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Octamer sequences with HEXplorer score amplitude of -10.35, –0.15 or +10.32

ABSTRACT: Correct pre-mRNA processing in higher eukaryotes vastly depends on splice site recognition. Beyond conserved 5’ss and 3’ss motifs, splicing regulatory elements (SREs) play a pivotal role in this recognition process. Here, we present in silico designed sequences with arbitrary a priori prescribed splicing regulatory HEXplorer properties that can be concatenated to arbitrary length without changing their regulatory properties. We performed pulldown analysis to identify RNA-binding proteins, that bind either sequence segments with a HEXplorer score amplitude of +10.32, –0.15 or -10.35.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Gereon Poschmann

LAB HEAD: Gereon Poschmann

PROVIDER: PXD030139 | Pride | 2022-10-13

REPOSITORIES: Pride

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	OE09619.raw	Raw
	OE09620.raw	Raw
	OE09621.raw	Raw
	OE09622.raw	Raw
	OE09623.raw	Raw

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Publications

Modeling splicing outcome by combining 5'ss strength and splicing regulatory elements.

Müller Lisa L Ptok Johannes J Nisar Azlan A Antemann Jennifer J Grothmann Ramona R Hillebrand Frank F Brillen Anna-Lena AL Ritchie Anastasia A Theiss Stephan S Schaal Heiner H

Nucleic acids research 20220801 15

Correct pre-mRNA processing in higher eukaryotes vastly depends on splice site recognition. Beyond conserved 5'ss and 3'ss motifs, splicing regulatory elements (SREs) play a pivotal role in this recognition process. Here, we present in silico designed sequences with arbitrary a priori prescribed splicing regulatory HEXplorer properties that can be concatenated to arbitrary length without changing their regulatory properties. We experimentally validated in silico predictions in a massively parall ...[more]

PMID: 35947702

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RNAseq of 76 samples from Uveal Melanoma tumors

Project description:Hotspot mutations in the spliceosome gene SF3B1 are reported in 20% of uveal melanomas. SF3B1 is involved in 3'-splice site (3'ss) recognition during RNA splicing; however, the molecular mechanisms of its mutation have remained unclear. Here we show, using RNA-Seq analyses of uveal melanoma, that the SF3B1 R625/K666 mutation results in deregulated splicing at a subset of junctions, mostly by the use of alternative 3'ss

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Selected humanization of yeast U1 snRNP leads to global suppression of pre-mRNA splicing and mitochondrial dysfunction in the budding yeast

Project description:The recognition of 5’ splice site (5’ ss) is one of the earliest steps of pre-mRNA splicing. To better understand the mechanism and regulation of 5’ ss recognition, we selectively humanized components of the yeast U1 snRNP to reveal the function of these components in 5’ ss recognition and splicing. We targeted U1C and Luc7, two proteins that interact with and stabilize the yeast U1 (yU1) snRNA and the 5’ ss RNA duplex. We replaced the Zinc-Finger (ZnF) domain of yU1C with its human counterpart, which resulted in cold-sensitive growth phenotype and moderate splicing defects. Next, we added an auxin-inducible degron to yLuc7 protein and found that Luc7-depleted yU1 snRNP resulted in the concomitant loss of PRP40 and Snu71 (two other essential yeast U1 snRNP proteins), and further biochemical analyses suggest a model of how these three proteins interact with each other in the U1 snRNP. The loss of these proteins resulted in a significant growth retardation accompanied by a global suppression of pre-mRNA splicing. The splicing suppression led to mitochondrial dysfunction as revealed by a release of Fe2+ into the growth medium and an induction of mitochondrial reactive oxygen species. Together, these observations indicate that the human U1C ZnF can substitute that of yeast, Luc7 is essential for the incorporation of the Luc7-Prp40-Snu71 trimer into yeast U1 snRNP, and splicing plays a major role in the regulation of mitochondria function in yeast.

2024-01-03 | GSE252348 | GEO

Cancer associated SF3B1 hotspot mutations induce cryptic 3' splice site selection through use of a different branch point

Project description:Recurrent mutations in the spliceosome are observed in several human cancers but their functional and therapeutic significance remain elusive. SF3B1, the most frequently mutated component of the spliceosome in cancer, is involved in the recognition of the branch point sequence (BPS) during selection of the 3’ splice site (ss) in RNA splicing. Here, we report that common and tumor-specific splicing aberrations are induced by SF3B1 mutations and establish aberrant 3’ ss selection as the most frequent splicing defect. Strikingly, mutant SF3B1 utilizes a BPS that differs from that used by wild-type SF3B1 and requires the canonical 3’ ss to enable aberrant splicing during the second step. Approximately 50% of the aberrantly spliced mRNAs are subjected to nonsense-mediated decay resulting in downregulation of gene and protein expression. These findings ascribe functional significance to the consequences of SF3B1 mutations in cancer. 72 samples, including two sets of patient data and cell lines with two additional technical replicates each

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Cancer associated SF3B1 hotspot mutations induce cryptic 3' splice site selection through use of a different branch point

