected with 17 μg of the corresponding shRNA pLKO.1 vector, pLCE-DDX39B or pLCE-GFP, 17 μg of packaging plasmid (pCMVR8.74) and 7 μg of VSV-G envelope plasmid (pMD2.G) in serum-free media and 160 μL of Lipofectamine 2000. The media was replaced with 20 mL fresh DMEM media 18 hr post-transfection. After 72 hr, supernatants were collected, filtered through 0.45 mm filters and concentrated to 6 mL in Amicon Ultra 100 K centrifugal filter units (Millipore Sigma). Concentrated lentiviral particles were used immediately or stored at –80 °C.Lentiviral packaging of shRNA constructs was conducted in 293T/17 cells using Lipofectamine 2000 (Thermo Fisher Scientific). In brief, 1.0×10+ T cells with either NTC or anti-DDX39B shRNAs was described previously.(6 activated primary CD4+ T cells from each donor were transduced in T25 flasks with lentiviruses encoding control (NTC) or DDX39B-targeting (Sh3 or Sh5) shRNAs for 3 days, and transduced cells were selected under medium supplemented with 1.5 μg/mL puromycin for 4 days. Cells were then cultured for 24 hr in the absence of puromycin and collected for functional analyses . Transduction of activated MT-2 cells (1.0×107 cells) was carried out as for primary CD4+ T cells. Activated primary Tregs (1.0×105 cells) were transduced in 96-well plates and cultured for 7 days with half media exchange every 2 days (without puromycin selection). Induced Tregs (1.0X105 cells) were transduced in 96 well plates with Sh3 and/or Sh5 for two days then subjected to puromycin selection for two days. Cells were then cultured for an additional 24 hr in the absence of puromycin and harvested for functional analyses. In all cell types, depletion of DDX39B protein was confirmed by western blot using anti-UAP56 (DDX39B) antibody  and α-Tubulin , PTBP1 (anti-PTB rabbit serum Transduction of human primary CD4eviously. In brief7 cells were co-transduced with the following combination of shRNAs and expression plasmids for 3 days: (i) NTC shRNA +pLCE GFP (control), (ii) Sh5 shRNA +pLCE GFP (knockdown), and (iii) Sh5 shRNA +pLCE-DDX39B (rescue). The cells were then selected with RPMI medium supplemented with 1.5 μg/mL puromycin for 2 days, followed by 24 hr culture in the absence of puromycin, and collection of cell lysates for RNA and protein analyses . We used the DDX39B shRNA Sh5 since it targets the 3’ untranslated region of DDX39B mRNA, and thus it depletes the endogenous DDX39B mRNAs but not the trans-gene DDX39B mRNAs. Depletion or rescue of DDX39B protein was confirmed by western blot using anti-UAP56 (DDX39B) antibody  and α-Tubulin  as loading control.To rescue DDX39B expression in MT-2 cells, 1.0×10+ T cells using ReliaPrep RNA Cell Miniprep System (Promega), and treated in-column with DNase I following the manufacturer’s recommendations. Poly-A+ RNA was enriched from 1 µg of total RNA and used as template to generate libraries using the Illumina TruSeq platform as recommended by the manufacturer. Libraries were sequenced on a 2x100 paired-end format on an Illumina Hi-Seq 1500. Reads were aligned to the human GRCh38 reference with program STAR version 2.5.2b, using the parameters recommended for the ENCODE consortium  or DDX39B-depleted (Sh3 or Sh5) CD4nsortium . The STAnsortium .https://github.com/vastgroup/vast-tools, copy archived at swh:1:rev:fb87ceb4379cd9fbd36fc402e4a3429567021548; Splicing analysis was carried out using Vast-tools program version 0.2.1  by alignThe study by the International Multiple Sclerosis Genetics (IMSG) Consortium  identifi1 genes from N, and counting the number of genes overlapping with each MS gene list n2 over 100,000 permutations. An empirical p value was calculated based on the fraction of instances that simulated (X) was greater than or equal to the actual observed value of k for each MS gene set.In order to estimate enrichment of differentially expressed genes (DEGs) in pre-determined MS gene sets, we used a simulation method. We determined DEGs in two donors with the shRNA that effectively knocked down DDX39B, Sh3, based on the Gfold method  with a cEEF1A1 was used as normalization control since its expression was not affected by DDX39B depletion.Total RNA was isolated from control or DDX39B-depleted cells using ReliaPrep RNA Cell Miniprep System (Promega) or Direct-zol RNA kit (Zymo Research), and treated in-column with DNase I following the manufacturer’s recommendations. Reverse transcription was conducted with random primers using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). DDX39B and FOXP3 RNA levels were measured by real-time quantitative PCR (RT-qPCR) using PowerUP SYBR Green Master Mix (Thermo Fisher Scientific) in