All ETDs from UAB

Advisory Committee Chair

David Schneider

Advisory Committee Members

Yvonne Edwards

Romi Gupta

Document Type

Thesis

Date of Award

2023

Degree Name by School

Master of Science (MS) Heersink School of Medicine

Abstract

RNA Polymerases are the molecular machines responsible for the synthesis of RNA from the DNA template. The presence of these machines is an absolute requirement for the function and replication of all cellular organisms. In eukaryotic organisms, there are a minimum of three RNA polymerases (Pols I, II, II). Despite shared subunits and homology, these three protein complexes are functionally distinct, with many unique sub-units and additional trans-acting factors. Pol I is responsible for the synthesis of three of the four ribosomal RNA species which is the first and rate limiting step of ribosome biogenesis. Pol I synthesizes the majority of RNA in growing cells despite only transcribing one gene. To improve our understanding of these machines we must characterize the role and function of not only the intrinsic sub-units of the polymerase but also any factors that may associate with the polymerase. The Spt4/5 complex is a putative Pol I trans-acting factor that has been well studied for its role in Pol II. Previous research from our group has shown that Spt4/5 also interacts with Pol I in budding yeast (Saccharomyces cerevisiae), and that mutations in the two subunits of the heterodimer, Spt5 or Spt4, cause defects in ribosome biogenesis. Unfortunately, in all of these models it is impossible to discount that the much more established role of Spt4/5 in Pol II is responsible for the phenotypes observed. To circumvent this issue, we used the auxin inducible degron system (AID) to deplete Spt5 to capture how its loss effected the organism in the short window of time before defects in Pol II start to impact ribosome biogenesis. We found that depletion of Spt5 leads to severe reduction in RNA synthesis starting at thirty minutes post induction of depletion. No significant changes were observed in Pol I occupancy using Pol I native elongating transcript sequencing (NET-seq) however the vehicle control differed significantly from previously published wildtype occupancy indicating the need for better controls. Depletion of Spt5 almost certainly impacts ribosome biogenesis and nominal cell growth however as of the completion of this Master’s project results remain inconclusive yet promising.

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