Identifying RNA-Protein Interaction Sites Throughout Eukaryotic Transcriptomes

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Doctor of Philosophy (PhD)
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Cell & Molecular Biology
RNA-binding protein
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Gene expression is regulated at both the transcriptional and post-transcriptional levels. While transcription controls only the rate of RNA production, numerous and diverse mechanisms regulate the processing, stability and translation of RNAs at the post-transcriptional level. At the heart of this regulation are RNA-binding proteins (RBPs) and their RNA targets. Thousands of RBPs are encoded in mammalian genomes, each with hundreds to thousands of RNA targets. Therefore, cataloging these interactions represents a significant challenge. Recent advances in high-throughput sequencing technologies have greatly expanded the toolkit that researchers have to probe RNA-protein interactions, but these technologies are still in their infancy and thus new methods and applications are required to move our understanding forward. We developed a novel, high-throughput approach to globally identify regions of RNAs that interact with proteins throughout a transcriptome of interest. We applied this technique to human HeLa cells and provide evidence that our approach captures both known and novel RNA-protein interaction sites. We identified global patterns of RNA-protein interactions, found evidence for co-binding of functionally related genes, and revealed that disease associated single-nucleotide polymorphisms are enriched within protein interaction sites. We also performed detailed analysis of the RNA targets for two specific RBPs; Poly(A)-binding protein cytoplasmic 1 (PABPC1) and Argonaute (AGO). First, we used CLIP-seq to generate a transcriptome-wide map of PABPC1 interaction sites in the mouse transcriptome. This analysis revealed that PABPC1 binds directly to the highly conserved polyadenylation signal sequence and to translation initiation and termination sites. We also showed that PABPC1 binds to A-rich regions in the 5’ untranslated region of a subset of messenger RNAs (mRNAs) and negatively regulates their gene expression. Finally, we applied a recently developed approach to isolate and sequence AGO-bound microRNA precursors (pre-miRNAs). We uncovered widespread trimming and tailing, identified novel intermediates and created an index for pre-miRNA processing efficiency. We discovered that numerous pre-miRNA-like elements are embedded within mRNAs, but do not produce functional small RNAs. In total, these studies provide several advances in our understanding of the global landscape of RNA-protein interactions and serve as a foundation for future mechanistic studies.

Brian D. Gregory
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