Stress-responsive maturation of Clk1/4 pre-mRNAs promotes phosphorylation of SR splicing factor

Ninomiya, K.; Kataoka, Naoyuki; Hagiwara, Masatoshi

doi:10.1083/jcb.201107093

Cited by 135 publications

(181 citation statements)

References 52 publications

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“…It will be interesting to examine whether pools of completed but unspliced transcripts can be differentially spliced depending on cellular conditions. It has been shown that a pool of partially spliced Clk1/4 mRNA is stored for later completion in response to stress, but this RNA does not appear to be at the Clk1/4 locus (Ninomiya et al 2011).…”

Section: Discussionmentioning

confidence: 99%

Splicing kinetics and transcript release from the chromatin compartment limit the rate of Lipid A-induced gene expression

Pandya-Jones

Bhatt

Lin

et al. 2013

RNA

102

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The expression of eukaryotic mRNAs is achieved though an intricate series of molecular processes that provide many steps for regulating the production of a final gene product. However, the relationships between individual steps in mRNA biosynthesis and the rates at which they occur are poorly understood. By applying RNA-seq to chromatin-associated and soluble nucleoplasmic fractions of RNA from Lipid A-stimulated macrophages, we examined the timing of exon ligation and transcript release from chromatin relative to the induction of transcription. We find that for a subset of genes in the Lipid A response, the ligation of certain exon pairs is delayed relative to the synthesis of the complete transcript. In contrast, 3′ end cleavage and polyadenylation occur rapidly once transcription extends through the cleavage site. Our data indicate that these transcripts with delayed splicing are not released from the chromatin fraction until all the introns have been excised. These unusual kinetics result in a chromatin-associated pool of completely transcribed and 3 ′ -processed transcripts that are not yet fully spliced. We also find that long introns containing repressed exons that will be excluded from the final mRNA are excised particularly slowly relative to other introns in a transcript. These results indicate that the kinetics of splicing and transcript release contribute to the timing of expression for multiple genes of the inflammatory response.

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Section: Discussionmentioning

confidence: 99%

Splicing kinetics and transcript release from the chromatin compartment limit the rate of Lipid A-induced gene expression

Pandya-Jones

Bhatt

Lin

et al. 2013

RNA

102

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“…Transcripts encoding the SR protein kinase Clk1 were previously shown to accumulate in the nucleus, fully spliced except for two introns; these introns were rapidly spliced to produce coding mRNA in response to heat or osmotic shock as well as upon inhibition of Clk kinase activity by a smallmolecule inhibitor (Ninomiya et al 2011). Clk kinase activity thus appears to promote the detention of these introns by inhibiting their splicing.…”

Section: Properties Of Dismentioning

confidence: 99%

“…adenylated pre-mRNAs encoding the SR protein kinase Clk1 contain two unspliced introns flanking a cassette exon. These intron-containing transcripts are retained in the nucleus until the cells experience heat or osmotic shock or treatment with a chemical inhibitor of the Clk kinases, at which point splicing occurs, allowing production of more protein (Ninomiya et al 2011). Intriguingly, Clk kinases are also implicated in the control of splicing of retained introns in platelets.…”

mentioning

confidence: 99%

Detained introns are a novel, widespread class of post-transcriptionally spliced introns

2015

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Deep sequencing of embryonic stem cell RNA revealed many specific internal introns that are significantly more abundant than the other introns within polyadenylated transcripts; we classified these as ''detained'' introns (DIs). We identified thousands of DIs, many of which are evolutionarily conserved, in human and mouse cell lines as well as the adult mouse liver. DIs can have half-lives of over an hour yet remain in the nucleus and are not subject to nonsense-mediated decay (NMD). Drug inhibition of Clk, a stress-responsive kinase, triggered rapid splicing changes for a specific subset of DIs; half showed increased splicing, and half showed increased intron detention, altering transcript pools of >300 genes. Srsf4, which undergoes a dramatic phosphorylation shift in response to Clk kinase inhibition, regulates the splicing of some DIs, particularly in genes encoding RNA processing and splicing factors. The splicing of some DIs-including those in Mdm4, a negative regulator of p53-was also altered following DNA damage. After 4 h of Clk inhibition, the expression of >400 genes changed significantly, and almost one-third of these are p53 transcriptional targets. These data suggest a widespread mechanism by which the rate of splicing of DIs contributes to the level of gene expression.

