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Mediator Complex Regulates Alternative mRNA Processing via the Med23 Subunit

ABSTRACT: Mediator complex is an integrative hub for transcriptional regulation. Here we show that Mediator regulates alternative mRNA processing via its Med23 subunit. Combining tandem affinity purification and mass spectrometry, we identified a number of mRNA processing factors that bind to a soluble recombinant Mediator subunit MED23 but not to several other Mediator components. One of these factors, hnRNP L, specifically interacts with MED23 in vitro and in vivo. Consistently, Mediator partially colocalizes with hnRNP L and the splicing machinery in the cell. Functionally Med23 regulates a subset of hnRNP L-targeted alternative splicing (AS) and alternative cleavage and polyadenylation (APA) events as shown by minigene reporters and exon array analysis. ChIP-seq analysis revealed that Med23 can regulate hnRNP L occupancy at their co-regulated genes. Taken together, these results demonstrate a crosstalk between Mediator and the splicing machinery, suggesting a novel mechanism for coupling mRNA processing to transcription.

ORGANISM(S): Homo sapiens

PROVIDER: GSE33771 | GEO | 2012/02/24

SECONDARY ACCESSION(S): PRJNA148383

REPOSITORIES: GEO

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Project description:We developed a high-throughput mutagenesis screen to comprehensively identify the cis-regulatory elements that control a target splicing event from the MST1R gene that codes for the RON receptor tyrosine kinase. Skipping of alternative exon 11 results in a constitutively active isoform that promotes epithelial to mesenchymal transition and thereby contributes to the invasive phenotype of tumors. First, we created a library of mutated minigenes via mutagenic PCR. Importantly, the reverse primer introduced a random barcode sequence, which labels the associated mutations. Next, the plasmid library was transfected as a pool and depending on the mutations, the transcripts exhibit changes in alternative splicing. The minigene library and the splicing outcome were analyzed by next-generation sequencing and subsequent integration of the datasets resulted in a map of splicing regulatory sites. The RNA binding protein hnRNP H was identified as an important regulator of RON exon 11 splicing. Thus, we performed RNA-seq experiments from minigene library transfected MCF7 cells under control and hnRNP H knockdown conditions in order to quantify the transcript isoforms originating from the minigene library. For preparation of high-throughput RNA sequencing (RNA-seq) libraries, MCF7 cells were first treated with single small interfering RNA (siRNA) against HNRNPH (5′-GGAGCUGGCUUUGAGAGGA[dT][dT] -3′, PMID: 21512137, Sigma-Aldrich) or non-targeting control siRNA (5′-UGGUUUACAUGUCGACUAA[dT][dT]-3′, Sigma-Aldrich) at a final concentration of 20 nM. Next, cells were transfected with the minigene library the day after. A day later, RNA was extracted and subsequently enriched for mRNA by performing polyA selection of 20 µg of total RNA using Dynabeads® Oligo (dT)25 beads (Invitrogen) according to the manufacturer’s protocol. Reverse transcription was carried out using 500 ng of enriched mRNA under the abovementioned conditions. To prevent the formation of chimeric amplicons, the libraries were amplified using emulsion PCR (PMID: 16791213), with Phusion DNA Polymerase (NEB) and cDNA derived from polyA-selected RNA. To amplify fragments for RNA-seq, the following primers containing Illumina sequencing adaptors were used: 5′-CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTNNNNNNNNNNGTTCCACTGAAGCCTGAG-3′ (forward primer) and 5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNNNATAGAATAGGGCCCTCTAGA-3′ (reverse primer). PCR products were purified using Agencourt AMPure XP beads (Beckman Coulter). Purified products were first analysed with the TapeStation 2200 capillary gel electrophoresis instrument (Agilent) and then fluorimetrically quantified using a Qubit fluorimeter (Thermo Scientific). Sequencing was carried out on the Illumina MiSeq platform using paired-end reads of 300 nt length and a 10% PhiX spike-in to increase sequence complexity.

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Mediator Complex Regulates Alternative mRNA Processing via the Med23 Subunit

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