a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific). + T cells were analyzed by RT-qPCR in control (NTC) or DDX39B-depleted (Sh3 and Sh5) primary CD4+ T cells (from 6 donors), MT-2 cells and primary Tregs (from 2 donors). Target genes and primers are shown in EEF1A1 expression. Differentially expressed genes were determined by comparing levels in DDX39B-depleted cells versus control cells.Genes whose expression was found to be differentially expressed upon DDX39B depletion in the RNAseq dataset in primary CD4Whole cell lysates from control (NTC) or DDX39B-depleted (Sh3 and Sh5) cells were collected in 1 X RIPA buffer  freshly supplemented with 1 X protease inhibitors (Roche). The cell lysates were quantified by bradford and equal amount of total protein was loaded per lane on NuPAGE 4–12% Bis-Tris pre-cast gels (Life Technologies), transferred to nitrocellulose membranes (Whatman), and blotted using standard protocols with antibodies against DDX39B , FOXP3 , and either PTB, α-Tubulin  or α-beta-actin  as loading control.+ T cells, we generated a gene list from the control condition  and a gene list from the DDX39B knockdown condition . All the genes that passed the initial cutoff (RPKM of ≥2 in both NTC libraries) were used. GSEA was conducted with the C2 curated genesets collections of Molecular Signatures Database (P<0.05). Of these, 648 and 82 genesets showed enrichment to NTC and KD groups, respectively . For the rescue experiments of endogenous DDX39B-depletion in MT-2 cells, cells transduced with NTC and GFP lentiviruses were used as controls. Detections of retained introns in FAM3A, PORCN, RBM10, RENBP, CFP and G6PD (C-rich py tracts) or FMR1 and DDX3X (U-rich py tracts) were conducted in the same manner. All primers used for this analysis are shown in To quantify intron retention events, we designed RT-qPCR primers to amplify total FOXP3 transcripts (constitutive exon 11), spliced FOXP3 transcripts (spanning the exon-exon junction between constitutive exons 10 and 11) and retained intron-containing FOXP3 transcripts: intron 2 (spanning the intron 2/exon 3 junction), intron 4 (spanning the intron 4/exon 5 junction), intron 6 (spanning the exon 6/intron 6 junction), intron 7 (spanning the intron 7/exon 8 junction), intron 9 (spanning the exon 9/intron 9 junction), or intron 11 (spanning the intron 11/exon 12 junction) . LikewisFOXP3 gene was inferred by using the Maximum Likelihood method based on the Tamura-Nei model .Likewise, the evolutionary history of the 5 HeLa cells were transfected in 12-well plates with control  or DDX39B-targeting  siRNAs using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific). After two days of culture, the Renilla luciferase (RLuc) splicing reporter plasmids were co-transfected with a Firefly luciferase plasmid  as transfection control. After 24 hr, cell lysates were collected for measurements of luciferase activity and RNA isolation. RLuc and FLuc activities were measured by Dual-Luciferase Reporter Assay System (Promega), and the data are presented as RLuc/FLuc.A total of 2.0×10To directly quantify the splicing efficiency of the reporters, RNA was isolated and used for reverse transcription as described above. Spliced and unspliced reporter transcripts were measured by endpoint PCR  with primers specific to the RLuc coding sequence. PCR amplicons were detected by electrophoresis on 6% non-denaturing polyacrylamide/TBE gels with SYBR Gold Nucleic Acid Gel Stain (Thermo Fisher Scientific). Splicing efficiency was measured by densitometry analysis of the PCR amplicons in image J .trans-genes were transfected with control siRNA (NTC) or DDX39B-targeting siRNA D13, which targets the 3’ untranslated region (3’UTR) of DDX39B mRNA. After two days of culture, expression of DDX39B trans-genes in NTC- or D13-treated cells was either induced by 1 µg/mL doxycycline or not (doxycycline withheld) to generate the following four conditions: NTC −doxycycline (control), NTC +doxycycline (overexpression), D13 −doxycycline (knockdown) and D13 +doxycycline (rescue). The cells were then transfected with pcDNA3.1-RLuc_FOXP3 Intron 11 and harvested 24 hr after for analyses. DDX39B protein expression was confirmed by western blot, and the splicing efficiency of the reporters was evaluated by endpoint PCR as described above.For the splicing-rescue assay, HeLa-Flp-In cells stably expressing either wild-type or mutant  DDX39B FOXP3, FOX family genes and X chromosome genes (2155 genes) were obtained from Human genome assembly (GRCh38.p13) using the UCSC table browser  were generated by WebLogo3 (5’ SS (from –3 to +6 nt at the 5’ Exon-Intron junction) and 3’ SS (from –20 to +3 nt at the 3’ Intron-Exon junction) sequences of  browser  and Gala browser . SequencWebLogo3 . SequencFor comparison of Py tracts of introns that were DDX39B-sensitive (retained in the knockdown) versus DDX39B-insensitive (unchanged in the knockdown), we compared the 402 introns that were significantly retained versus 500 events randomly selected from the total unchanged introns. 