Post-Transcriptional Gene Regulation: Roles For Micrornas In Cd8+ T Cells And Discovery Of Novel Cis-Regulatory Elements
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Post-transcriptional events control the amount of protein produced by an mRNA transcript. In mammals, the sequences that regulate post-transcriptional events are predominantly located within a transcript's 3′ untranslated region (UTR), and these cisregulatory elements alter rates of mRNA decay and translation via interactions with trans-factors, including microRNAs and RNA binding proteins. It is challenging to identify the sequences within 3′ UTRs that have regulatory function and to determine the importance of cis-regulatory elements to cellular phenotypes. In this work, I discovered hundreds of functional sequences within 3′ UTRs by creating a fluorescence-based reporter assay that tested in parallel thousands of possible small sequences' regulatory potential. These functional sequences included both elements able to increase and to decrease expression of the reporter gene, demonstrating that 3′ UTRs can have activating and repressive roles. Importantly, sequences discovered from this screen were able to regulate expression of endogenous 3′ UTRs. Smaller sub-motifs are enriched within activating and repressive elements, suggesting that sequences 3-5 nucleotides in length can confer post-transcriptional regulation. The novel cis-regulatory elements discovered in this screen will greatly increase our understanding of how mRNA transcripts and their translation are regulated. Additionally, I examined the role of microRNAs in CD8+ T cells, which allowed me to investigate how cis-regulatory elements can affect the adaptive immune system. CD8+ T cells are responsible for recognizing and killing cells that have been infected with intracellular pathogens, and they require miRNAs, a class of post-transcriptional trans-factors, in order to reach sites of infection and kill cells efficiently. I found that a specific microRNA, miR-150, is highly expressed in CD8+ T cells, and in its absence, CD8+ T cells have widespread misregulation of genes, including a failure to produce genes needed for proliferation and response to infection. I also identified microRNAs that are differentially expressed in adult and neonatal CD8+ T cells, and these microRNAs may underlie the inability of neonatal cells to survive an infection. The targets of two differentially expressed miRNAs, miR-29 and miR-130, are themselves differentially expressed in neonatal CD8+ T cells. These results demonstrate that posttranscriptional events are required for CD8+ T cells to correctly respond to infection.
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Rudd,Brian D.
Soloway,Paul