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

Transfer RNAs (tRNAs) are key molecules participating in protein synthesis. To augment their functionality they undergo extensive post-transcriptional modifications and, as such, are subject to regulation at multiple levels including transcription, transcript processing, localization and ribonucleoside base modification. Post-transcriptional enzyme-catalyzed modification of tRNA occurs at a number of base and sugar positions and influences specific anticodon-codon interactions and regulates translation, its efficiency and fidelity. This phenomenon of nucleoside modification is most remarkable and results in a rich structural diversity of tRNA of which over 100 modified nucleosides have been characterized. Most often these hypermodified nucleosides are found in the wobble position of tRNAs, where they play a direct role in codon recognition as well as in maintaining translational efficiency and fidelity, etc. Several recent studies have pointed to a link between defects in tRNA modifications and human diseases including neurological disorders. Therefore, defects in tRNA modifications in humans need intensive characterization at the enzymatic and mechanistic level in order to pave the way to understand how lack of such modifications are associated with neurological disorders with the ultimate goal of gaining insights into therapeutic interventions.

• Klíčová slova
• Q-tRNA, modified nucleosides, neurological disease, queuosine, transfer RNA modifications,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

The Nrd1-Nab3-Sen1 (NNS) complex is essential for controlling pervasive transcription and generating sn/snoRNAs in S. cerevisiae. The NNS complex terminates transcription of noncoding RNA genes and promotes exosome-dependent processing/degradation of the released transcripts. The Trf4-Air2-Mtr4 (TRAMP) complex polyadenylates NNS target RNAs and favors their degradation. NNS-dependent termination and degradation are coupled, but the mechanism underlying this coupling remains enigmatic. Here we provide structural and functional evidence demonstrating that the same domain of Nrd1p interacts with RNA polymerase II and Trf4p in a mutually exclusive manner, thus defining two alternative forms of the NNS complex, one involved in termination and the other in degradation. We show that the Nrd1-Trf4 interaction is required for optimal exosome activity in vivo and for the stimulation of polyadenylation of NNS targets by TRAMP in vitro. We propose that transcription termination and RNA degradation are coordinated by switching between two alternative partners of the NNS complex.

• MeSH
• DNA-dependentní DNA-polymerasy chemie   metabolismus   MeSH
• exozómy metabolismus   MeSH
• fungální RNA metabolismus   MeSH
• konformace nukleové kyseliny MeSH
• magnetická rezonanční spektroskopie MeSH
• molekulární modely MeSH
• nekódující RNA metabolismus   MeSH
• polyadenylace MeSH
• proteiny vázající RNA chemie   metabolismus   MeSH
• RNA-polymerasa II metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny chemie   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   MeSH
• stabilita RNA MeSH
• terminace genetické transkripce * MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA-dependentní DNA-polymerasy MeSH
• fungální RNA MeSH
• nekódující RNA MeSH
• NRD1 protein, S cerevisiae MeSH Prohlížeč
• PAP2 protein, S cerevisiae MeSH Prohlížeč
• proteiny vázající RNA MeSH
• RNA-polymerasa II MeSH
• Saccharomyces cerevisiae - proteiny 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