Expression profiling by high throughput sequencing
Summary
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
Overall design
RAW 264.7 macrophage mRNA profiles of SCR (control) and hnRNP or SR shRNA knockown cells
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