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Ubiquitin-like processing of the conserved splicing regulator Sde2 promotes telomeric silencing and genome stability

ABSTRACT: Intron-containing gene expression in eukaryotes proceeds through the process of RNA splicing to generate protein-coding messenger RNAs (mRNAs). Herein a large and dynamic ribonucleoprotein complex — the spliceosome — removes non-coding introns from pre-mRNAs and joins exons. Spliceosomes must also ensure accurate and timely removal of diverse and highly prevalent introns. Here we show that Sde2 is a conserved splicing regulator, contains a ubiquitin fold, and supports splicing of a subset of pre-mRNAs in an intron-specific manner in Schizosaccharomyces pombe. Its orthologs in S. pombe and humans are synthesized as precursors harboring a ubiquitin fold, followed by an invariant GGKGG motif and an uncharacterized C-terminal domain (referred to as Sde2-C). The precursor must be cleaved at GG^K by the ubiquitin specific proteases Ubp5 and Ubp15 to produce the Sde2-C protein containing a lysine residue at its C-terminus, and is a substrate of the N-end rule pathway of proteasomal degradation. The truncated Sde2-C functions as a component of the spliceosome, and loss of Sde2-C results in inefficient splicing of selected introns from target genes having functions in DNA replication, transcription and telomeric silencing. Thus, the ubiquitin-like processing of Sde2 — associated with its regulation by the N-end rule pathway — contributes to genomic stability in S. pombe through specific pre-mRNA splicing events.

ORGANISM(S): Schizosaccharomyces pombe

PROVIDER: GSE97097 | GEO | 2017/09/22

SECONDARY ACCESSION(S): PRJNA380667

REPOSITORIES: GEO

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Project description:The spliceosome is a dynamic macromolecular machine that catalyzes the removal of introns from pre-mRNA to make mature message. Schizosaccharomyces pombe Cwf10 (homolog Saccharomyces cerevisiae Snu114 and of Human U5-116K), an integral member of the U5 snRNP, is a GTPase that shares sequence homology with the eukaryotic translation elongation factor EF2. Cwf10 is required for pre-mRNA splicing; however, its mechanism(s) of action is still not understood. Cwf10/Snu114 family members contain a conserved N-terminal extension (NTE) that lacks homology with EF2 and has been predicted to be an intrinsically unfolded domain. Using S. pombe as a model system, we show that the NTE is not essential, but cells lacking this domain are defective in pre-mRNA splicing at all temperatures. Genetic interactions between cwf10-M-NM-^TNTE and other pre-mRNA splicing mutants are consistent with a role for the NTE in spliceosome activation. Characterization of Cwf10-NTE by various biophysical techniques shows the NTE contains both regions of structure and disorder in solution. The first twenty-three highly-conserved amino acids of the NTE are essential for its role in splicing, but are not sufficient to restore pre-mRNA splicing to wild-type levels in cwf10-M-bM-^HM-^FNTE cells. When the NTE is overexpressed in the cwf10-M-NM-^TNTE background, it can complement the truncated Cwf10 protein in trans, and it also immunoprecipitates a complex similar in composition to the late-stage U5.U2/U6 spliceosome. These data show that the structurally flexible NTE is capable of making specific contacts within the spliceosome that may facilitate Cwf10M-bM-^@M-^Ys overall role facilitating spliceosome rearrangements. Interrogation of the S. pombe transcriptome using poly-A enriched RNA sequencing (Illumina HiSeq 2500) in wild type and cwf10-M-NM-^TNTE cultures. A total of 4 samples were analysed: two biological repeats of wild-type strain and two biological repeats of cwf10-M-NM-^TNTE

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Ubiquitin-like processing of the conserved splicing regulator Sde2 promotes telomeric silencing and genome stability

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