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U2AF1 plays a critical role in global co-transcriptional splicing (Fraction chrRNA-seq)

ABSTRACT: U2AF1 plays a critical role in global co-transcriptional splicing (Fraction chrRNA-seq)

PROVIDER: PRJNA733172 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR14668213_1.fastq.gz	Fastqsanger.gz
	SRR14668213_2.fastq.gz	Fastqsanger.gz
	SRR14668214_1.fastq.gz	Fastqsanger.gz
	SRR14668214_2.fastq.gz	Fastqsanger.gz
	SRR14668215_1.fastq.gz	Fastqsanger.gz

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U2AF1 plays a critical role in global co-transcriptional splicing (Fraction chrRNA-seq)

Project description:In order to measure the impact of U2AF1 on co-transcriptional splicing, we treated HeLa cells with control siRNA or U2AF1 siRNA. To measure co-transcriptional splicing, we fractionated HeLa cells into cytosolic (Cyt), nucleoplasm (NP), and chromatin/particle (Chr) fractions. We purified total RNA from Chr compartment, spiked in with in vitro transcribed EGFP and FireFly luciferase RNA for data normalization, depleted ribosomal RNAs, and constructed two independent libraries for RNA-seq.

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The splicing factor U2AF1 contributes to cancer progression through a non-canonical role in translation regulation

Project description:Somatic mutations in the spliceosome have emerged in recent years as oncogenes in human cancer. These mutations are in the factors involved in splice site selection, including a missense mutation (Ser34Phe) in a conserved nucleic acid binding domain of the spicing factor U2AF1. This protein plays a critical role in recognition of the 3'-splice site and assembly of the pre-spliceosomal complex. However, the role that this mutation plays in oncogenesis is still unknown. Here, we have uncovered a non-canonical function of U2AF1. PAR-CLIP and RIP data show that U2AF1 directly binds mature mRNA in the cytoplasm and that binding on or near the start codon results in translational repression. This splicing-independent translational regulatory role of U2AF1 is altered by the S34F mutation, leading to elevated translation of hundreds of mRNA, as revealed by polysome profiling.

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Project description:To define the function of Rpb9 on co-transcriptional splicing, we used auxin-inducible Rpb9 degron system in mouse embryonic stem cells (mESCs) with auxin treatment for 3 hours to sufficiently deplete Rpb9. To measure co-transcriptional splicing, we fractionated mESCs cells (spiked in with Drosophila S2 cells for data normalization) into cytosolic (Cyt), nucleoplasm (NP), and chromatin/particle (Chr) fractions. We purified total RNA from Chr compartment, depleted ribosomal RNAs, and constructed two independent libraries for RNA-seq, which showed excellent reproducibility.

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U2AF1 mutations alter splice site recognition in hematological malignancies

Project description:Whole-exome sequencing studies have identified common mutations affecting genes encoding components of the RNA splicing machinery in hematological malignancies. Here, we sought to determine how mutations affecting the 3' splice site recognition factor U2AF1 altered its normal role in RNA splicing. We find that U2AF1 mutations influence the similarity of splicing programs in leukemias, but do not give rise to widespread splicing failure. U2AF1 mutations cause differential splicing of hundreds of genes, affecting biological pathways implicated in myeloid disease such as DNA methylation (DNMT3B), X chromosome inactivation (H2AFY), the DNA damage response (ATR, FANCA), and apoptosis (CASP8). We show that U2AF1 mutations alter the preferred 3' splice site motif in vivo, in cell culture, and in vitro. Mutations affecting the first and second zinc fingers give rise to different alterations in splice site preference and largely distinct downstream splicing programs. These allele-specific effects are consistent with a computationally predicted model of U2AF1 in complex with RNA. Our findings suggest that U2AF1 mutations contribute to pathogenesis by causing quantitative changes in splicing that affect diverse cellular pathways, and give insight into the normal function of U2AF1’s zinc finger domains. mRNA profiles of K562 cells expressing U2AF1 WT, mutants and knockdown of U2AF1 generated by deep sequencing.

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2017-04-25 | GSE94153 | GEO

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