5’ SS and 3’ SS sequences for these events were obtained using the Galaxy platform, and MaxEntScore for these were determined by MaxEntScan . The nuc+ T cells from individual donors, One-way repeated measures ANOVA was used. To specifically compare each experimental (Sh3 and Sh5) group to control (NTC) after a significant Omnibus f test, Dunnett’s test was performed to control family-wise type I error rate due to multiple comparisons. For the experiments other than CD4+ T cells, One-way ANOVA model followed by aforementioned Dunnett’s tests were conducted to compare between experimental (Sh3 and Sh5) and control (NTC). For experiments done using iTregs from Donor 10, no statistical methods were used due to the difference in number of replicates for each experimental condition . For the rescue assay of splicing, One-way ANOVA models followed by Tukey’s tests were used to perform pairwise comparisons among all tested conditions. For the sequence analysis of retained introns, outliers of Max entropy scores were identified and removed   unless otherwise noted. Asterisks denote level of statistical significance: * p<0.05, ** p<0.01, *** p<0.001 and **** p<0.0001. Statistical analyses were conducted in Prism8 (GraphPad Software) or SAS (SAS Institute). For the statistical analysis of experiments using CD4 Q=0.5%) . Then, M+ T cell were carried out using a linear mixed model on the delta CT values (dCT). For each gene, the linear mixed model includes treatment  as fixed effect, and random intercept to account for the heterogeneity of the individuals and correlations among repeated measures from the same individual. The fold-change differences  and their standard error (SE) between treatment groups versus the control were estimated from the model. The p-values were adjusted by False Discovery Rate (FDR) method, which controls the false discovery rate due to multiplicity in hypothesis testing. To facilitate the interpretation using relative expression, the point estimate of the ddCT values were converted to 2-ddCT.Statistical analysis of RT-qPCR data in CD4 DDX39B is a helicase with known functions in mRNA splicing and nuclear export. This important study provides convincing evidence that DDBX39B regulates Foxp3, a lineage marker for T-regulatory cells in the immune system. The work provides a detailed analysis of the post-transcriptional regulation of Foxp3, and also positions DDX39B more broadly, as an important player in the regulation of autoimmune responses. The work will be of interest to RNA biologists, immunologists, and those studying autoimmune disorders. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Satyajit Rath as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "The RNA helicase DDX39B activates FOXP3 RNA splicing to control T regulatory cell fate" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:Specific concerns and suggested experiments are as below.1. Figure 1E: Data from more than one donor should be shown and individual donors linked as Figure 1C. The same applies to Figure 2 C-F.The inclusion of non-transduced Tregs, as an internal control from the same culture conditions is an important control. One way to do this is as follows: If pCLE-GFP was the marker for transduced cells, Foxp3 MFI should be reported in GFP- and GFP+ gates, to demonstrate that Foxp3 expression is indeed reduced upon knock-down of DDX39B.2. Figure 2: Is the GSEA analysis performed from only DDX39B knocked-down Treg dataset? Or from all the T helper types?Foxp3 dependent gene expression should be maximally affected in the Tregs upon DDX39B knockdown if it is an effect mediated through Foxp3. If the same extent of reduction is observed in both CD4 cells (Tregs and non-Tregs), then that would suggest that many of these genes are themselves under DDX39B control, independent of Foxp3. In fact, this second possibility seems to be likely, especially for IL10, due to two reasons. First, the extent of its downregulation upon DDX39B knock-down appears to be the same in pan CD4 T cells and primary Tregs, if anything, it is much lower in pan T-cells. Second, IL10 is not a direct target of Foxp3, since in Foxp3 deficient Foxp3GFPKO mice, IL10 expression is not affected . So the conclusion that IL10 downregulation is indicative of