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Stalled spliceosomes are a signal for RNAi-mediated genome defense

ABSTRACT: Using the yeast Cryptococcus neoformans, we describe a mechanism by which transposons are initially targeted for RNAi-mediated genome defense. We show that intron-containing mRNA precursors template siRNA synthesis. We identify a Spliceosome-Coupled And Nuclear RNAi (SCANR) complex required for siRNA synthesis and demonstrate that it physically associates with the spliceosome. We find that RNAi target transcripts are distinguished by suboptimal introns and abnormally high occupancy on spliceosomes. Functional investigations demonstrate that the stalling of mRNA precursors on spliceosomes is required for siRNA accumulation. Lariat debranching enzyme is also necessary for siRNA production, suggesting a requirement for processing of stalled splicing intermediates. We propose that recognition of mRNA precursors by the SCANR complex is in kinetic competition with splicing, thereby promoting siRNA production from transposon transcripts stalled on spliceosomes. Disparity in the strength of gene expression signals encoded by transposons versus genes offers an avenue for the evolution of genome defense.

ORGANISM(S): Cryptococcus neoformans

PROVIDER: GSE43363 | GEO | 2013/01/21

SECONDARY ACCESSION(S): PRJNA185599

REPOSITORIES: GEO

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Stalled spliceosomes are a signal for RNAi-mediated genome defense

Project description:Using the yeast Cryptococcus neoformans, we describe a mechanism by which transposons are initially targeted for RNAi-mediated genome defense. We show that intron-containing mRNA precursors template siRNA synthesis. We identify a Spliceosome-Coupled And Nuclear RNAi (SCANR) complex required for siRNA synthesis and demonstrate that it physically associates with the spliceosome. We find that RNAi target transcripts are distinguished by suboptimal introns and abnormally high occupancy on spliceosomes. Functional investigations demonstrate that the stalling of mRNA precursors on spliceosomes is required for siRNA accumulation. Lariat debranching enzyme is also necessary for siRNA production, suggesting a requirement for processing of stalled splicing intermediates. We propose that recognition of mRNA precursors by the SCANR complex is in kinetic competition with splicing, thereby promoting siRNA production from transposon transcripts stalled on spliceosomes. Disparity in the strength of gene expression signals encoded by transposons versus genes offers an avenue for the evolution of genome defense. Small RNA profile of wild-type Cryptococcus neoformans was determined by deep sequencing using the Illumina SBS platform

2013-01-21 | E-GEOD-43363 | biostudies-arrayexpress

Cryptococcus neoformans

Project description:Stalled spliceosomes are a signal for RNAi-mediated genome defense

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CDK11 phosphorylates SF3B1 and is essential for constitutive splicing (iCLIP)

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CDK11 phosphorylates SF3B1 and is essential for constitutive splicing (4sU-seq)

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CDK11 phosphorylates SF3B1 and is essential for constitutive splicing (ChIP-seq)

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Endogenous siRNAs derived from transposons and mRNAs in Drosophila somatic cells

Project description:Small interfering RNAs (siRNAs) direct RNA interference (RNAi) in eukaryotes. In flies, somatic cells produce siRNAs from exogenous double-stranded RNA as a defense against viral infection. Here, we identify 21-nt long, endogenous siRNAs (endo-siRNAs) corresponding to transposons and heterochromatic sequences in the somatic cells of Drosophila melanogaster. We also detected endo-siRNAs complementary to mRNAs: these siRNAs disproportionately mapped to the complementary regions of overlapping mRNAs predicted to form dsRNA in vivo. Normal accumulation of somatic endo-siRNAs requires the siRNA-generating ribonuclease, Dicer-2, and the RNAi effector protein, Ago2. We propose that endo-siRNAs generated by the fly RNAi pathway silence selfish genetic elements in the soma much as piRNAs do in the germ line. Keywords: Small RNA detection and quantification.

