A Gene-Centered Method For Mapping 3’UTR-RBP Interactions: A Dissertation

Interactions between 3´ untranslated regions (UTRs) and RNA-binding proteins (RBPs) play critical roles in post-transcriptional gene regulation. Metazoan genomes encode hundreds of RBPs and thousands of 3’ UTRs have been experimentally identified, yet the spectrum of interactions between 3´UTRs and RBPs remains largely unknown. Several methods are available to map these interactions, including protein-centered methods such as RBP immunoprecipitation (RIP) and cross-link immunoprecipitation (CLIP), yeast three-hybrid assays and RNAcompete. However, there is a paucity of RNA-centered approaches for assaying an RNA element of interest against multiple RBPs in a parallel, scalable manner. Here, I present a strategy for delineating protein-RNA interaction networks using a gene centered approach. This approach includes annotating RBPs and identifying physical interactions between an RNA of interest and these RBPs using the Protein-RNA Interaction Mapping Assay (PRIMA). Few RBPs have been experimentally determined in most eukaryotic organisms. Therefore I show that existing RBP annotations can be supplemented using computational predictions of RNA binding domains (RBD) from protein sequences. A single RNA of interest can be tested using PRIMA against a library of RBPs constructed from these annotations. PRIMA utilizes the green fluorescent protein (GFP) in yeast as a reporter. PRIMA is based on reconstitution of the interaction between the 5´ and 3´ ends of an mRNA, which increases mRNA stability and enhances translation. PRIMA recapitulates known and uncovers new interactions involving RBPs from human, Caenorhabditis elegans and bacteriophage with short RNA fragments and full-length 3´UTRs. The development of RBP prey libraries will enable the testing of 3´UTRs against the hundreds of RBPs, which is essential to gain broad insights into post-transcriptional gene regulation at a systems level

A compendium of Caenorhabditis elegans RNA binding proteins predicts extensive regulation at multiple levels

Gene expression is regulated at multiple levels, including transcription and translation, as well as mRNA and protein stability. Although systems-level functions of transcription factors and microRNAs are rapidly being characterized, few studies have focused on the posttranscriptional gene regulation by RNA binding proteins (RBPs). RBPs are important to many aspects of gene regulation. Thus, it is essential to know which genes encode RBPs, which RBPs regulate which gene(s), and how RBP genes are themselves regulated. Here we provide a comprehensive compendium of RBPs from the nematode Caenorhabditis elegans (wRBP1.0). We predict that as many as 887 (4.4%) of C. elegans genes may encode RBPs ~250 of which likely function in a gene-specific manner. In addition, we find that RBPs, and most notably gene-specific RBPs, are themselves enriched for binding and modification by regulatory proteins, indicating the potential for extensive regulation of RBPs at many different levels. wRBP1.0 will provide a significant contribution toward the comprehensive delineation of posttranscriptional regulatory networks and will provide a resource for further studies regulation by RBPs

Transcription factor binding to Caenorhabditis elegans first introns reveals lack of redundancy with gene promoters

Gene expression is controlled through the binding of transcription factors (TFs) to regulatory genomic regions. First introns are longer than other introns in multiple eukaryotic species and are under selective constraint. Here we explore the importance of first introns in TF binding in the nematode Caenorhabditis elegans by combining computational predictions and experimentally derived TF-DNA interaction data. We found that first introns of C. elegans genes, particularly those for families enriched in long first introns, are more conserved in length, have more conserved predicted TF interactions and are bound by more TFs than other introns. We detected a significant positive correlation between first intron size and the number of TF interactions obtained from chromatin immunoprecipitation assays or determined by yeast one-hybrid assays. TFs that bind first introns are largely different from those binding promoters, suggesting that the different interactions are complementary rather than redundant. By combining first intron and promoter interactions, we found that genes that share a large fraction of TF interactions are more likely to be co-expressed than when only TF interactions with promoters are considered. Altogether, our data suggest that C. elegans gene regulation may be additive through the combined effects of multiple regulatory regions

PRIMA: a gene-centered, RNA-to-protein method for mapping RNA-protein interactions

Interactions between RNA binding proteins (RBPs) and mRNAs are critical to post-transcriptional gene regulation. Eukaryotic genomes encode thousands of mRNAs and hundreds of RBPs. However, in contrast to interactions between transcription factors (TFs) and DNA, the interactome between RBPs and RNA has been explored for only a small number of proteins and RNAs. This is largely because the focus has been on using \u27protein-centered\u27 (RBP-to-RNA) interaction mapping methods that identify the RNAs with which an individual RBP interacts. While powerful, these methods cannot as of yet be applied to the entire RBPome. Moreover, it may be desirable for a researcher to identify the repertoire of RBPs that can interact with an mRNA of interest-in a \u27gene-centered\u27 manner-yet few such techniques are available. Here, we present Protein-RNA Interaction Mapping Assay (PRIMA) with which an RNA \u27bait\u27 can be tested versus multiple RBP \u27preys\u27 in a single experiment. PRIMA is a translation-based assay that examines interactions in the yeast cytoplasm, the cellular location of mRNA translation. We show that PRIMA can be used with small RNA elements, as well as with full-length Caenorhabditis elegans 3\u27 UTRs. PRIMA faithfully recapitulated numerous well-characterized RNA-RBP interactions and also identified novel interactions, some of which were confirmed in vivo. We envision that PRIMA will provide a complementary tool to expand the depth and scale with which the RNA-RBP interactome can be explored