2015-09-08 | GSE72790 | GEO

Illumina mRNA-Seq of control and hnRNP H knockdown in 293T cells

Project description:Pre-mRNA splicing is regulated through combinatorial activity of RNA motifs including splice sites and splicing regulatory elements (SREs). Here, we show that the activity of a major class of mammalian SREs is highly sensitive to the strength of the adjacent 5' splice site (5'ss) sequence, and that this has important functional and evolutionary implications. Activity of G-run SREs was higher for intermediate strength 5'ss by ~4-fold relative to weak 5'ss, and by ~1.3-fold relative to strong 5'ss. The dependence on 5'ss strength was supported both by comparative genomics and by microarray and Illumina mRNA-Seq analyses of splicing changes following RNAi against the splicing factor heterogeneous nuclear ribonucleoprotein (hnRNP) H, which binds G-runs. This dependence implies that the responsiveness of exons to changes in hnRNP H levels is a bivariate function of both SRE abundance and 5'ss strength; this relationship may hold also for other splicing factors. This pattern of activity enables G-runs and hnRNP H to buffer the effects of 5'ss mutations, augmenting both the frequency of 5'ss polymorphism and the evolution of new splicing patterns.

2009-09-13 | GSE16642 | GEO

Illumina mRNA-Seq of control and hnRNP H knockdown in 293T cells

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Affymetrix exon array analysis of control and hnRNP H knockdown in 293T cells

Project description:Splice site strength and the presence of splicing regulatory elements (SREs) play central roles in the control of pre-mRNA splicing1-3 and in exon evolution4. However, the degree to which SRE function is dependent or independent of the strength of nearby splice sites is not well understood. Here, we show that the activity of a major class of mammalian SREs is highly sensitive to the strength of the adjacent 5' splice site (5'ss) sequence, with important functional and evolutionary implications. Using extensive splicing reporter assays, we found that activity of identical 'G-run' intronic splicing enhancers (ISEs)5, 6 was higher by ~4-fold for 5'ss of intermediate strength relative to weak 5'ss, and higher by ~1.3-fold relative to strong 5'ss, based on established 5'ss scoring criteria7. This dependence on 5'ss strength was supported both by comparative genomic analyses and by high-throughput transcriptome sequencing analyses of splicing changes following RNAi against the G-run-binding factor heterogeneous nuclear ribonucleoprotein (hnRNP) H. This pattern and an inverse pattern of 5'ss-dependent activity observed for G-run exonic splicing silencers (ESSs) enables G-runs and hnRNP H to buffer 5'ss mutations and to function as effective evolutionary capacitors8 of splicing change. Human exons flanked by G-runs exhibited increased 5'ss polymorphism and a ~30% increased frequency of evolutionary change between constitutive and alternative splicing, supporting a role in facilitating splicing-level evolution. Evolutionary conservation indicated a substantially greater role for intronic elements generally in splicing of exons with weak and intermediate 5'ss, and supported 5'ss strength-dependent activity for several other intronic motifs, suggesting widespread functional and evolutionary differences between exons of differing 5'ss strength. Keywords: gene expression array-based, exon and protein coding gene analysis

2009-09-13 | GSE12386 | GEO

Novel SF3B1 Deletion Mutations Result in Aberrant RNA Splicing in CLL Patients

Project description:Recurrent mutations in RNA splicing factors SF3B1, U2AF1, and SRSF2 have been reported in hematologic cancers including myelodysplastic syndromes (MDS) and chronic lymphocytic leukemia (CLL). However, SF3B1 is the only splicing associated gene to be found mutated in CLL and has been shown to induce aberrant splicing. To investigate if any other genomic aberration caused similar transcriptome changes, we clustered RNASeq samples based on an alternative 3’ splice site (ss) pattern previously identified in SF3B1-mutant CLL patients. Out of 215 samples, we identified 37 (17%) with alternative 3’ ss usage, the majority of which harbored known SF3B1 hotspot mutations. Interestingly, 3 patient samples carried previously unreported in-frame deletions in SF3B1 around K700, the most frequent mutation hotspot. To study the functional effects of these deletions, we used various minigenes demonstrating that recognition of canonical 3’ ss and alternative branchsite are required for aberrant splicing, as observed for SF3B1 p.K700E. The common mechanism of action of these deletions and substitutions result in similar sensitivity of primary cells towards splicing inhibitor E7107. Altogether, these data demonstrate that novel SF3B1 in-frame deletion events identified in CLL result in aberrant splicing, a common biomarker in spliceosome-mutant cancers.

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mRNA splicing is modulated by intronic microRNAs

Project description:Splicing of transcripts is catalyzed by the spliceosome, a mega-complex consisting of hundreds of proteins and five snRNAs, which employs direct interactions. When U1 snRNA forms high affinity binding, namely more than eight base-pairs, with the 5’SS, the result is usually a suppressing effect on the splicing activity. This likely occurs due to the inefficient unwinding of U1/5’SS base-pairing, or other regulatory obstructions. Here we show in vitro and in patient-derived cell lines that pre-microRNAs can modulate the splicing reaction by interacting with U1 snRNA. This leads to reduced binding affinity to the 5’SS, and hence promotes the inclusion of exons containing 5’SS, despite sequence-based high affinity to U1. Application of the mechanism resulted in correction of the splicing defect in the disease-causing VCAN gene from an individual with Wagner syndrome. This pre-miRNA/U1 interaction can regulate the expression of alternatively spliced exons, thus extending the scope of mechanisms regulating splicing.

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