Eric Nguyen

RNA-binding proteins play a number of important roles throughout the cell. In order to more closely investigate their activity, we have adapted high-throughput techniques to characterize their activity across the transcriptome. We have previously identified heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 as a potential adaptor protein for interactions between the chromatin silencing complex PRC2 and the RNA HOTAIR. We used enhanced cross- linking immunoprecipitation (eCLIP) to map the complete set of direct interactions between hnRNP A2/B1 and RNA in breast cancer cells. Surprisingly, a strong A2/B1 binding site occurs in the third intron of HOTAIR, which interrupts a known RNA- RNA interaction hotspot and is retained at a higher frequency than other HOTAIR introns. In vitro eCLIP experiments suggest that A2/B1 may redistribute to exonic binding sites once this intron is spliced. A2/B1 associates with multiple lncRNAs at regions that may contribute to regulation. Finally, we performed cellular fractionation to characterize the pattern of RNA association of A2/B1 in chromatin, nucleoplasm, and cytoplasm. We also examined the potential relevance of hnRNP A2/B1 in myogenesis. As A2/B1 has been associated with a number of muscle diseases, we performed eCLIP on A2/B1 in both undifferentiated mouse myoblasts and differentiated myotubes. We found that A2/B1 binds the 3′ UTR of transcripts in differentiated cells, and that these transcripts tended to be protein-coding. We also performed eCLIP on another protein, TDP-43, that has been shown to directly interact with A2/B1 and be dysregulated in many of the same diseases. This experiment identified a number of exonic binding sites in myogenesis-associated transcripts, indicative of a role for TDP-43 in the nuclear export of long RNAs during myogenesis. Comparison of A2/B1 and TDP-43-bound transcripts shows some overlap, suggesting that they may act cooperatively in RNA regulation during myogenesis. Finally, we developed a novel method to investigate heterochromatin- associated RNA called hmRIP-seq, which was designed to differentiate between RNA-enzyme interactions leading to heterochromatin formation, and those that do not. This method identified a potential heterochromatin-interacting noncoding RNA, MTRNR2L12, that may direct silencing towards repetitive elements with similar sequence.

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