Spliceosomal snRNPs are multicomponent particles that undergo a complex maturation pathway. Human Sm-class snRNAs are generated as 3'-end extended precursors, which are exported to the cytoplasm and assembled together with Sm proteins into core RNPs by the SMN complex. Here, we provide evidence that these pre-snRNA substrates contain compact, evolutionarily conserved secondary structures that overlap with the Sm binding site. These structural motifs in pre-snRNAs are predicted to interfere with Sm core assembly. We model structural rearrangements that lead to an open pre-snRNA conformation compatible with Sm protein interaction. The predicted rearrangement pathway is conserved in Metazoa and requires an external factor that initiates snRNA remodeling. We show that the essential helicase Gemin3, which is a component of the SMN complex, is crucial for snRNA structural rearrangements during snRNP maturation. The SMN complex thus facilitates ATP-driven structural changes in snRNAs that expose the Sm site and enable Sm protein binding.

• MeSH
• HeLa Cells MeSH
• snRNP Core Proteins genetics   MeSH
• Humans MeSH
• RNA Precursors * metabolism   MeSH
• SMN Complex Proteins metabolism   MeSH
• Ribonucleoproteins, Small Nuclear metabolism   MeSH
• RNA, Small Nuclear * metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• snRNP Core Proteins MeSH
• RNA Precursors * MeSH
• SMN Complex Proteins MeSH
• Ribonucleoproteins, Small Nuclear MeSH
• RNA, Small Nuclear * 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.

• 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

Spliceosomal small nuclear ribonucleoprotein particles (snRNPs) undergo a complex maturation pathway containing multiple steps in the nucleus and in the cytoplasm. snRNP biogenesis is strictly proofread and several quality control checkpoints are placed along the pathway. Here, we analyzed the fate of small nuclear RNAs (snRNAs) that are unable to acquire a ring of Sm proteins. We showed that snRNAs lacking the Sm ring are unstable and accumulate in P-bodies in an LSm1-dependent manner. We further provide evidence that defective snRNAs without the Sm binding site are uridylated at the 3' end and associate with DIS3L2 3'→5' exoribonuclease and LSm proteins. Finally, inhibition of 5'→3' exoribonuclease XRN1 increases association of ΔSm snRNAs with DIS3L2, which indicates competition and compensation between these two degradation enzymes. Together, we provide evidence that defective snRNAs without the Sm ring are uridylated and degraded by alternative pathways involving either DIS3L2 or LSm proteins and XRN1.

• MeSH
• Exoribonucleases metabolism   MeSH
• HeLa Cells MeSH
• Nucleic Acid Conformation * MeSH
• Humans MeSH
• Organelles metabolism   MeSH
• SMN Complex Proteins metabolism   MeSH
• RNA-Binding Proteins metabolism   MeSH
• Proto-Oncogene Proteins metabolism   MeSH
• RNA, Small Nuclear chemistry   metabolism   MeSH
• Base Sequence MeSH
• RNA Stability MeSH
• RNA Transport * MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DIS3L2 protein, human MeSH Browser
• Exoribonucleases MeSH
• GEMIN5 protein, human MeSH Browser
• LSM1 protein, human MeSH Browser
• SMN Complex Proteins MeSH
• RNA-Binding Proteins MeSH
• Proto-Oncogene Proteins MeSH
• RNA, Small Nuclear 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'.

• Keywords
• RNA degradation, RNA modification, RNA processing, RNA surveillance, RNA uridylation, tutase,
• MeSH
• Eukaryota MeSH
• Eukaryotic Cells physiology   MeSH
• Humans MeSH
• RNA metabolism   MeSH
• RNA 3' End Processing * MeSH
• Uridine metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• RNA MeSH
• Uridine MeSH

Spliceosomal snRNPs are complex particles that proceed through a fascinating maturation pathway. Several steps of this pathway are closely linked to nuclear non-membrane structures called Cajal bodies. In this review, I summarize the last 20 y of research in this field. I primarily focus on snRNP biogenesis, specifically on the steps that involve Cajal bodies. I also evaluate the contribution of the Cajal body in snRNP quality control and discuss the role of snRNPs in Cajal body formation.

• MeSH
• Coiled Bodies metabolism   MeSH
• Transcription, Genetic MeSH
• Humans MeSH
• RNA Processing, Post-Transcriptional MeSH
• Ribonucleoproteins, Small Nuclear genetics   metabolism   MeSH
• Spliceosomes MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Ribonucleoproteins, Small Nuclear MeSH