The RNA exosome processes a wide variety of RNA and mediates RNA maturation, quality control and decay. In marked contrast to its high processivity in vivo, the purified exosome exhibits only weak activity on RNA substrates in vitro. Its activity is regulated by several auxiliary proteins, and protein complexes. In budding yeast, the activity of exosome is enhanced by the polyadenylation complex referred to as TRAMP. TRAMP oligoadenylates precursors and aberrant forms of RNAs to promote their trimming or complete degradation by exosomes. This chapter provides protocols for the purification of TRAMP and exosome complexes from yeast and the in vitro evaluation of exosome activation by the TRAMP complex. The protocols can be used for different purposes, such as the assessment of the role of individual subunits, protein domains or particular mutations in TRAMP-exosome RNA processing in vitro.

• Keywords
• Air1, Air2, Degradation assay, Mtr4, Noncanonical poly(A) polymerase, Noncoding RNAs, Polyadenylation assay, RNA exosome, RNA quality control, Rrp6, TAP purification, TRAMP4, Trf4,
• MeSH
• Cell Nucleus metabolism   MeSH
• Exosome Multienzyme Ribonuclease Complex metabolism   MeSH
• Exosomes metabolism   MeSH
• Polyadenylation physiology   MeSH
• RNA metabolism   MeSH
• Saccharomyces cerevisiae Proteins metabolism   MeSH
• Saccharomyces cerevisiae metabolism   MeSH
• Serine Endopeptidases metabolism   MeSH
• RNA Stability physiology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Exosome Multienzyme Ribonuclease Complex MeSH
• RNA MeSH
• Saccharomyces cerevisiae Proteins MeSH
• Serine Endopeptidases MeSH
• tunicate retinoic acid-inducible modular protease MeSH Browser

The Nuclear Exosome Targeting (NEXT) complex is a key cofactor of the mammalian nuclear exosome in the removal of Promoter Upstream Transcripts (PROMPTs) and potentially aberrant forms of other noncoding RNAs, such as snRNAs. NEXT is composed of three subunits SKIV2L2, ZCCHC8 and RBM7. We have recently identified the NEXT complex in our screen for oligo(U) RNA-binding factors. Here, we demonstrate that NEXT displays preference for U-rich pyrimidine sequences and this RNA binding is mediated by the RNA recognition motif (RRM) of the RBM7 subunit. We solved the structure of RBM7 RRM and identified two phenylalanine residues that are critical for interaction with RNA. Furthermore, we showed that these residues are required for the NEXT interaction with snRNAs in vivo. Finally, we show that depletion of components of the NEXT complex alone or together with exosome nucleases resulted in the accumulation of mature as well as extended forms of snRNAs. Thus, our data suggest a new scenario in which the NEXT complex is involved in the surveillance of snRNAs and/or biogenesis of snRNPs.

• MeSH
• Amino Acid Motifs MeSH
• HEK293 Cells MeSH
• HeLa Cells MeSH
• Humans MeSH
• Oligoribonucleotides metabolism   MeSH
• Protein Subunits chemistry   metabolism   MeSH
• RNA-Binding Proteins analysis   chemistry   metabolism   MeSH
• RNA, Small Nuclear chemistry   metabolism   MeSH
• Base Sequence MeSH
• Uracil Nucleotides metabolism   MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• oligo(U) MeSH Browser
• Oligoribonucleotides MeSH
• Protein Subunits MeSH
• RNA-Binding Proteins MeSH
• RBM7 protein, human MeSH Browser
• RNA, Small Nuclear MeSH
• Uracil Nucleotides MeSH