reduced Foxp3 function merits re-examination. The possibility that IL10 itself is a target of DDX39B, which may contribute to MS susceptibility may also be considered. Treg functionality upon DDX39B knockdown can be assessed by in vitro suppression assay. If indeed Foxp3 downregulation is the consequence, this is expected to directly affect Treg mediated suppressive capacity.3. Figure 3: Key conclusions be validated in in vitro TGF β generated iTreg cells, where enough cells are expected to be generated for FACS analyses and real-time PCR mediated intron retention assays.4. Figure 6: Β-globin intron, as mentioned by authors, is unaffected. Does splitting and the luciferase signal remain unchanged upon DDX39B knockdown. These experiments would be most convincing if done using iTregs5. The lack of mechanistic understanding of why certain C-rich introns are sensitive to DDX39B is a key gap. However, the authors can address whether DDX39B binds better to introns it regulates vs. others – either in vitro (EMSA) or by CLIP-Seq.Comments not requiring new experiments:1. Figure 3: At first, the dramatic reduction of Fox3p in the cytoplasm in Sh3/5-treated cells seems to suggest a role of DDX39B in nuclear export of Fox3p . Upon further reflection, the argument against this conclusion is that the nucleoplasmic pool is not increased significantly – rather only the chromatin-associated pool. The authors should clarify this point.In the description in line numbers 208-213, Figure S3C, which cells are used for this experiment?2. Figure 4: The rescue experiments are commendable and convincing. However, the transgene clearly doesn't recover full splicing or expression of Fox3p even though higher than endogenous levels of DDX39B are achieved. Some comment on what this might be due to or imply would be useful.The authors may consider making a subfigure after customizing the height of the tracks so that the difference between control and DDX39B knockdown becomes more apparent.3. Figure 5A: The rationale for comparing Fox3p exons to those of other genes specifically on the X chromosome is unclear. Would a different result be obtained if compared to all genes? Essential revisions:Specific concerns and suggested experiments are as below.1. Figure 1E: Data from more than one donor should be shown and individual donors linked as Figure 1C. The same applies to Figure 2 C-F.In response to concern about original Figure 1E and based on comments below re the advantage of using iTreg cells, we repeated critical experiments in iTregs from two additional human donors. Data from Tregs from four human donors are now shown in new Figure 2.The inclusion of non-transduced Tregs, as an internal control from the same culture conditions is an important control. One way to do this is as follows: If pCLE-GFP was the marker for transduced cells, Foxp3 MFI should be reported in GFP- and GFP+ gates, to demonstrate that Foxp3 expression is indeed reduced upon knock-down of DDX39B.We respectfully disagree with the reviewer on the use of non-transduced Tregs since it is known that transduction can lead to spurious effects that must be controlled. For transduction experiments, including those shown in original Figure 2D, we used lentivirus coding non-targeting shRNA as control, thus, we cannot use the GFP signal as a transduction control. Also, we selected the cells with puromycin after transduction, so it is unlikely to be biased by the non-transduced cells. Orginal Figure 2D showed IL2RA, not FOXP3.2. Figure 2: Is the GSEA analysis performed from only DDX39B knocked-down Treg dataset? Or from all the T helper types?+ Tcells. To make sure this is clear we carefully edited the Results section and the legend to new Figure 3.The GSEA analysis, which is now shown in new Figure 3, was derived from data from DDX39B kd vs control in CD4Foxp3 dependent gene expression should be maximally affected in the Tregs upon DDX39B knockdown if it is an effect mediated through Foxp3. If the same extent of reduction is observed in both CD4 cells (Tregs and non-Tregs), then that would suggest that many of these genes are themselves under DDX39B control, independent of Foxp3. In fact, this second possibility seems to be likely, especially for IL10, due to two reasons. First, the extent of its downregulation upon DDX39B knock-down appears to be the same in pan CD4 T cells and primary Tregs, if anything, it is much lower in pan T-cells. Second, IL10 is not a direct target of Foxp3, since in Foxp3 deficient Foxp3GFPKO mice, IL10 expression is not affected . So the conclusion that IL10 downregulation is indicative of reduced Foxp3 function merits re-examination. The