Date of Award

August 2019

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Biological Sciences

First Advisor

Madhusudan Dey

Abstract

Protein synthesis is a fundamental life process. Protein synthesis regulates cellular metabolism, cellular growth, the cell cycle, and cell morphogenesis. Technical advances in molecular biology, advanced high-throughput sequencing technologies and recent developments in crystallographic methodologies have aided in better understanding of the process of protein synthesis. Current knowledge of protein synthesis provides us with an overview of the initiation, elongation, and termination steps, general regulatory mechanisms, and molecular functions of the ribosomal rRNA and proteins. However, even with all this information we are far from understanding the detailed sequence of molecular interactions involved in the process of protein synthesis. We have snapshots of different stages of protein synthesis (initiation, elongation, and termination) but, we are still missing the intricate details responsible for linking these snapshots and creating a complete picture depicting the mechanism of protein synthesis. This thesis is focused on understanding the mechanism of protein synthesis using the yeast HAC1 mRNA as a model mRNA in yeast Saccharomyces cerevisiae.

The messenger RNA (mRNA) bears the genetic information that is decoded in the process of protein synthesis. The fate of a messenger mRNA is decided by regulatory elements present in the mRNA that are known as cis-acting elements. They influence not only translation of the mRNA, but also mRNA splicing, mRNA localization, mRNA processing, and mRNA degradation. One of the cis-regulating factors is the RNA secondary structure. The RNA secondary structure controls the translation of an mRNA. The precise mechanism of which is not clearly understood. This thesis is aimed at uncovering the regulation of protein synthesis by the RNA secondary structure.

Proteins in their native conformation either spontaneously attain the folded conformation or they are folded by chaperone proteins in the cytoplasm or in the endoplasmic reticulum (ER). Sometimes the ER experiences an overload of unfolded proteins which results in a condition termed as “ER stress”. Under the stress conditions, unfolded protein response (UPR) pathways are activated which result in expression of assorted transcription factors. These transcription factors modulate the cellular transcriptome and proteome to alleviate the ER stress conditions.

Our model mRNA from Saccharomyces cerevisiae encodes a transcription factor that is expressed under conditions of ER stress. HAC1 mRNA contains a cytoplasmic intron (252 nucleotides) that base-pairs with the 5’- untranslated region (5’-UTR) of the mRNA. This base-pairing interaction inhibits the translation of the HAC1 mRNA under physiological conditions. We use HAC1 mRNA and the inherent base-pairing interaction to uncover new insights into the mechanism of translational regulation by the RNA secondary structure.

HAC1 pre-mRNA is composed of a 5’-untranslated region {(5’-UTR, 68-nucleotides (nt)}, an exon1 (661-nt), an intron (252-nt), an exon2 (57-nt), and a 3’-UTR (462-nt). Under conditions of ER stress, an endonuclease Ire1 cleaves the intron from the HAC1 pre-mRNA to relieve the translational block of the mRNA. The two exons are spliced by tRNA ligase and the mature HAC1 mRNA produces Hac1 protein which is an active transcription factor. We have shown that the base pairing interaction between 5’-UTR and intron inhibits translation initiation of HAC1 mRNA [1].

We performed a random genetic screen to identify an intragenic suppressor mutation (s) that would overcome the translational block in HAC1 mRNA. We identified a point mutation in the base-pairing interaction that relieved the translational block in HAC1 mRNA. Further mutational analyses of the base-pairing interaction suggested that it is critical for the regulation of HAC1 mRNA translation. We also showed that insertion of an in-frame AUG start codon upstream of the RNA secondary structure releases the translational block, demonstrating that an elongating ribosome can disrupt the interaction. Moreover, overexpression of translation initiation factor eIF4A, a helicase, enhances production of Hac1 from an mRNA containing an upstream AUG start codon at the beginning of the base-paired region. Together, we showed that the RNA secondary structure regulates translation initiation of HAC1 mRNA [1].

To dissect the translation initiation block further we shifted the RNA secondary structure from its normal position (which is cap-proximal) to one away from the mRNA cap structure (a cap-distal position) by inserting an unstructured RNA sequence. We observed that genetically engineered HAC1 mRNA with the cap-distal secondary structure resulted in translation of the Hac1 protein. This result suggested that the cap-proximal RNA secondary structure possibly inhibits the interaction of the pre-initiation complex (PIC) with the HAC1 mRNA.

Further analyses of the yeast transcriptome and translatome suggest that the HAC1 gene locus expresses two overlapping transcripts; referred to here as “HAC1a” like the one described above, and “HAC1b”, a newly identified variant. Interestingly, the newly identified HAC1b mRNA overlaps with the exon1 of HAC1a. Yeast transcriptome analyses show that it is composed of a long 5’-UTR (~400-nt), an open reading frame (693-nt) and a short 3’-UTR (124-nt). We characterized the role of the HAC1b transcript in the context of the ER stress response. We observed that HAC1b, like HAC1a mRNA, remains translationally silent. However, HAC1b can activate the ER stress response as a functional Hac1 protein is synthesized in the absence of “Duh1” protein. “Duh1” is a component of the proteasome complex. These observations are consistent with the previous report that Duh1 targets the protein product from the un-spliced HAC1 mRNA for degradation.

Taken together, our results provide mechanistic insights into the translational regulation of HAC1 mRNA. In addition, we provide evidence that the transcript isoform of HAC1 mRNA might play a role in the ER stress response in yeast, S. cerevisiae.

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