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.

• Klíčová slova
• Air1, Air2, Degradation assay, Mtr4, Noncanonical poly(A) polymerase, Noncoding RNAs, Polyadenylation assay, RNA exosome, RNA quality control, Rrp6, TAP purification, TRAMP4, Trf4,
• MeSH
• buněčné jádro metabolismus   MeSH
• exozom metabolismus   MeSH
• exozómy metabolismus   MeSH
• polyadenylace fyziologie   MeSH
• RNA metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny metabolismus   MeSH
• Saccharomyces cerevisiae metabolismus   MeSH
• serinové endopeptidasy metabolismus   MeSH
• stabilita RNA fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• exozom MeSH
• RNA MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• serinové endopeptidasy MeSH
• tunicate retinoic acid-inducible modular protease MeSH Prohlížeč

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
• aminokyselinové motivy MeSH
• HEK293 buňky MeSH
• HeLa buňky MeSH
• lidé MeSH
• oligoribonukleotidy metabolismus   MeSH
• podjednotky proteinů chemie   metabolismus   MeSH
• proteiny vázající RNA analýza   chemie   metabolismus   MeSH
• RNA malá jaderná chemie   metabolismus   MeSH
• sekvence nukleotidů MeSH
• uracilnukleotidy metabolismus   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• oligo(U) MeSH Prohlížeč
• oligoribonukleotidy MeSH
• podjednotky proteinů MeSH
• proteiny vázající RNA MeSH
• RBM7 protein, human MeSH Prohlížeč
• RNA malá jaderná MeSH
• uracilnukleotidy MeSH

RNA metabolism is altered following DNA damage, but the underlying mechanisms are not well understood. Through a 14-3-3 interaction screen for DNA damage-induced protein interactions in human cells, we identified protein complexes connected to RNA biology. These include the nuclear exosome targeting (NEXT) complex that regulates turnover of noncoding RNAs termed promoter upstream transcripts (PROMPTs). We show that the NEXT subunit RBM7 is phosphorylated upon DNA damage by the MAPKAPK2 kinase and establish that this mediates 14-3-3 binding and decreases PROMPT binding. These findings and our observation that cells lacking RBM7 display DNA damage hypersensitivity link PROMPT turnover to the DNA damage response.

• Klíčová slova
• 14-3-3, DNA damage response, MAPKAPK2, UV, nuclear exosome,
• MeSH
• exozom metabolismus   MeSH
• fosforylace MeSH
• intracelulární signální peptidy a proteiny metabolismus   MeSH
• lidé MeSH
• MAP kinasový signální systém MeSH
• nekódující RNA metabolismus   MeSH
• poškození DNA fyziologie   MeSH
• protein-serin-threoninkinasy metabolismus   MeSH
• proteiny 14-3-3 metabolismus   MeSH
• ultrafialové záření MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• exozom MeSH
• intracelulární signální peptidy a proteiny MeSH
• MAP-kinase-activated kinase 2 MeSH Prohlížeč
• nekódující RNA MeSH
• protein-serin-threoninkinasy MeSH
• proteiny 14-3-3 MeSH

Trf4/5p-Air1/2p-Mtr4p polyadenylation complex (TRAMP) is an essential component of nuclear RNA surveillance in yeast. It recognizes a variety of nuclear transcripts produced by all three RNA polymerases, adds short poly(A) tails to aberrant or unstable RNAs and activates the exosome for their degradation. Despite the advances in understanding the structural features of the isolated complex subunits or their fragments, the details of complex assembly, RNA recognition and exosome activation remain poorly understood. Here we provide the first understanding of the RNA binding mode of the complex. We show that Air2p is an RNA-binding subunit of TRAMP. We identify the zinc knuckles (ZnK) 2, 3 and 4 as the RNA-binding domains, and reveal the essentiality of ZnK4 for TRAMP4 polyadenylation activity. Furthermore, we identify Air2p as the key component of TRAMP4 assembly providing bridging between Mtr4p and Trf4p. The former is bound via the N-terminus of Air2p, while the latter is bound via ZnK5, the linker between ZnK4 and 5 and the C-terminus of the protein. Finally, we uncover the RNA binding part of the Mtr4p arch, the KOW domain, as the essential component for TRAMP-mediated exosome activation.

• MeSH
• adaptorové proteiny signální transdukční chemie   metabolismus   MeSH
• DEAD-box RNA-helikasy chemie   metabolismus   MeSH
• DNA-dependentní DNA-polymerasy chemie   metabolismus   MeSH
• interakční proteinové domény a motivy MeSH
• podjednotky proteinů chemie   metabolismus   MeSH
• proteiny vázající RNA chemie   metabolismus   MeSH
• ribonukleasy metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny chemie   metabolismus   MeSH
• terciární struktura proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adaptorové proteiny signální transdukční MeSH
• Air2 protein, S cerevisiae MeSH Prohlížeč
• DEAD-box RNA-helikasy MeSH
• DNA-dependentní DNA-polymerasy MeSH
• MTR4 protein, S cerevisiae MeSH Prohlížeč
• PAP2 protein, S cerevisiae MeSH Prohlížeč
• podjednotky proteinů MeSH
• proteiny vázající RNA MeSH
• ribonukleasy MeSH
• Saccharomyces cerevisiae - proteiny MeSH