Doctor of Philosophy in Regulatory Biology, Cleveland State University, 2019, 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 (open full item for complete abstract)

Committee: 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) Subjects: Developmental Biology; Molecular Biology

Doctor of Philosophy in Regulatory Biology, Cleveland State University, 2014, College of Sciences and Health Professions

Inflammation is an obligatory attempt of the host immune system to protect the body against infection. However, unregulated synthesis of pro-inflammatory products can have detrimental effects. Though mechanisms which contribute to inflammation are well appreciated, those that resolve inflammation are poorly understood. Therefore, understanding the molecular basis of such pathways will provide an entirely novel approach to treat and prevent inflammatory diseases. Transcript-selective translational control can regulate the expression of a set of inflammatory genes. We have identified one such mechanism in a novel animal model which relies on the abrogation of ribosomal protein L13a-dependent translational silencing by creating macrophage-specific L13a-knockout mice where resolution of inflammation is severely compromised. We have used these knockout mice to study two different kinds of inflammation: LPS induced systemic inflammation and dextran sodium sulphate (DSS) induced experimental colitis. Upon LPS induced endotoxemia, these mice displayed high mortality rates and severe symptoms of inflammation such as infiltration of immune cells in the peritoneum and major organs leading to tissue destruction. These animals also exhibited high serum levels of TNF-a, blood urea nitrogen (BUN), aspartate aminotransferase (AST) and several other markers of inflammation. Macrophages from these knockouts showed unregulated synthesis of several chemokines (e.g., CXCL13, CCL22, CCL8 and CCR3) and increased polysomal abundance of these mRNAs due to the abrogation of their translational silencing. Upon DSS induced colitis, these knockout mice demonstrated higher susceptibility to colitis displaying reduced survival, significant weight loss, enhanced rectal bleeding and diarrhea. Histopathology analysis of tissue sections from the knockouts showed disruption of epithelial crypts in the colon with infiltration of macrophages in colon and spleen sections. Additionally, elevated levels of (open full item for complete abstract)

Committee: Barsanjit Mazumder Ph.D. (Advisor); Crystal Weyman Ph.D. (Committee Member); Anton Komar Ph.D. (Committee Member); William Baldwin M.D., Ph.D. (Committee Member); Roman Kondratov Ph.D. (Committee Member); Aimin Zhou Ph.D. (Committee Member) Subjects: Animals; Biology; Immunology; Molecular Biology

Doctor of Philosophy in Regulatory Biology, Cleveland State University, 2012, College of Sciences and Health Professions

Ribosome biogenesis, a fundamental process, occurs in the nucleolus. It involves incorporation and association of ribosomal proteins (r-proteins) in the ribosomal subunit. Despite its obligatory and critical role in cellular function, the explicit mechanism of incorporation of different r-proteins into the pre-ribosome is not well understood. Using mammalian cell and r-protein L13a as our model, this study addresses the function and mechanism of r-protein incorporation during ribosome maturation. Published results from our laboratory showed the requirement of the release of L13a from the 60S ribosome to silence a cohort of inflammatory proteins directly at the level of translation thus showing the significant potential of this mechanism to resolve inflammation. To get further insight into the mechanism of its release, it is essential to identify the domain of L13a and the subcellular site required for ribosome incorporation. Homology modeling of human L13a with the crystal structure of prokaryotic L13, predicted some amino acid residues that could bind to ribosomal RNA (rRNA). Consistent with this model, a combined experimental approach involving ribosome incorporation assay of recombinant L13a and RNA immunoprecipitation have identified Arg at position 68 and Arg-Lys-Arg at position 59-60-61 as potential interaction site with 60S subunit. We have performed immunofluorescence studies to test whether the incorporation defective mutant L13a failed to translocate to the nucleolus, the site of ribosome biogenesis. L13a harboring the mutation of Arg at position 68 to Ala translocate to the nucleolus, but however alters the nucleolar morphology. In contrast L13a with the mutation of Arg-Lys-Arg at position 59-60-61 to Ala-Ala-Ala is nucleolar translocation incompetent. These studies also identified that incorporation of L13a during ribosome biogenesis occurs at the stage of 90S pre-ribosome formation. Previous studies from our laboratory showed an essential role of L13a i (open full item for complete abstract)

Committee: Barsanjit Mazumder Ph.D (Advisor); Crystal Weyman Ph.D (Committee Member); Anton Komar Ph.D (Committee Member); Jaharul Haque Ph.D. (Committee Member); Girish Shukla Ph.D. (Committee Member); Amin Zhou Ph.D. (Committee Member) Subjects: Molecular Biology

Master of Science in Biology, Cleveland State University, 2008, College of Science

Transcript-specific translational control restricts macrophage inflammatory gene expression. The pero-inflammatory cytokine IFN-γ induces the phosphorylation of human ribosomal protein L13a and its subsequent release from 60S ribosome. L13a is a component of the interferon-gamma-activated inhibitor of translation (GAIT). The GAIT complex binds a defined element in the 3'-untranslated region (UTR) of ceruloplasmin (Cp) mRNA and causes delayed silencing of translation. In this research, we elucidate the molecular mechanism underlying L13a translational silencing activity. L13a mediates translational silencing particularly, when driven by internal ribosome entry sites (IRESs) that requires the initiation factor eIF4G, but is resistant to silencing when driven by eIF4F- independent IRESs. This demonstrates a critical role of the scaffold protein eIF4G. Global inhibition of protein synthesis by targeting eIF4G is well appreciated in virus infection and apoptosis; however interaction of L13a with eIF4G blocks the 43S complex recruitment showing a unique role of eIF4G in gene specific translational silencing.

Committee: Barsanjit Mazumder PhD (Advisor); Anton Komar PhD (Committee Member); Crystal Weyman PhD (Committee Member) Subjects: Cellular Biology; Molecular Biology