RESUMO
Intron diversity facilitates regulated gene expression and alternative splicing. Spliceosomes excise introns after recognizing their splicing signals: the 5'-splice site (5'ss), branchpoint (BP) and 3'-splice site (3'ss). The latter two signals are recognized by U2 small nuclear ribonucleoprotein (snRNP) and its accessory factors (U2AFs), but longer spacings between them result in weaker splicing. Here, we show that excision of introns with a BP-distant 3'ss (e.g. rap1 intron 2) requires the ubiquitin-fold-activated splicing regulator Sde2 in Schizosaccharomyces pombe. By monitoring splicing-specific ura4 reporters in a collection of S. pombe mutants, Cay1 and Tls1 were identified as additional regulators of this process. The role of Sde2, Cay1 and Tls1 was further confirmed by increasing BP-3'ss spacings in a canonical tho5 intron. We also examined BP-distant exons spliced independently of these factors and observed that RNA secondary structures possibly bridged the gap between the two signals. These proteins may guide the 3'ss towards the spliceosome's catalytic centre by folding the RNA between the BP and 3'ss. Orthologues of Sde2, Cay1 and Tls1, although missing in the intron-poor Saccharomyces cerevisiae, are present in intron-rich eukaryotes, including humans. This type of intron-specific pre-mRNA splicing appears to have evolved for regulated gene expression and alternative splicing of key heterochromatin factors.
Assuntos
Precursores de RNA , Schizosaccharomyces , Processamento Alternativo , Proteínas de Transporte , Proteínas de Ligação a DNA/genética , Éxons , Heterocromatina , Humanos , Íntrons/genética , Proteínas Nucleares/metabolismo , Precursores de RNA/metabolismo , Sítios de Splice de RNA , Splicing de RNA , Ribonucleoproteína Nuclear Pequena U2/genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe , Complexo Shelterina , Proteínas de Ligação a Telômeros , Ubiquitina/genética , Ubiquitina/metabolismoRESUMO
The expression of intron-containing genes in eukaryotes requires generation of protein-coding messenger RNAs (mRNAs) via RNA splicing, whereby the spliceosome removes non-coding introns from pre-mRNAs and joins exons. Spliceosomes must ensure accurate removal of highly diverse introns. We show that Sde2 is a ubiquitin-fold-containing splicing regulator that supports splicing of selected pre-mRNAs in an intron-specific manner in Schizosaccharomyces pombe Both fission yeast and human Sde2 are translated as inactive precursor proteins harbouring the ubiquitin-fold domain linked through an invariant GGKGG motif to a C-terminal domain (referred to as Sde2-C). Precursor processing after the first di-glycine motif by the ubiquitin-specific proteases Ubp5 and Ubp15 generates a short-lived activated Sde2-C fragment with an N-terminal lysine residue, which subsequently gets incorporated into spliceosomes. Absence of Sde2 or defects in Sde2 activation both result in inefficient excision of selected introns from a subset of pre-mRNAs. Sde2 facilitates spliceosomal association of Cactin/Cay1, with a functional link between Sde2 and Cactin further supported by genetic interactions and pre-mRNA splicing assays. These findings suggest that ubiquitin-like processing of Sde2 into a short-lived activated form may function as a checkpoint to ensure proper splicing of certain pre-mRNAs in fission yeast.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Íntrons , Splicing de RNA , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Ubiquitina/metabolismo , Proteínas de Ligação a DNA/genética , Instabilidade Genômica , Humanos , Precursores de RNA/genética , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , SpliceossomosRESUMO
The occurrence of spliceosomal introns in eukaryotic genomes is highly diverse and ranges from few introns in an organism to multiple introns per gene. Introns vary with respect to their lengths, strengths of splicing signals, and position in resident genes. Higher intronic density and diversity in genetically complex organisms relies on increased efficiency and accuracy of spliceosomes for pre-mRNA splicing. Since intron diversity is critical for functions in RNA stability, regulation of gene expression and alternative splicing, RNA-binding proteins, spliceosomal regulatory factors and post-translational modifications of splicing factors ought to make the splicing process intron-specific. We recently reported function and regulation of a ubiquitin fold harboring splicing regulator, Sde2, which following activation by ubiquitin-specific proteases facilitates excision of selected introns from a subset of multi-intronic genes in Schizosaccharomyces pombe (Thakran et al. EMBO J, https://doi.org/10.15252/embj.201796751 , 2017). By reviewing our findings with understandings of intron functions and regulated splicing processes, we propose possible functions and mechanism of intron-specific pre-mRNA splicing and suggest that this process is crucial to highlight importance of introns in eukaryotic genomes.