Investigating the function of novel cis-regulatory elements in 3'UTRs of mRNAs and their molecular mechanisms in post-transcriptional gene regulation.
Final Report Abstract
Post-transcriptional gene expression is mainly regulated by cis-regulatory RNA elements located in 5ꞌ and 3ꞌ untranslated regions (UTRs). Whereas elements in 5ꞌUTRs predominantly balance translation, motifs in 3ꞌUTRs regulate mRNA processing, transport, decay and translation. Many regulatory motifs are known and correspond to miRNA target sites, cleavage and polyadenylation signals or splice sites, however, there are hundreds of yet unstudied motifs with potential functions. These novel elements presumably function by recruiting trans-acting factors, whose identities are also unknown. The present work aimed at uncovering the underlying mechanisms of novel identified cisregulatory elements and their functions in post-transcriptional gene regulation. These novel motifs are highly conserved across species and located in hundreds of different 3ꞌUTRs. Initial luciferase reporter experiments revealed two similar motifs mediating strong repression in all tested 3ꞌUTRs. The downregulation is comparable to strong miRNA target sites. These two elements were further selected and characterized in terms of their mechanisms of action. To our knowledge, this is the first study where functional elements could be identified and characterized solely based on their conservation. Gene ontology analyses of genes harboring those two elements indicate enrichments for transcription factors. Interestingly, many genes also belong to interleukin signaling pathways and contain strong functional elements. The biological significance in interleukin signaling was confirmed using cell proliferation assays. Lower secreted interleukin levels could be observed, when the interleukin gene contains the wild type motif in comparison to the mutated version. Therefore, misregulation might indicate great importance to human diseases. Characterization of individual steps in post-transcriptional gene regulation indicates that both elements promote mRNA decay. Reduced mRNA levels could already be observed in the nucleus indicating a nuclear decay pathway. This hypothesis is currently tested by analyzing decay rates in the cytoplasm and nucleus. To identify trans-acting factors, pull-down experiments with RNAs containing multiple copies of these motifs have been performed. These experiments have identified hnRNP A2/B1 and hnRNP A1 as RNA-binding proteins. The motif-dependent regulatory potential of hnRNP A2/B1 and A1 was confirmed by knockdown experiments. Surprisingly, double knockdowns of hnRNP A2/B1 and A1 show less regulation by the element than single knockdowns. Analysis of an endogenous transcript containing a motif in its 3ꞌUTR also revealed differentially regulated expression by comparing single and double knockdown cells. Based on these results genomewide studies to identify all 3ꞌUTRs that contain functional elements are in progress.
