Nuclear RNA Exosome and Pervasive Transcription: Dual Sculptors of Genome Function

The genome is pervasively transcribed across various species, yielding numerous non-coding RNAs. As a counterbalance for pervasive transcription, various organisms have a nuclear RNA exosome complex, whose structure is well conserved between yeast and mammalian cells. The RNA exosome not only regulates the processing of stable RNA species, such as rRNAs, tRNAs, small nucleolar RNAs, and small nuclear RNAs, but also plays a central role in RNA surveillance by degrading many unstable RNAs and misprocessed pre-mRNAs. In addition, associated cofactors of RNA exosome direct the exosome to distinct classes of RNA substrates, suggesting divergent and/or multi-layer control of RNA quality in the cell. While the RNA exosome is essential for cell viability and influences various cellular processes, mutations and alterations in the RNA exosome components are linked to the collection of rare diseases and various diseases including cancer, respectively. The present review summarizes the relationships between pervasive transcription and RNA exosome, including evolutionary crosstalk, mechanisms of RNA exosome-mediated RNA surveillance, and physiopathological effects of perturbation of RNA exosome.

The Potential of U6 and Its Copies in the Regulation of the Human Genome

Non-coding RNAs are conformed by a large repertoire of RNA molecules with unimaginable tridimensional structures and functions. Small nuclear RNAs are an essential part of the spliceosome machinery, which is crucial for proper mRNA maturation. It is important to add that U6, one of the four snRNAs forming the spliceosome has been extensively studied. Full-length U6 (U6-1) loci are widely dispersed throughout the genome (200-900 copies), but a few U6 full-length loci have been identified to date as potentially active genes. The importance of U6 to carry out, together with other snRNAs, the catalytic activity and recognition of annealing target sequences, its evolution in the genome and the fact that the genome has many U6 copies and pseudogenes, its association with retrotransposition, as well as their implication in diseases is discussed in this review.

Nopp140-chaperoned 2’-O-methylation of small nuclear RNAs in Cajal bodies ensures splicing fidelity

ABSTRACTSpliceosomal small nuclear RNAs (snRNAs) are modified by small Cajal body (CB) specific ribonucleoproteins (scaRNPs) to ensure snRNP biogenesis and pre-mRNA splicing. However, the function and subcellular site of snRNA modification are largely unknown. We show that CB localization of the protein Nopp140 is essential for concentration of scaRNPs in that nuclear condensate; and that phosphorylation by casein kinase 2 (CK2) at some 80 serines targets Nopp140 to CBs. Transiting through CBs, snRNAs are apparently modified by scaRNPs. Indeed, Nopp140 knockdown-mediated release of scaRNPs from CBs severely compromises 2’-O-methylation of spliceosomal snRNAs, identifying CBs as the site of scaRNP catalysis. Additionally, alternative splicing patterns change indicating that these modifications in U1, U2, U5, and U12 snRNAs safeguard splicing fidelity. Given the importance of CK2 in this pathway, compromised splicing could underlie the mode of action of small molecule CK2 inhibitors currently considered for therapy in cholangiocarcinoma, hematological malignancies, and COVID-19.

Spliceosomal snRNA Epitranscriptomics

Small nuclear RNAs (snRNAs) are critical components of the spliceosome that catalyze the splicing of pre-mRNA. snRNAs are each complexed with many proteins to form RNA-protein complexes, termed as small nuclear ribonucleoproteins (snRNPs), in the cell nucleus. snRNPs participate in pre-mRNA splicing by recognizing the critical sequence elements present in the introns, thereby forming active spliceosomes. The recognition is achieved primarily by base-pairing interactions (or nucleotide-nucleotide contact) between snRNAs and pre-mRNA. Notably, snRNAs are extensively modified with different RNA modifications, which confer unique properties to the RNAs. Here, we review the current knowledge of the mechanisms and functions of snRNA modifications and their biological relevance in the splicing process.

The integrity of the U12 snRNA 3′ stem–loop is necessary for its overall stability

Disruption of minor spliceosome functions underlies several genetic diseases with mutations in the minor spliceosome-specific small nuclear RNAs (snRNAs) and proteins. Here, we define the molecular outcome of the U12 snRNA mutation (84C>U) resulting in an early-onset form of cerebellar ataxia. To understand the molecular consequences of the U12 snRNA mutation, we created cell lines harboring the 84C>T mutation in the U12 snRNA gene (RNU12). We show that the 84C>U mutation leads to accelerated decay of the snRNA, resulting in significantly reduced steady-state U12 snRNA levels. Additionally, the mutation leads to accumulation of 3′-truncated forms of U12 snRNA, which have undergone the cytoplasmic steps of snRNP biogenesis. Our data suggests that the 84C>U-mutant snRNA is targeted for decay following reimport into the nucleus, and that the U12 snRNA fragments are decay intermediates that result from the stalling of a 3′-to-5′ exonuclease. Finally, we show that several other single-nucleotide variants in the 3′ stem-loop of U12 snRNA that are segregating in the human population are also highly destabilizing. This suggests that the 3′ stem-loop is important for the overall stability of the U12 snRNA and that additional disease-causing mutations are likely to exist in this region.

What’s all the phos about? Insights into the phosphorylation state of the RNA polymerase II C-terminal domain via mass spectrometry

RNA polymerase II (RNAP II) is one of the primary enzymes responsible for expressing protein-encoding genes and some small nuclear RNAs. The enigmatic carboxy-terminal domain (CTD) of RNAP II and...

Exploration of the cyanidioschyzon merolae intron landscape

The eukaryotic process of pre-mRNA splicing involves the removal of noncoding intron sequences and the fusion of the remaining protein-coding exon sequences. The splicing reaction is catalyzed by the spliceosome, a dynamic multi-megadalton ribonucleoprotein complex that, in humans, is composed of 5 small nuclear RNAs (snRNAs) and over 200 associated proteins acting on more that 200,000 introns present within 25,000 genes. The unicellular red alga Cyanidioschyzon merolae possesses a more tractable splicing environment, with only 4 snRNAs and 75 associated proteins interacting with 27 annotated introns found in 26 our of 5,331 genes. Intron-rich genomes can confer benefits to their host species such as improved gene expression, incredible proteomic diversity, and increased genetic stability. This raises the question of why intron-poor C. merolae has retained such a small number of introns and a dramatically reduced spliceosome. A comprehensive investigation into the precise role that introns play in C. merolae would require the systematic removal of introns and an analysis of the effects thereof. The ability to elucidate the role of splicing in C. merolae via genome-wide intron deletion, however, hinges on the feasibility of establishing the efficiently scalable CRISPR genome engineering tool in C. merolae. It also follows that such an endeavour would require an accurate picture of the intron landscape of C. merolae, and since the number of annotated introns in C. merolae is relatively small, it is especially vital to determine whether any introns are missing from the C. merolae annotation. To that end, a stable and inducible Cas9-expressing strain of C. merolae was successfully developed. Transcriptome analysis using RNA-seq data revealed the discovery of 11 novel introns and 1 misannotated intron, as well as the presence of alternative splicing in the form of alternative splice site usage.