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Project description:The minor spliceosome is evolutionarily conserved in higher eukaryotes, but its biological significance remains poorly understood. Here, by precise CRISPR/Cas9-mediated disruption of the U12 and U6atac snRNAs, we report that a defective minor spliceosome is responsible for spinal muscular atrophy (SMA) associated phenotypes in Drosophila. Using a newly developed bioinformatic approach, we identified a large set of minor spliceosome-sensitive splicing events and demonstrate that three sensitive intron-containing neural genes, Pcyt2, Zmynd10, and Fas3, directly contribute to disease development as evidenced by the ability of their cDNAs to rescue the SMA-associated phenotypes in muscle development, neuromuscular junctions, and locomotion. Interestingly, many splice sites in sensitive introns are recognizable by both minor and major spliceosomes, suggesting a new mechanism of splicing regulation through competition between minor and major spliceosomes. These findings reveal a vital contribution of the minor spliceosome to SMA and to regulated splicing in animals.

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CDK11 regulates pre-mRNA splicing by phosphorylation of SF3B1

Project description:RNA splicing, the process of intron removal from pre-mRNA, is essential for the regulation of gene expression. It is controlled by the spliceosome, a megadalton RNA-protein complex that assembles de novo on each pre-mRNA intron via an ordered assembly of intermediate complexes. Spliceosome activation is a major control step requiring dramatic protein and RNA rearrangements leading to a catalytically active complex. Splicing factor 3B subunit 1 (SF3B1) protein, a subunit of the U2 snRNP, is phosphorylated during spliceosome activation, but the responsible kinase has not been identified. Here we show that cyclin-dependent kinase 11 (CDK11) associates with SF3B1 and phosphorylates threonine residues at its N-terminus during spliceosome activation. The phosphorylation is important for association of SF3B1 with U5 and U6 snRNAs in spliceosome activated Bact complex and it can be blocked by OTS964, a potent and selective inhibitor of CDK11. CDK11 inhibition prevents spliceosomal transition from the precatalytic complex B to the activated complex Bact and leads to widespread intron retention and accumulation of non-functional spliceosomes on pre-mRNAs and chromatin. We characterize OTS964 as a quality chemical biology probe for CDK11 and demonstrate a central role of CDK11 in spliceosome assembly and splicing regulation.

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The histone variant H2A.Z promotes efficient co-transcriptional splicing in S. cerevisiae

Project description:In eukaryotes, a dynamic ribonucleic protein machine known as the spliceosome catalyzes the removal of introns from pre-messenger RNA (pre-mRNA). Recent studies show the process of RNA-synthesis and RNA-processing to be spatio-temporally coordinated, indicating that RNA splicing takes place in the context of chromatin. H2A.Z is a highly conserved histone variant of the canonical histone H2A. In S. cerevisiae, H2A.Z is deposited into chromatin by the SWR1-complex, is found near the 5’ ends of protein-coding genes, and has been implicated in transcription regulation. Here we show that splicing of intron-containing genes in cells lacking H2A.Z is impaired, particularly under suboptimal splicing conditions. Cells lacking H2A.Z are especially dependent on a functional U2 snRNP, as H2A.Z shows extensive genetic interactions with U2 snRNP associated proteins, and RNA-seq reveals introns with non-consensus branch points are particularly sensitive to H2A.Z loss. Consistently, H2A.Z promotes efficient spliceosomal rearrangements involving the U2 snRNP, as H2A.Z loss results in persistent U2 snRNP association and decreased recruitment of downstream snRNPs to nascent RNA. H2A.Z impairs transcription elongation, suggesting that spliceosome rearrangements are tied to H2A.Z’s role in elongation. Depletion of disassembly factor Prp43 suppresses H2A.Z-mediated splice defects, indicating that, in the absence of H2A.Z, stalled spliceosomes are disassembled and unspliced RNAs are released. Together these data demonstrate that H2A.Z is required for efficient pre-mRNA splicing and indicate a role for H2A.Z in coordinating the kinetics of transcription elongation and splicing.

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