possibility that IL10 itself is a target of DDX39B, which may contribute to MS susceptibility may also be considered. Treg functionality upon DDX39B knockdown can be assessed by in vitro suppression assay. If indeed Foxp3 downregulation is the consequence, this is expected to directly affect Treg mediated suppressive capacity.We thank the reviewer for this comment, with which we agree. We have revised the Results section and new Figure 3 carefully to make this point clear.3. Figure 3: Key conclusions be validated in in vitro TGF β generated iTreg cells, where enough cells are expected to be generated for FACS analyses and real-time PCR mediated intron retention assays.FOXP3 intron retention is exacerbated by DDX39B knockdown in Tregs/iTregs from all four human donors as shown in the new Figure 5—figure supplement 2. The result that several FOXP3 introns are highly susceptible to DDX39B levels was observed in CD4+ Tcells, the Treg-like MT-2 cells and in primary Tregs/iTregs, therefore we believe this critical conclusion is very strongly supported.We thank the reviewer for this suggestion on data from original Figure 3  and indeed used iTregs from two human donors to repeat critical experiments done with Treg cells isolated from the first two human donors. 4. Figure 6: Β-globin intron, as mentioned by authors, is unaffected. Does splitting and the luciferase signal remain unchanged upon DDX39B knockdown. These experiments would be most convincing if done using iTregsFOXP3 introns in primary Tregs and iTregs form four human donors , we did not repeat the reporter assays in iTregs. Since increased intron retention effect was observed in endogenous 5. The lack of mechanistic understanding of why certain C-rich introns are sensitive to DDX39B is a key gap. However, the authors can address whether DDX39B binds better to introns it regulates vs. others – either in vitro (EMSA) or by CLIP-Seq.We agree with the reviewer about the importance of a mechanistic understanding of why certain introns with C-rich polypyrimidine tracts are more sensitive to DDX39B knockdown and indeed there are multiple projects in the laboratory investigating this issue. We respectfully submit that further investigation of this issue goes beyond the scope of the current manuscript. We point out that introns that bind DDX39B poorly will actually be the most sensitive to DDX39B depletion. It is these low affinity binding sites that are disproportionally affected when the nuclear DDX39B concentration is reduced by knockdown.Comments not requiring new experiments:1. Figure 3: At first, the dramatic reduction of Fox3p in the cytoplasm in Sh3/5-treated cells seems to suggest a role of DDX39B in nuclear export of Fox3p . Upon further reflection, the argument against this conclusion is that the nucleoplasmic pool is not increased significantly – rather only the chromatin-associated pool. The authors should clarify this point.We have edited the text in a way that now should be much clearer .In the description in line numbers 208-213, Figure S3C, which cells are used for this experiment?+ T cells.Figure S3C showed the result of analysis of NGS of CD42. Figure 4: The rescue experiments are commendable and convincing. However, the transgene clearly doesn't recover full splicing or expression of Fox3p even though higher than endogenous levels of DDX39B are achieved. Some comment on what this might be due to or imply would be useful.We thank the reviewer and note that these rescue experiments are indeed challenging . In the revised manuscript we have included an explanation in the Results section – we believe that once FOXP3 levels are reduced this leads to the disruption of epigenetic maintenance that makes recovery of FOXP3 expression difficult to achieve even when FOXP3 intron retention is rescued by DDX39B overexpression.The authors may consider making a subfigure after customizing the height of the tracks so that the difference between control and DDX39B knockdown becomes more apparent.Assuming this refers to original Figure 4  we respectfully suggest that the difference is clear as shown.3. Figure 5A: The rationale for comparing Fox3p exons to those of other genes specifically on the X chromosome is unclear. Would a different result be obtained if compared to all genes?FOXP3 is on the X chromosome and we compared its introns to those of other protein-coding genes on the same chromosome. In the revised manuscript we re-wrote this to make the rationale clear. Based on work of many others that examined sequence composition of py tracts genomewide  leads to the same result as we observe in X chromosome genes. This is noted in the Results section associated with the data described in original Figure 5 .We apologize for not making the rationale clear.


