Competition between maturation and degradation drives human snRNA 3' end quality control.

Polymerases and exonucleases act on 3' ends of nascent RNAs to promote their maturation or degradation but how the balance between these activities is controlled to dictate the fates of cellular RNAs remains poorly understood. Here, we identify a central role for the human DEDD deadenylase TOE1 in distinguishing the fates of small nuclear (sn)RNAs of the spliceosome from unstable genome-encoded snRNA variants. We found that TOE1 promotes maturation of all regular RNA polymerase II transcribed snRNAs of the major and minor spliceosomes by removing posttranscriptional oligo(A) tails, trimming 3' ends, and preventing nuclear exosome targeting. In contrast, TOE1 promotes little to no maturation of tested U1 variant snRNAs, which are instead targeted by the nuclear exosome. These observations suggest that TOE1 is positioned at the center of a 3' end quality control pathway that selectively promotes maturation and stability of regular snRNAs while leaving snRNA variants unprocessed and exposed to degradation in what could be a widespread mechanism of RNA quality control given the large number of noncoding RNAs processed by DEDD deadenylases.

Human spliceosomal snRNA sequence variants generate variant spliceosomes.

Human pre-mRNA splicing is primarily catalyzed by the major spliceosome, comprising five small nuclear ribonucleoprotein complexes, U1, U2, U4, U5, and U6 snRNPs, each of which contains the corresponding U-rich snRNA. These snRNAs are encoded by large gene families exhibiting significant sequence variation, but it remains unknown if most human snRNA genes are untranscribed pseudogenes or produce variant snRNAs with the potential to differentially influence splicing. Since gene duplication and variation are powerful mechanisms of evolutionary adaptation, we sought to address this knowledge gap by systematically profiling human U1, U2, U4, and U5 snRNA variant gene transcripts. We identified 55 transcripts that are detectably expressed in human cells, 38 of which incorporate into snRNPs and spliceosomes in 293T cells. All U1 snRNA variants are more than 1000-fold less abundant in spliceosomes than the canonical U1, whereas at least 1% of spliceosomes contain a variant of U2 or U4. In contrast, eight U5 snRNA sequence variants occupy spliceosomes at levels of 1% to 46%. Furthermore, snRNA variants display distinct expression patterns across five human cell lines and adult and fetal tissues. Different RNA degradation rates contribute to the diverse steady state levels of snRNA variants. Our findings suggest that variant spliceosomes containing noncanonical snRNAs may contribute to different tissue- and cell-type-specific alternative splicing patterns.

The nuclear cap-binding complex interacts with the U4/U6·U5 tri-snRNP and promotes spliceosome assembly in mammalian cells.

The nuclear cap-binding complex (CBC) binds to the 7-methyl guanosine cap present on every RNA polymerase II transcript. CBC has been implicated in many aspects of RNA biogenesis; in addition to roles in miRNA biogenesis, nonsense-mediated decay, 3'-end formation, and snRNA export from the nucleus, CBC promotes pre-mRNA splicing. An unresolved question is how CBC participates in splicing. To investigate CBC's role in splicing, we used mass spectrometry to identify proteins that copurify with mammalian CBC. Numerous components of spliceosomal snRNPs were specifically detected. Among these, three U4/U6·U5 snRNP proteins (hBrr2, hPrp4, and hPrp31) copurified with CBC in an RNA-independent fashion, suggesting that a significant fraction of CBC forms a complex with the U4/U6·U5 snRNP and that the activity of CBC might be associated with snRNP recruitment to pre-mRNA. To test this possibility, CBC was depleted from HeLa cells by RNAi. Chromatin immunoprecipitation and live-cell imaging assays revealed decreased cotranscriptional accumulation of U4/U6·U5 snRNPs on active transcription units, consistent with a requirement for CBC in cotranscriptional spliceosome assembly. Surprisingly, recruitment of U1 and U2 snRNPs was also affected, indicating that RNA-mediated interactions between CBC and snRNPs contribute to splicing. On the other hand, CBC depletion did not impair snRNP biogenesis, ruling out the possibility that decreased snRNP recruitment was due to changes in nuclear snRNP concentration. Taken together, the data support a model whereby CBC promotes pre-mRNA splicing through a network of interactions with and among spliceosomal snRNPs during cotranscriptional spliceosome assembly.

Spliceosomal snRNAs, the Essential Players in pre-mRNA Processing in Eukaryotic Nucleus: From Biogenesis to Functions and Spatiotemporal Characteristics.

Spliceosomal small nuclear RNAs (snRNAs) are a fundamental class of non-coding small RNAs abundant in the nucleoplasm of eukaryotic cells, playing a crucial role in splicing precursor messenger RNAs (pre-mRNAs). They are transcribed by DNA-dependent RNA polymerase II (Pol II) or III (Pol III), and undergo subsequent processing and 3' end cleavage to become mature snRNAs. Numerous protein factors are involved in the transcription initiation, elongation, termination, splicing, cellular localization, and terminal modification processes of snRNAs. The transcription and processing of snRNAs are regulated spatiotemporally by various mechanisms, and the homeostatic balance of snRNAs within cells is of great significance for the growth and development of organisms. snRNAs assemble with specific accessory proteins to form small nuclear ribonucleoprotein particles (snRNPs) that are the basal components of spliceosomes responsible for pre-mRNA maturation. This article provides an overview of the biological functions, biosynthesis, terminal structure, and tissue-specific regulation of snRNAs.