In Vitro Characterization of the Activity of the Mammalian RNA Exosome on mRNAs in Ribosomal Translation Complexes.
The RNA exosome is a multisubunit protein complex that exhibits a 3' to 5' exoribonuclease activity, endoribonuclease activity, and participates in a variety of RNA processing and degradation pathways in both the nucleus and the cytoplasm. Exosomes interact with various cofactors which target them to specific RNA substrates and processes. Investigation of the mechanisms by which mammalian RNA exosomes are targeted to specific RNA substrates requires the development of in vitro approaches for purification of exosomes and their co-factors, assembly of substrates and monitoring of the exosomal activity. Here, we describe protocols for in vitro reconstitution of ribosomal 80S elongation complexes on cap-labeled mRNAs and for assaying exosomal degradation of mRNAs in such complexes depending on the presence of GTPBP1, which has previously been implicated in directing the exosome to mRNA targets.
['Zinoviev|Alexandra|A|', 'Hellen|Christopher U T|CUT|', 'Pestova|Tatyana V|TV|']
D000818:Animals D002460:Cell Line D002467:Cell Nucleus D004926:Escherichia coli D063326:Exosome Multienzyme Ribonuclease Complex D055354:Exosomes D057809:HEK293 Cells D006801:Humans D008322:Mammals D012313:RNA D020871:RNA Stability D012333:RNA, Messenger D011817:Rabbits D012270:Ribosomes D015003:Yeasts
RNA exosome GTPBP1 Cap labeling of mRNA In vitro reconstitution of mammalian ribosomal complexes Toeprinting mRNA degradation assay