PPP2R1A variants were reviewed. We reported five unrelated individuals with PPP2R1A variants, including two novel missense variants and one frameshift variant. The protein expression of the Arg498Leu variant was less than that of the wild-type protein, the frameshift variant Asn282Argfs*14 was not decreased but truncated, and these two variants impaired the interactions with endogenous PPP25RD and PPP2CA. Furthermore, we found that pathogenic variants clustered in HEAT repeats V, VI and VII, and patients with the Met180Val/Thr variants had macrocephaly, severe ID and hypotonia, but no epilepsy, whereas those with Arg258 amino acid changes had microcephaly, while a few had epilepsy or feeding problems. In this study, we reported five NDD patients with PPP2R1A gene variants and expanded PPP2R1A pathogenic variant spectrum. The genotype and phenotype association findings provide reminders regarding the prognostication and evidence for genetic counseling.Neurodevelopmental disorders (NDDs) are a group of high-incidence rare diseases with genetic heterogeneity. PPP2R1A, the regulatory subunit of protein phosphatase 2A, is a recently discovered gene associated with NDDs. Whole/clinical exome sequencing was performed in five patients with a family with NDDs. In vitro experiments were performed to evaluate the mutants’ expression and interactions with the complex. The genotype–phenotype correlations of reported cases as well as our patients with  Neurodevelopmental disorders (NDDs) are a group of rare diseases with high-incidence genetic heterogeneity. The genetic diagnostic rates of NDD and intellectual disability (ID) are about 30% and 40%, respectively, in the UK 100,000 genome project . PP2A plde novo pathogenic variants of PPP2R1A and PPP2R5D genes [In 2015, the Decipher Developmental Disorder (DDD) group reported seven patients with ID with 5D genes . Present5D genes , with cl5D genes ,13,14,155D genes ,10.PPP2R1A gene is located in chromosome 19q13.33, encoding PP2A scaffolding Aα subunit and a total of 589 amino acids. The gene is widely expressed and highly conserved. Aα has 15 tandem Huntington elongation A-subunit TOR (HEAT) repeats (HRs). Each HR is composed of two α-helices connected by an intra-repeat loop. These HRs form a B-subunit-binding and a C-subunit-binding domain. Currently, the reported variants of PPP2R1A have all been localized in the B-subunit-binding domain. PP2A-Aα is an all-helical structural protein that keeps the phosphatase complex together [The together . Mice witogether . Some patogether .PPP2R1A gene variants, including two reported missenses, two novel missenses and one frameshift variation. We performed in vitro experiments to validate the pathogenicity of the novel variants. We also summarized the patients’ clinical features and genetic profiles, and analyzed correlations between the genotypes and phenotypes.In this study, we report five NDD patients with Genomic DNA was extracted from the peripheral blood collected from the patients and their parents using the QIAamp DNA Blood Mini kit, following the manufacturer’s instructions. The library was constructed with the Agilent SureSelect XT Human all-exon 50 Mb kit and sequenced as 150 bp paired-end runs on the Illumina X Ten platform. The sequencing was conducted following the protocols described in our published study . The canhttp://gnomad-sg.org/) (accessed on 12 June 2020), the ExAC database and our in-house database , and the variants were evaluated using SIFT (http://sift.jcvi.org/) (accessed on 24 January 2017), PolyPhen2.2 (http://genetics.bwh.harvard.edu/pph2/) (accessed on 24 January 2017), MutationTaster (http://www.mutationtaster.org/) (accessed on 24 January 2017) and CADD. Our data interpretation followed the American College of Medical Genetics and Genomics, the Association for Molecular Pathology (ACMG/AMP) guidelines and our previously published research.The sequence data were mapped to the human reference genome (GRCh37/hg19). Variant calling was performed using the Genome Analysis Toolkit Best Practices Pipeline (Version 3.2.2). The allelic frequencies were annotated from gnomAD  were constructed with the KOD-Plus-Mutagenesis Kit (Code No. SMK-101) from Toyobo . The constructs were sequenced to confirm that no secondary mutation was introduced. HEK293T cells were cultured in high-glucose DMEM  supplemented with 10% fetal bovine serum  and 1% (50 μg/mL) penicillin/streptomycin . Once the cells had grown to 70–80% confluence, they were separately transfected with PPP2R1AWT and