Translation is a crucial mechanism for generating proteins to carry out cellular processes and for ensuring proper cell functions. Ribosomes are at the center of translation and are complex pieces of machinery. They consist of at least 80 core eukaryotic ribosomal proteins, which are conserved from prokaryotes, and four ribosomal RNAs (rRNAs): 18S, 28S, 5,8A 5S. In addition, numerous translation factors aid the ribosome in protein production. While ribosomes are typically described by these core features, they are known to exist in a heterogenous pool with variations in protein composition, modifications of rRNA, and an assortment of non-ribosomal proteins that can associate with the translation complex. This variety is thought to allow for preferential translation of subsets of mRNAs depending on RNA elements and their recruitment for certain translation factors.
Two proteins, Receptor for Activated C Kinase 1 (RACK1) and glycogen synthase 1 (GYS1), both associate with the ribosome and have been shown to dictate the translation of specific mRNAs. In this dissertation, I will first describe how RACK1 plays a role in viral Internal Ribosomal Entry Site (IRES)-mediated translation. During viral infection, viruses need to gain access to cellular ribosomes to efficiently translate their genome. Therefore, some viruses employ IRES elements to commandeer the ribosome and bypass canonical translation initiation. For certain classes of IRESs, such as the type 1 IRES within the poliovirus genome (PV), the presence of RACK1 in the cell facilitates viral protein production and the viral life cycle. Since we saw RACK1 played a role in viral IRES-mediated translation, we were also curious about the role of RACK1 in cellular IRES-mediated translation. Several eukaryotic mRNAs have been described to contain IRES elements to recruit certain translation initiation factors when cap-dependent translation is compromised. We tested the translation efficiency of a variety of cellular genes previously described to contain IRES when RACK1 was depleted in cells and amongst them, the oncogenic c-Myc showed a decrease in translation when RACK1 was depleted in cells. Identifying cellular proteins that facilitate IRES-mediated translation can hold promising therapeutic potential. In the case of viruses, protein biosynthesis is essential to viral success and revealing host factors such as RACK1 that viruses rely on might hold promise for future antiviral drug targets. For cellular IRESs such as Myc which is highly expressed in cancer, we may further uncover the cellular requirements necessary for cancer cell growth.
As researchers investigate the mechanisms of IRES-mediated translation and other modes of specialized translation, we are critical of strategies used to uncover their findings. In the case of GYS1, we used CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) genome technology to deplete GYS1 expression in HeLa cells to uncover other novel functions of the metabolic protein. However, upon attempting to rescue GYS1 expression in a lentivirus addback prepared cell line, we were unable to rescue the phenotype in the GYS1 knockout cells. This lack of rescue in knockout cells has been described by other researchers who also found that genome editing might alter the state of the cell due to indirect changes. As we probe the eukaryotic ribosome for answers regarding specialized modes of translation, we use GYS1 as a lesson to be cautious of unforeseen changes in cell state which may further convolute promising findings. Overall, this work describes how certain proteins, RACK1 and GYS1, associate with the ribosome to influence translation of a subset of mRNAs and investigations such as the ones described in this dissertation provide further understanding of sophisticated translation mechanisms.
|Commitee:||Larsen, Melinda, Pager, Cara, Wade, Joseph|
|School:||State University of New York at Albany|
|School Location:||United States -- New York|
|Source:||DAI-B 82/2(E), Dissertation Abstracts International|
|Subjects:||Biology, Molecular biology, Virology|
|Keywords:||GYS1, IRES, RACK1, Ribosome heterogeneity, Translation, Translational control|
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