Staufen1 (STAU1) is a dsRNA binding protein mediating mRNA transport and localization, translational control and STAU1-mediated mRNA decay (SMD). The STAU1 binding site (SBS) within human ADP-ribosylation factor1 (ARF1) 3'UTR binds STAU1 and this downregulates ARF1 cytoplasmic mRNA levels by SMD. However, how STAU1 recognizes specific mRNA targets is still under debate. Our structure of the ARF1 SBS-STAU1 complex uncovers target recognition by STAU1. STAU1 dsRNA binding domain (dsRBD) 4 interacts with two pyrimidines and one purine from the minor groove side via helix α1, the β1-β2 loop anchors the dsRBD at the end of the dsRNA and lysines in helix α2 bind to the phosphodiester backbone from the major groove side. STAU1 dsRBD3 displays the same binding mode with specific recognition of one guanine base. Mutants disrupting minor groove recognition of ARF1 SBS affect in vitro binding and reduce SMD in vivo. Our data thus reveal how STAU1 recognizes minor groove features in dsRNA relevant for target selection.

• MeSH
• ADP-ribosylační faktor 1 chemie   genetika   MeSH
• cytoplazma chemie   genetika   MeSH
• cytoskeletální proteiny chemie   genetika   MeSH
• dvouvláknová RNA chemie   genetika   MeSH
• konformace proteinů MeSH
• lidé MeSH
• proteiny vázající RNA chemie   genetika   MeSH
• stabilita RNA genetika   MeSH
• vazebná místa genetika   MeSH
• vazebný motiv pro dvoušroubovici RNA genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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.

• 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

Most eukaryotic RNAs are posttranscriptionally modified. The majority of modifications promote RNA maturation, others may regulate function and stability. The 3' terminal non-templated oligouridylation is a widespread modification affecting many cellular RNAs at some stage of their life cycle. It has diverse roles in RNA metabolism. The most prevalent is the regulation of stability and quality control. On the cellular and organismal level, it plays a critical role in a number of pathways, such as cell cycle regulation, cell death, development or viral infection. Defects in uridylation have been linked to several diseases. This review summarizes the current knowledge about the role of the 3' terminal oligo(U)-tailing in biology of various RNAs in eukaryotes and describes key factors involved in these pathways.This article is part of the theme issue '5' and 3' modifications controlling RNA degradation'.

• MeSH
• Eukaryota MeSH
• eukaryotické buňky fyziologie   MeSH
• lidé MeSH
• RNA metabolismus   MeSH
• úpravy 3' konce RNA * MeSH
• uridin metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Uridylation of various cellular RNA species at the 3' end has been generally linked to RNA degradation. In mammals, uridylated pre-let-7 miRNAs and mRNAs are targeted by the 3' to 5' exoribonuclease DIS3L2. Mutations in DIS3L2 have been associated with Perlman syndrome and with Wilms tumor susceptibility. Using in vivo cross-linking and immunoprecipitation (CLIP) method, we discovered the DIS3L2-dependent cytoplasmic uridylome of human cells. We found a broad spectrum of uridylated RNAs including rRNAs, snRNAs, snoRNAs, tRNAs, vault, 7SL, Y RNAs, mRNAs, lncRNAs, and transcripts from pseudogenes. The unifying features of most of these identified RNAs are aberrant processing and the presence of stable secondary structures. Most importantly, we demonstrate that uridylation mediates DIS3L2 degradation of short RNA polymerase II-derived RNAs. Our findings establish the role of DIS3L2 and oligouridylation as the cytoplasmic quality control for highly structured ncRNAs.

• MeSH
• buněčné linie MeSH
• exoribonukleasy genetika   metabolismus   MeSH
• imunoprecipitace MeSH
• lidé MeSH
• nekódující RNA metabolismus   MeSH
• nukleotidyltransferasy metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH