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Collaborative interactions of heterogenous ribonucleoproteins contribute to transcriptional regulation of sterol metabolism

ABSTRACT: Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a group of functionally versatile proteins that play critical roles in the biogenesis, cellular localization and transport of RNA. Although several lines of evidence link hnRNP abnormalities to neurodegenerative diseases and cancer their contribution to metabolic control remains unexplored. Here, we outline a role for hnRNPs in regulatory circuits controlling sterol homoeostasis. Specifically, we find that tissue-selective loss of the conserved hnRNP RALY enriches for metabolic pathways arguing that hnRNPs can discriminately influence regulated gene expression programs. Liver-specific deletion of RALY alters hepatic lipid content and serum cholesterol level. In vivo interrogation of chromatin architecture and genome-wide RALY binding pattern reveal insights into its cooperative interactions and mode of action in regulating cholesterogenesis. Interestingly, we find that RALY binds the promoter region of the master metabolic regulator Srebp2 and show that it directly interacts with coactivator NFY to influence cholesterogenic gene expression. Our work offers new insights into mechanisms orchestrating selective promoter activation in metabolic control and a model by which hnRNPs can impact metabolic health and disease states.

ORGANISM(S): Mus musculus

PROVIDER: GSE133166 | GEO | 2020/01/07

REPOSITORIES: GEO

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Project description:Purpose: The goal of this study is to measure how steady state RNA and alternatively spliced transcript abundance levels are impacted by loss of specific RNA-binding proteins as part of macrophage transcriptional activation upon pathogen sensing. Methods: SRSF 1, 2, 6, 7, 9, and hnRNP C, F, K, U were knockdown using shRNA in RAW 264.7 macrophages. Macrophages were collected as uninfected (resting) cells and infected with Salmonella Typhimuirum for 4HR. mRNA profiles of uninfected (UN) and Salmonella infected (SAL) hnRNP C, F, K, U and SRSF 1, 2, 6, 7, 9 were generated by deep sequencing, in triplicate, using Illumina. The sequence reads that passed quality filters were analyzed at the gene expression and transcript expression level with CLC Genomics Workbench. qRT–PCR validation was performed using SYBR Green assays. Results: We identified thousands of transcripts whose abundance is increased or decreased by SR/hnRNP knockdown in macrophages. We observed that different SR/hnRNPs control the ex-pression of distinct gene regulons in uninfected and Salmonella-infected macrophages, with sev-eral key innate immune genes (Nos2, Mx1, Il1a) relying heavily on multiple SR/hnRNPs to main-tain proper induction and/or repression. Knockdown of SR/hnRNPs promoted differential isoform usage (DIU) for a number of key immune sensors and signaling molecules and many of these splicing changes were distinct in uninfected and Salmonella-infected macrophages. After observ-ing a surprising degree of similarity between the differentially expressed genes (DEGs) and DIUs in hnRNP K and U knockdown macrophages, we found that these cells are better able to restrict vesicular stomatitis virus repli-cation than control cells, supporting a role for these hnRNPs in controlling infection outcomes in macrophages ex vivo. Based on these findings, we conclude that many innate immune genes have evolved to rely on one or more splicing regulatory factors to ensure the proper timing and magnitude of their induction, bolstering a model wherein pre-mRNA splicing is a critical regulatory node in the innate immune response. Conclusions: SR and hnRNP proteins have profound impacts on the abundance of innate immune transcripts and seem to contribute to macrophage gene expression via distinct mechanisms