Award Date

12-2011

Degree Type

Dissertation

Degree Name

Doctor of Philosophy in Biological Sciences

Department

Life Sciences

First Committee Member

Frank van Breukelen, Chair

Second Committee Member

Andrew Andres

Third Committee Member

Jeffrey Shen

Fourth Committee Member

Allen Gibbs

Graduate Faculty Representative

Ernesto Abel-­‐Santos

Number of Pages

125

Abstract

Mammalian hibernation is a highly dynamic physiological process that is composed of a series of torpor bouts, wherein hibernators oscillate between periods of torpor and interbout arousal. Although normally vital to homeostasis, many energetically consumptive processes such as translation or protein synthesis are virtually ceased during hibernation. Earlier studies indicated that protein synthesis had fallen to almost negligible levels. Cap-dependent initiation of translation is well regulated by eukaryotic translation initiation factor 4E (eIF4E) and its binding partner eIF4E-binding protein 1 (4E-BP1) when hibernators cycle in and out the torpor state. Herein, I investigated well-characterized regulatory mechanisms of global and specific control of protein synthesis in hibernators, with the ultimate goal of understanding how hibernators regulate protein synthesis in an extremely dynamic physiological process of hibernation.

Given that cap-dependent initiation of translation was actively regulated during hibernation in golden-mantled ground squirrels, Spermophilas lateralis, I was particularly intrigued by the role of cap-independent initiation of translation, more specifically, the role of internal ribosomal entry site mediated initiation of translation (IRESmt), which allows for internal initiation of translation. I utilized quantitative real time PCR (qRT-PCR) to assess the association of both IRES and non-IRES containing transcripts with ribosomes throughout the entire sucrose gradients as a function of torpor state. Data indicate that mRNAs harboring IRES elements are preferentially associated with ribosomes as a torpor bout progresses. Naturally aroused squirrels have a higher IRES preference index than those animals that are prematurely aroused from torpor. Furthermore, data indicate that this change in IRES preference is the result of changes in mRNA association instead of mRNA abundance. Thus, ribosomes are preferentially loaded with IRES-containing transcripts when squirrels arouse from torpor and translation resumes. Differential translation of preexisting mRNAs may allow for the preferential synthesis of key stress proteins critical for survival of physiological hardships of hibernation that are lethal to non-hibernating mammals.

microRNAs (miRNAs) are a class of conserved ∼ 22 nucleotide-long non-coding RNA molecules. miRNAs have been demonstrated to play critical roles in controlling protein synthesis by targeting mRNA transcripts for translational repression in many systems, e.g. miRNAs control translation initiation by inhibiting eIF4E. However, it was largely unknown as to what role miRNAs may play during hibernation. I screened for changes in miRNA populations in livers of golden-mantled ground squirrels as a function of torpor state by utilizing a miRNA microarray. Data indicate that there were no changes in the four major miRNA species (miRNA-122, miRNA-15, miRNA-21, and miRNA-146) as a function of torpor state. These findings suggest that the potential control of protein synthesis exerted by miRNAs may be limited during hibernation.

p53 is a specific regulator of protein synthesis, e. g. p53 activation can cause dephosphorylation of 4E-BP1 and therefore repress translation initiation. However, p53 is best recognized as a transcription factor that regulates the expression of stress response genes involved in a variety of biological progresses. Earlier studies have shown that transcription is significantly depressed during hibernation. However, some reports in the literature revealed transcription factor movements during torpor, e.g. nuclear factor kappa B (NF-κB) enters the nucleus and p53 is excluded from nucleus. The presumption is that transcription factors function during hibernation and that these changes have an impact on the physiology of the animal. An earlier study claimed that nuclear p53 protein concentration was significantly reduced 4-fold in torpid ground squirrels. I exploited a variety of techniques to further study the role of p53 during hibernation. My data reveal that p53 mRNA and protein levels are not related, but that p53 protein concentrations were elevated during late torpor and interbout aroused states. Data also indicate that expression levels of several known regulators of p53 are consistent with the activation of p53 during hibernation. Taken at face value, these data might suggest that p53 works as a transcription factor to induce preferential transcription of target genes. My data indicate that nuclear p53 levels are ∼ 2 fold higher in the late torpor state. Furthermore, p53 protein binds to known target genes. However, nuclear run-on data as well as quantitative assessment of downstream target genes indicate a very modest role for p53 as a transcription factor. These data are important in that formulated assumptions using lessons learned from active steady state conditions should be applied cautiously in the setting of a depressed metabolic state.

My research investigates the global and specific controls of protein synthesis in hibernation by studying the basic roles of IRES mediated initiation of translation, miRNAs, and transcription factor p53 use in hibernating goldenmantled ground squirrels. These data provide compelling evidence that hibernators actively regulate translation initiation and therefore preferentially produce key stress proteins geared towards enhanced survivorship. However, miRNAs likely play a limited role in the control of protein synthesis in hibernating ground squirrels. Finally, my data on p53 metabolism also underscore the value of cautious data interpretation of regulatory mechanisms at a time of suspended regulation.

Keywords

Chromatin immunoprecipitation; Golden-mantled ground squirrel; Hibernation; Immunohistochemistry; Internal ribosome entry site; Microrna; Nuclear run on assay; P53; Proteins — Synthesis; Ribosomes; RNA; Spermophilas lateralis

Disciplines

Animal Sciences | Genetics and Genomics | Molecular Biology | Systems Biology | Zoology

Language

English


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