Functional and structural mapping of 5' UTR cis-regulatory elements that control translation

Translation can be critically and pervasively regulated in a transcript-specific manner to modulate protein synthesis. However, it is poorly understood which and how cis-regulatory features of the messenger ribonucleic acid (mRNA) encode variable transcript-specific translation rates to impact spatiotemporal gene expression patterns. Here, I address the role of 5' untranslated (5' UTR) in regulating mRNA translation in vertebrate species. In the first part of the dissertation, I explore the phenomenon of extreme non-coding sequence conservation in vertebrate genomes at the level of RNA function in 5' UTRs. Extreme conservation is a fascinating mystery in comparative genomics in which sequence conservation, at levels often greater than coding regions with invariant polypeptide sequences, stretches on for hundreds of nucleotides in the non-coding regions of the genome. While extreme conservation has been extensively studied for its role in transcriptional regulation, its RNA-level function in translational regulation remains largely unknown. This work reveals the role of extremely conserved 5' UTRs in translational regulation of genes linked to the emergence of essential developmental features in vertebrate species. Extremely conserved 5' UTRs are found to contain cis-elements that promote cell-type specific non-canonical translation initiation. As these elements function as RNA molecules, an understanding of their structures is essential. To this end, I develop in-cell mutate-and-map (icM2), a methodology that maps RNA structure using high-throughput mutational analysis inside cells. icM2 maps the ensemble of multiple conformations in an extremely conserved 5' UTR which is found to be important for its translational regulatory function. I find that active RNA structural remodeling inside cells by RNA helicase activity maintains the relative balance of the conformations. Furthermore, cellular structural remodeling occurs frequently in the most conserved regions of the 5' UTRs. I propose a structural explanation for extreme conservation at the level of RNA and highlight the importance of comparative genomics and of RNA structure in understanding 5' UTR function and evolution. In the second part of the dissertation, I explore the genome-wide role of mammalian ribosome expansion segments (ES) in interacting with 5' UTRs to regulate mRNA translation. ESs are eukaryote-specific insertions to the ribosomal ribonucleic acid (rRNA). They are positioned mostly at the exterior surface of the ribosome structure, extending from the core of the ribosome like flexible tentacles. It has been recently shown that the mammalian ES9S in 18S rRNA directly interacts with a Hox gene 5' UTR element to promote its translation. Direct rRNA-mRNA interaction is not a widely recognized paradigm for translation initiation in eukaryotes. However, addressing the potential genome-wide significance of such a mechanism mediated by ribosome ESs and mRNA 5' UTRs is challenging. Since rRNAs are transcribed from tandem repeats of ribosomal DNA units that can range up to hundreds of thousands of copies, it has not been possible to directly manipulate ESs in most species. To solve this problem, I develop VELCRO-IP RNA-seq: variable expansion segment-ligand chimeric ribosome immunoprecipitation RNA sequencing. VELCRO-IP RNA-seq interrogates the interaction of a ribosome ES with mRNA elements genome-wide by combining yeast genetics, in vitro biochemical pull-down and high-throughput sequencing. By applying VELCRO-IP RNA-seq on mammalian ES9S, hundreds of mRNA regions that interact with the ribosome via specific interaction with the ES are identified. Furthermore, the 5' UTR targets of ES9S are found to promote non-canonical translation. A number of short k-mers that have Watson-Crick complementary to ES9S subsequences are overrepresented in its targets, suggesting potential importance of canonical base-pairing. These results provide evidence for the usage of direct mRNA-rRNA interaction as a mechanism of translation initiation on a wider scale than previously imagined for vertebrate species. Finally, in the last chapter, I discuss how my findings can shape future investigations into the remaining unanswered questions on 5' UTR regulation of translation. Altogether, this dissertation takes us a step closer to the ultimate goal of deciphering the code for translational regulation

Keywords: Genetics.
Advisor: Barna Maria (Professor of developmental biology)
Bassik Michael
Das Rhiju
Steinmetz Lars