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Insights into the ribosomal, extra-ribosomal and developmental role of RP L13a in mammalian model

Kour, Ravinder
http://rave.ohiolink.edu/etdc/view?acc_num=csu1572548728931568

Abstract Details

2019, Doctor of Philosophy in Regulatory Biology, Cleveland State University, College of Sciences and Health Professions.
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 the prokaryotic L13 structure with human L13a, which depicted the presence of an α-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. CryoEM studies of the human ribosome showed the interaction of the amino acids at position 185(V), 189(I) and 196(L) of L13a 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) 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.
Barsanjit Mazumder (Advisor)
Anton A. Komar (Committee Member)
Crystal M. Weyman (Committee Member)
William M. Baldwin (Committee Member)
Girish Shukla (Committee Member)
William C. Merrick (Committee Member)
137 p.
Developmental Biology; Molecular Biology
GAIT; Ribosomal protein L13a; rRNA; ribosomes; Inflammation; extra-ribosomal functions; embryonic development; embryonic lethality

Recommended Citations

Citations

  • Kour, R. (2019). Insights into the ribosomal, extra-ribosomal and developmental role of RP L13a in mammalian model [Doctoral dissertation, Cleveland State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=csu1572548728931568

    APA Style (7th edition)

  • Kour, Ravinder. Insights into the ribosomal, extra-ribosomal and developmental role of RP L13a in mammalian model. 2019. Cleveland State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=csu1572548728931568.

    MLA Style (8th edition)

  • Kour, Ravinder. "Insights into the ribosomal, extra-ribosomal and developmental role of RP L13a in mammalian model." Doctoral dissertation, Cleveland State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=csu1572548728931568

    Chicago Manual of Style (17th edition)

csu1572548728931568
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This open access ETD is published by Cleveland State University and OhioLINK.