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“…The skipped exon 4 and intron retention variants are either both inactive or rapidly cleared by nonsense-mediated decay (63,69). The loss of CLK1 activity may disrupt a feedback loop because its kinase activity is critical for the phosphorylation of multiple SR proteins that regulate splicing (63,70,71). The inclusion of CASP3 exon 2 with EWS-FLI1 reduction places an internal ribosome entry site into the 5′-UTR of the mRNA (72).…”

Section: Yk-4-279 Alters Splicing Rather Than Direct Transcriptionalmentioning

confidence: 99%

“…One prior report identified EWS-FLI1 increasing TERT activity independently of DNA binding; however, no isoform analysis was performed (56). These four genes were recognized as putative contributors to oncogenesis based on the published literature (57)(58)(59)(60)(61)(62)(63)(64)(65). None of these genes were alternatively spliced by WT EWS reduction.…”

Section: Yk-4-279 Alters Splicing Rather Than Direct Transcriptionalmentioning

confidence: 99%

Oncogenic fusion protein EWS-FLI1 is a network hub that regulates alternative splicing

Selvanathan

Graham

Erkizan

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

118

135

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The synthesis and processing of mRNA, from transcription to translation initiation, often requires splicing of intragenic material. The final mRNA composition varies based on proteins that modulate splice site selection. EWS-FLI1 is an Ewing sarcoma (ES) oncoprotein with an interactome that we demonstrate to have multiple partners in spliceosomal complexes. We evaluate the effect of EWS-FLI1 on posttranscriptional gene regulation using both exon array and RNAseq. Genes that potentially regulate oncogenesis, including CLK1, CASP3, PPFIBP1, and TERT, validate as alternatively spliced by EWS-FLI1. In a CLIP-seq experiment, we find that EWS-FLI1 RNA-binding motifs most frequently occur adjacent to intron-exon boundaries. EWS-FLI1 also alters splicing by directly binding to known splicing factors including DDX5, hnRNP K, and PRPF6. Reduction of EWS-FLI1 produces an isoform of γ-TERT that has increased telomerase activity compared with wild-type (WT) TERT. The small molecule YK-4-279 is an inhibitor of EWS-FLI1 oncogenic function that disrupts specific protein interactions, including helicases DDX5 and RNA helicase A (RHA) that alters RNA-splicing ratios. As such, YK-4-279 validates the splicing mechanism of EWS-FLI1, showing alternatively spliced gene patterns that significantly overlap with EWS-FLI1 reduction and WT human mesenchymal stem cells (hMSC). Exon array analysis of 75 ES patient samples shows similar isoform expression patterns to cell line models expressing EWS-FLI1, supporting the clinical relevance of our findings. These experiments establish systemic alternative splicing as an oncogenic process modulated by EWS-FLI1. EWS-FLI1 modulation of mRNA splicing may provide insight into the contribution of splicing toward oncogenesis, and, reciprocally, EWS-FLI1 interactions with splicing proteins may inform the splicing code.T he alternative splicing of mRNA expands the diversity of the human proteome through evolution and ontogeny (1, 2). Spliceosomal network interactions, including proteins that recognize splice enhancer and silencer regions, are critical for the regulation of alternative splicing leading to protein isoforms with disparate functionality (3). Alternative splicing provides both a method by which to categorize subsets of cancers and an avenue for more effective targeted treatments (4). However, the spliceosomal protein interaction networks that are specific to cancer have not been systematically defined; alternative splicing can also change protein-protein interactions within networks (5). A systems biology approach can be used to study the relationship between splicing and oncogenesis by using tumor models with chromosomal translocations whose expressed fusion proteins have putative roles in splicing (6, 7).Fusion proteins produced by chromosomal translocations in sarcomas often contain the amino-terminal portion of EWS and are classified as transcription factors due to the presence of a canonical carboxyl-terminal DNA-binding domain (6). EWS-FLI1 is a well-established ES oncoprotein and is re...

show abstract

Stress-responsive maturation of Clk1/4 pre-mRNAs promotes phosphorylation of SR splicing factor

Abstract: A nuclear pool of partially spliced Clk1/4 pre-mRNAs matures in response to stress and induces SR protein phosphorylation and activation.

Cited by 135 publications

References 52 publications

Splicing kinetics and transcript release from the chromatin compartment limit the rate of Lipid A-induced gene expression

Splicing kinetics and transcript release from the chromatin compartment limit the rate of Lipid A-induced gene expression

Detained introns are a novel, widespread class of post-transcriptionally spliced introns

Oncogenic fusion protein EWS-FLI1 is a network hub that regulates alternative splicing

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