Global analysis of T cell post-transcriptional regulatory elements

University Of California, San Francisco, San Francisco CA

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Abstract

Project Summary: Global analysis of T cell post-transcriptional regulatory elements T cells are key cellular players of the immune system, and their activity is regulated by widespread modulation of gene expression. Genes are regulated at both the transcriptional and post-transcriptional level, but the post-transcriptional regulatory landscape is comparatively understudied. We propose to perform a global profiling of the post-transcriptional regulatory elements operating in T cells. To achieve this, we will use novel biochemical techniques to clone all protein-bound pieces of messenger RNAs in human T cells grown in vitro into libraries suitable for high throughput sequencing. We will compare this data set to comparable data from mouse T cells that we have already generated. For both of these data sets, we will proceed to functionally annotate the tens of thousands of protein binding sites in messenger RNAs for their effect on transcript stability and translational efficiency. To achieve this, we will clone libraries of these putative cis-regulatory elements into the 3? untranslated region of a heterologous reporter construct, and compare the relative stability and translation of reporter transcripts to measure the functional impact of each of protein binding sequences. These experiments will generate a transcriptome-wide map of active post-transcriptional cis-regulatory sequences in mouse and human T cells. In addition, we will mine these datasets with bioinformatic analyses to identify novel regulatory motifs associated with gene regulation in activated T cells or in specific T cell subsets, and to determine the evolutionary conservation of post-transcriptional regulatory sequences and mechanisms in mouse and human T cells. We expect this analysis to reveal generalizable information about the global mechanics of post-transcriptional gene regulation.