RNA-Protein interactions at AU-rich elements

The expression of many proto-oncogenes, nuclear transcription factors and cytokines is regulated post-transcriptionally by specific interactions between distinct cis-elements within the mRNA and trans-acting factors that bind to these elements and thereby either promote or inhibit the rapid decay of the mRNA. This kind of regulation enables the cell to rapidly respond to changes in the cellular environment. These elements are usually located within the 3’UTR of the mRNA, rich in adenylate and uridylate and called AU-rich elements or AREs. To date, many of these trans-acting factors have been determined and among the best studied are the stabilizing HuR, the rather destabilizing AUF1 and the destabilizing KSRP and TTP. Previous studies have suggested that these ARE-BPs as well as yet unidentified factors might have overlapping target specificities and bind either in a concurrent, concomitant or maybe an even cooperative fashion to their targets to regulate mRNA half-life. However, the exact manner of how these factors might interact and how these interactions might be influenced, is largely unknown. Thus, my first project aimed to study the interactions between AUF1, KSRP and HuR in the context of the 3’UTRs of RANKL and Bcl-6 that were recently identified as AUF1 targets with a short half-life. By combining ARE-BP immunoprecipitations with qRT-PCR analysis of co-precipitated reporter RNAs, we were able to gain interesting insight into the RNA-protein interactions at AU-rich elements. However, this approach is very laborious and can study RNA-protein interactions only within a limited context. Yet, the pools of mRNA targets as well as binding factors are very multifaceted and likely depend on the cell type and condition as well as the cellular environment. Thus, a much broader approach is necessary to reveal the entire network of RNA-protein and protein-protein interactions at AU-rich elements. A promising technique is the combination of ARE-BP immunoprecipitation and high-throughput sequencing to analyze co-precipitated RNA. This approach allows for the extraction of a lot of important data from a single experiment as for example the virtually complete pool of target mRNAs under the studied conditions, the binding sites within these mRNAs as well as the frequency with which these sequences were detected. Based on these data, it would be possible to make suggestions about the binding specificities as well as potential, yet un-identified targets. If accumulating data sets for different cellular or environmental conditions, we might gain insight into the regulation of these RNA-protein interactions. In a similar way, the comparison of data sets for different ARE-binding proteins would give us further insight into the protein-protein interactions at and around AU-rich elements. Therefore, my second project focused on the development of a novel protocol that allows for the rapid, reliable and reproducible generation of high-throughput sequencing samples based on co-immunoprecipitated RNA fragments.