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Theses Doctoral

How to steal ribosomes: structural studies of two different internal ribosome entry sites

Neupane, Ritam

Taking control of the protein production machinery of the host cell is a required step in the life cycle of viruses. Towards this end, viruses have evolved diverse strategies of cellular mimicry and deception to hijack and steal host cell ribosomes for viral protein production. In higher eukaryotes, where translation is sophisticated and access to ribosomes intricately regulated, numerous positive strand RNA viruses have evolved structured RNA sequences to evade translation regulation mechanisms. These RNA sequences, called Internal Ribosomal Entry Sites (IRESs), use their RNA structure to hijack the eukaryotic host cell ribosomes during the highly regulated initiation phase of translation. While a select few of such IRESs have been both biochemically and structurally characterized, the diversity of IRESs isn’t fully explored. Structural basis for the working mechanism of intergenic IRESs such as the Israeli Acute Paralysis Virus IRES (IAPV-IRES) with unique RNA features and expanded coding capacity is unavailable. Similarly, structural and biochemical understanding of newly described IRESs such as the complex IRES located at the 5′ untranslated region of the Cricket Paralysis Virus (CrPV 5′-UTR-IRES) is also unavailable. This body of work uses cryo-electron microscopy (cryo-EM) and biochemistry to characterize these two IRESs.Here, we show how the IAPV-IRES uses its unique features to exploit novel binding sites and commits the IRES-ribosome complexes towards a global pre-translocation mimicry. We trace a complete path of the IRES from its initial binding with the small subunit to its formation of an elongation-ready ribosome. We show that its mechanism of ribosome hijacking is different from currently accepted mechanistic paradigm for other IRESs from viruses similar to IAPV-IRES.

We also identify another divergent mechanism of ribosome hijacking used by a different type of IRES. We show that the CrPV 5′-UTR-IRES features a novel, extended, and multi-domain architecture unlike any of the previously characterized IRESs from the group it belongs to. We also show that this IRES uses its novel structure and a minimal set of initiation factors to assemble a canonical-like pre-initiation complex on the small subunit of the ribosome at an upstream start-stop open reading frame.
This body of work underscores the unexplored diversity in IRESs found in single stranded positive sense viral RNA genomes, invites re-visiting of the currently standing mechanisms of cap-independent initiation carried out by IRESs, and sheds light on a possible evolutionary past where IRESs could have given rise to the current eukaryotic translation initiation system.

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More About This Work

Academic Units
Biological Sciences
Thesis Advisors
Fernandez, Israel S.
Degree
Ph.D., Columbia University
Published Here
January 11, 2021