mutants. The cells were transfected with recombinant DNA plasmids for transient expression of the proteins using Lipofectamine 3000 reagent  according to the manufacturer’s instructions.The wild-type plasmid tagged with 3*Flag in the N terminal of PPP2R1A (EX-C0281-M12) was purchased from GeneCopoeiag for 5 min, and the supernatant was collected and added to the LDS sample buffer. The protein extracts were separated using SDS–PAGE electrophoresis and transferred to the nitrocellulose membrane. The membrane was incubated with primary anti-Flag (CST) antibody, anti-GAPDH (CST), anti-vinculin (CST), anti-PPP2R5D , anti-PPP2CA  and the secondary anti-rabbit antibody. Detection was performed using the BioRad ChemiDoc System (Version 2.4. 0.03).For Western blot analysis, the cells were lysed with RIPA lysis buffer with 1 mM phenylmethanesulfonylfluoride (PMSF) and a protease inhibitor cocktail, both from Cell Signaling Technology (CST) , for 15 min on ice. The cell lysates were clarified by centrifugation at 12,000× Co-immunoprecipitation (Co-IP) was performed as a routine process. The HEK293T cells were transiently transfected with the mammalian expression vectors encoding Flag-PPP2R1A or variants and HA-PPP2R5D using Lipofectamine 3000. After 48 h of transfection, the cells were collected for the experiment via a cell lysis buffer with PMSF and protease inhibitor. The cell lysates were incubated with the Flag beads  overnight at 4 °C with rotation. The interactions between the endogenous PPP2R5D and PPP2CA with the wild-type or mutant PPP2R1A protein were detected with Western blotting. PPP2R1A gene P/LP variants was also conducted using the online database ; ClinVar, https://www.ncibi.nlm.nih.gov/clinvar/ (accessed on 1 May 2023); and Decipher, https://www.deciphergenomics.org/ (accessed on 31 January 2023)). We excluded duplicate cases.PubMed and Web of Science were searched using the following terms: “(mutation OR variant) AND (PPP2R1A) AND (patient)”. Twenty-six publications were carefully checked, and the neurodevelopmental disorders of patients with PPP2R1A pathogenic/likely pathogenic (P/LP) variations were summarized. A search for Patient 1 was a five-month-old girl. Ultrasound showed that she had a large head circumference at five months of fetal age. The birth weight was 3250 g without any abnormalities in the delivery process. She could not lift her head and was diagnosed with hypotonia when she was five months old. The brain MRI showed brain dysplasia, dilated Virchow-Robin spaces, less white matter and a thin corpus callosum. A skeletal X-ray did not show any hip dislocation or abnormalities in the thoracolumbar spine. When she was 5 months and 22 days old, the Gesell scores of adaptive behavior, gross motor, fine motor, language development, individual and society ability were 70, 50, 68, 85 and 73, respectively. And her lower extremities showed dystrophy. At the last follow-up, when she was eight months old, her head circumference was 52 cm; however, she still could not hold her head steadily.Patient 2 was a girl aged one year and two months. She was born at full-term gestation via cesarean delivery to a G2P1 mother. The first fetal arrest occurred in early pregnancy. Her birth weight was 3600 g. The fetal ultrasound showed bilateral ventriculomegaly and subarachnoid effusion. The brain MRI showed bilateral ventriculomegaly and brain asymmetry when she was three months old. She could raise her head unsteadily, but could not turn over at six months old; she was then diagnosed with hypotonia. She suffered from respiratory tract infection and pneumonia repeatedly. She could sit at one year old, and could say “baba, mama” at one year and five months old, at which point her head circumference was 50.5 cm.Patient 3 was a boy aged one year and six months. He was born full-term but small for gestational age (SGA) after an uncomplicated pregnancy. He had his first seizure at seven days after birth, and epilepsy occurred four times within two months at one year and five months old. He could walk at 13 months old, and could say “baba, mama” at one year and six months old, at which point his head circumference was 45 cm. He was diagnosed with hypospadias .Patient 4 was a girl of two years and four months. She could not say any words or walk steadily. The brain MRI showed myelination delays. She had seizures two or three times per year. She was diagnosed with hyperactivity disorder. Her right-ear hearing was 40 dB.Patient 5 was a boy of one year and three months old. He was born at term after an uncomplicated pregnancy to a G2P1 mother. His birth weight was 3300 g. He had his first seizure at ten