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Prp4 Kinase Grants the License to Splice: Control of Weak Splice Sites during Spliceosome Activation

ABSTRACT: The genome of the fission yeast Schizosaccharomyces pombe encodes 17 kinases that are essential for cell growth. These include the cell-cycle regulator Cdc2, as well as several kinases that coordinate cell growth, polarity, and morphogenesis during the cell cycle. In this study, we further characterized another of these essential kinases, Prp4, and showed that the splicing of many introns is dependent on Prp4 kinase activity. For detailed characterization, we chose the genes res1 and ppk8, each of which contains one intron of typical size and position. Splicing of the res1 intron was dependent on Prp4 kinase activity, whereas splicing of the ppk8 intron was not. Extensive mutational analyses of the 5’ splice site of both genes revealed that proper transient interaction with the 5’ end of snRNA U1 governs the dependence of splicing on Prp4 kinase activity. Proper transient interaction between the branch sequence and snRNA U2 was also important. Therefore, the Prp4 kinase is required for recognition and efficient splicing of introns displaying weak exon1/5’ splice sites and weak branch sequences.

ORGANISM(S): Schizosaccharomyces pombe

PROVIDER: GSE75517 | GEO | 2015/12/01

SECONDARY ACCESSION(S): PRJNA304451

REPOSITORIES: GEO

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Novel RNA-binding Proteins Bind U1 snRNA to Promote Splicing of Weak 5' Splice Sites

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2023-09-07 | PXD029392 | Pride

Novel RNA-binding Proteins Bind U1 snRNA to Promote Splicing of Weak 5' Splice Sites

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Schizosaccharomyces pombe splicing microarray

Project description:Analysis of splicing defects in Schizosaccharomyces pombe upon chemical genetic inhibition of splicing kinases dsk1, lkh1, and prp4, as well as alanine-mutation of phosphorylated residues in the splicing factors bpb1, prp2, rsd1, srp1, srp2, usp101, usp103, sum3, prp22, cdc5, and cwf22. This study shows the splicing kinase dsk1 modulates splicing efficiency of introns with non-consensus splice sites, likely through phosphorylation of bpb1. Modulation of splicing efficiency of transcripts through kinase signaling pathways may afford the necessary flexibility to tune the gene expression profile in response to environmental and developmental cues. Experiments were conducted as direct two-color designs with 2-3 biological replicates per genotype pairing. Raw microarray data was normalized with loess normalization using the R package limma. Log2-fold changes (perturbation over reference) are reported. Each splicing event on the custom-designed splicing microarray was monitored with an exon probe reading out mRNA changes, an intron probe for unspliced pre-mRNA, and a splice junction probe spanning the junction between two spliced exons. For the analysis of the splicing efficiency for a given intron, a score was calculated as exon*intron/junction.

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Identification of New Branch Points and Unconventional Introns in Saccharomyces cerevisiae

Project description:Spliced messages constitute one-fourth of expressed mRNAs in the yeast Saccharomyces cerevisiae, and most mRNAs in metazoans. Splicing requires 5' splice site (5'SS), branch point (BP), and 3' splice site (3'SS) elements, but the role of the BP in splicing control is poorly understood because BP identification remains difficult. We developed a high-throughput method, Branch-seq, to map BP and 5'SS of isolated RNA lariats. Applied to S. cerevisiae, Branch-seq detected 76% of expressed, annotated BPs and identified a comparable number of novel BPs. We used RNA-seq to confirm associated 3'SS locations, identifying some 200 novel splice junctions, including an AT-AC intron. We show that several yeast introns use two or even three different BPs, with effects on 3'SS choice, protein coding potential, or RNA stability and identify novel introns whose splicing changes during meiosis or in response to stress. Together, these findings reveal BP-based regulation and demonstrate unanticipated complexity of splicing in yeast. 1 Lariat-seq experiment library. 3 barcoded Branch-seq libraries that make up one experiment. 26 RNA-seq samples, 2 biological replicates of each.

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Intron looping is mediated by U1 snRNP and RNA polymerase II co-transcriptionally [ChIP-seq]

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FUBP1 is a core splicing factor that facilitates 3' splice site recognition and splicing of long introns [RNA-Seq]

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