Date of Award

Winter 1-2019

Degree Type

Thesis

Degree Name

Doctor of Philosophy In Regulatory Biology Degree

Department

Biological, Geological and Environmental Sciences

First Advisor

Mazumder, Barsanjit

Second Advisor

Dr. Anton A. Komar, GRHD/BGES, Cleveland State University

Third Advisor

Dr. Girish Shukla, GRHD/BGES, Cleveland State University

Abstract

Ribosomal protein L13a plays an extra-ribosomal function in translational silencing of GAIT (IFN-gamma-activated inhibitor of translation) element bearing mRNAs encoding inflammatory proteins but the underlying molecular mechanism of translational silencing and ribosomal incorporation of L13a remains poorly understood. Also, our laboratory showed that L13a acts as a physiological defense against uncontrolled inflammation in macrophage-specific knockout (KO) mice. However, the consequence of a total knockout of L13a in mammals remains unexplored. Therefore, our current study is focused on (i) identifying the amino acid residue(s) of L13a essential for incorporation and translational silencing of target mRNAs and (ii) studying the consequences of systemic loss of L13a in a mouse model. To address the first question, we compared prokaryotic L13 structure with human L13a, which depicted the presence of an a- helical extension of ~55 amino acids at the C-terminal end of human L13a. We observed that deletion of this helix impairs ribosomal incorporation and the translational silencing ability of L13a. We have identified the amino acids within this helix at position 159(K) and 161(K) that are essential for ribosomal incorporation. Cryo EM studies of the human ribosome showed the interaction of the amino acids at position 185(V), 189(I) and 196(L) ofL13a with RP L14. We found that mutating these residues abrogates the ribosomal incorporation of L13a. Importantly, we also showed that mutation of the amino acids at position 169(R), 170(K) vii and 171(K) to Ala abrogate translational silencing activity, but not ribosomal incorporation, showing mutually exclusive ribosome incorporation and translational silencing domain. To address the second question, we generated heterozygous L13a mice (L13a+/-). However, the homozygous KO (L13a-/-) mice are embryonically lethal at an early stage. We have identified the KO embryos in the pre-implantation (morula) stage, suggesting an essential role of L13a in early embryonic development. Next Generation Sequencing (NGS) analysis of morula stage embryos harvested from L13a+/- heterozygous parents, identified several potential targets with altered expression. Together, these studies provide a comprehensive analysis of the amino acid residues of L13a essential for ribosome incorporation and translational silencing activity and its essential role in early embryonic development in mammals.

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