months old, and repeated seizures from 10 months old to one year and three months old. He could walk at 11 months old, but could not say any words. The Gesell test showed that his adaptive behavior score was 10.5 months old, gross motor quotient was 14.7 months old, fine motor quotient was 11.67 months old, language developmental quotient was 8.63 months old, and individual and society quotient was 14.23 months old at one year and three months old. The brain MRI showed a large, patchy, abnormal signal shadow in the right temporo-occipital lobe and frontotemporal ventriculomegaly. He was diagnosed with Sturge–Weber syndrome, focal epilepsy and glaucoma.PPP2R1A (NM_014225.6) gene were identified in the patients mentioned above; four of them were confirmed to be de novo variants  and one frameshift  variant of the variants A. Three variants B. The povariants C. Two vavariants C.WT and mutants  with HA-PPP2R5D plasmids were transfected in the HEK293T cell line to investigate the effect of the identified PPP2R1A variants on protein expression and binding to over-expressed PPP2R5D. Western blot results showed that the expressions of the PPP2R1AM180T and PPP2R1AV470A proteins were similar to the wild-type PPP2R1A, while the expression of the PPP2R1AR498L protein was relatively less than the wild-type protein. The protein level of the frameshift variant PPP2R1AD282Rfs*14 was not decreased but showed a truncated size (from 65 kDa to 33 kDa) (The Flag-PPP2R1A 33 kDa) A. The Co 33 kDa) A.PPP2R1A gene variants to endogenous PPP2R5D (B’δ subunit) and PPP2CA (C subunit) by transfecting the Flag-PPP2R1A plasmids in the HEK293T cell line. A very weak band of PPP2R5D and no PPP2CA band was detected in the Arg498Leu-PPP2R1A-transfected cell lines; neither band of these two subunits was detected in the Asn282Argfs*14-PPP2R1A-transfected cell lines; a weak band of PPP2CA and a very weak band of PPP2R5D protein were detected in the Met180Thr-PPP2R1A-transfected cell lines. While no binding difference in the B’δ and C subunit was detected between the Val470Ala-PPP2R1A- and WT-PPP2R1A-transfected cell lines  . Of thesOf the 64 patients (2 fetus cases were excluded), 25 were female and 35 were male, and 4 were unknown (4 recorded in ClinVar without sex information). The age at diagnosis ranged from neonate to 27 years old. The common features of these individuals were language delay (100%), intellectual disability/developmental delay , hypotonia  and motor delay . Among them, 54 patients had head circumference information . Interestingly, the clinical manifestation differed in patients with microcephaly and macrocephaly . In the PPP2R1A gene variants. The clinical features were compared in different variant sites. The variant sites were grouped into five clusters, named Thr178/Pro179 , Met180 , Arg182/Arg183 , Ser219/Val220 , and Arg258 (including Arg258Ser and Arg258His). From the perspective of the patients’ head circumference, all 9 patients with the Arg258 site variants had microcephaly, and 10 of 12 (83.3%) patients with the Met180 site variants had macrocephaly  (We sought to examine the genotype–phenotype correlations of patients with ference) . From thference) B. We alsference) C. Notablference) .PPP2R1A [PPP2R5B, PPP2R5C, PPP2R5D [PPP2CA [SET, SETBP1, BOD1 and CIP2A [PPP2R1A, PPP2CA and PPP2R5D [PP2A is a serine–threonine phosphatase that plays an essential regulatory role in cell signaling and physiology, including brain development and function, tumorigenesis and autoimmune diseases ,18. ThisPPP2R1A , PPP2R5B PPP2R5D ,21 and P [PPP2CA , and PP2 PPP2R5D ,10,11,21PPP2R1A gene pathogenic variations. These patients showed different clinical manifestations such as opposite abnormal head circumference types, and microcephaly and macrocephaly. To identify the correlations between the different variations of PPP2R1A with clinical features, we collected and reviewed the clinical and genetic information of 64 NDD patients  [PPP2R1A-related disorder is now known as Houge–Janssen syndrome 2 (MIM:616362). Currently, over 60 patients have been reported to have ncluded) ,14,15,22PPP2R1A variations disrupt the binding ability to the B and C subunits of PP2A ,10. The Additionally, variations in impaired function can cause the insufficient dephosphorylation of PP2A substrates ,18. VariPPP2R1A gene variants with patients’ clinical features was shown to have important implications for clinical follow-up and prognostic assessment.Our study reported five patients with PPP2R1A gene variants and provided functional evidence; one of them was the first-reported variant in the C-subunit-binding domain. In addition, the relationship of