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HNRNPA2B1 regulates alternative RNA processing in the nervous system and accumulates in granules in ALS IPSC-derived motor neurons [iCLIP-seq]

ABSTRACT: HnRNPA2B1 encodes an RNA binding protein associated with neurodegenerative disorders. However, its function in the nervous system is unclear. Transcriptome-wide cross-linking and immunoprecipitation in mouse spinal cord discover UAGG motifs enriched within ~2,500 hnRNP A2/B1 binding sites and an unexpected role for hnRNP A2/B1 in alternative polyadenylation. Loss of hnRNP A2/B1 results in alternative splicing, including skipping of an exon in amyotrophic lateral sclerosis (ALS)-associated D-amino acid oxidase (DAO) that reduces D-serine metabolism. Inclusion of the DAO exon is also reduced in transgenic ALS mice models. ALS-associated hnRNP A2/B1 D290V mutant patient fibroblasts and motor neurons differentiated from induced pluripotent stem cells demonstrate gain-of-mutant-dependent splicing differences. Mutant motor neurons also exhibit increased hnRNP A2/B1 localization to cytoplasmic granules during stress, which are abrogated by a small molecule CA43. Our findings and cellular resource identify RNA networks affected in loss of normal and mutated hnRNP A2/B1 with broad relevance to neurodegeneration.

ORGANISM(S): Mus musculus

PROVIDER: GSE86041 | GEO | 2016/10/20

SECONDARY ACCESSION(S): PRJNA341994

REPOSITORIES: GEO

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Project description:Limited knowledge of the downstream targets of hnRNP A2/B1 has, however, precluded a clear understanding of their roles in cancer cell growth. To define the pathways in which this protein acts we have now carried out microarray experiments with total RNA from Colo16 epithelial cells transfected with an shRNA that markedly suppresses hnRNP A2/B1 expression. The microarray data identified 123 genes, among 22283 human gene probe sets, with altered expression levels in hnRNP A2/B1-depleted cells. Ontological analysis showed that many of these downstream targets are involved in regulation of the cell cycle and cell proliferation and that this group of proteins is significantly over-represented amongst the affected proteins. The changes detected in the microarray experiments were confirmed by real-time PCR for a subset of proliferation-related genes. Immunoprecipitation-RT-PCR demonstrated that hnRNP A2/B1 formed complexes with the transcripts of many of the verified downstream genes, suggesting that hnRNP A2/B1 contributes to the regulation of these genes. Experiment Overall Design: Western blotting was performed to confirm the efficient suppression of hnRNP A2/B1 by shRNA, which was endogenously generated from a pSUPER vector system. A vector carrying a target sequence of firefly was used as a control. The specificity of the hnRNP A2/B1 targeting vector has been established in our previous study [Yaowu He, Melissa A. Brown, Joseph A. Rothnagel, Nicholas A. Saunders, and Ross Smith. Roles of heterogeneous nuclear ribonucleoprotein A/B in cell proliferation. Journal of Cell Science. 2005.118(14):3173-83] Experiment Overall Design: For microarray hybridization, the GeneChip Human Genome U133A platform (Affymetrix) used in this study was printed with 22,283 oligonucleotide probe sets representing 18,400 transcripts and variants, including 14,500 known genes. For a probe set, there were typically 8 probe pairs, each containing a perfect match and a mismatch oligonucleotide. Two chips were prepared for each treatment, allowing 4 comparisons between the hnRNP A2-suppressed and the control cells (samples were not paired).

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HNRNPA2B1 regulates alternative RNA processing in the nervous system and accumulates in granules in ALS IPSC-derived motor neurons [iCLIP-seq]

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