Date of Award

2020-01-01

Degree Name

Master of Science

Department

Biological Sciences

Advisor(s)

Manuel Llano

Abstract

Schlafen13 (Slfn13) is an enzyme that belongs to the Schlafen family whose expression and function is not very well characterized. The N-terminal has a pseudo dimer structure that contains its catalytic site. There is no characterization functionally or structurally of the C-terminal of Slfn13 other than the prediction of a region with helicase activity. The objective of my Thesis was to increase our understanding of the Slfn family of proteins. Currently, Slfn13 is reported to play a role in the differentiation of monocytes and to function as an endoribonuclease that cleaves tRNA and rRNA molecules in a site-dependent sequence-independent manner. Further analysis of the anti-viral activity is hampered by the poor expression of the full-length protein. We have been unable to detect Slfn13 by western blot using different antibodies in different cell lines which were successfully transfected either stably or transiently. Our data indicate that the regulation of Slfn13 expression is at a post-transcriptional level. We demonstrated the robust expression of endogenous and exogenous mRNA encoding Slfn13 in different cell lines by means of RT-PCR. This suggests a potential instability and/or toxicity of Slfn13. We investigated whether regulation is at the post-translational level by using compounds that inhibit proteasomes and lysosomes. Despite obstructing these major pathways of protein degradation, we were unable to rescue Slfn13 expression. It is reported that type I-interferons play a role in the induction of Slfn proteins. Intrinsically, we theorized that perhaps an interferon-induced chaperon was required for Slfn13 expression but interferon α failed to stabilize this protein. In contrast to Slfn13, the family member Slfn11 is widely expressed at very high levels in multiple cell lines. We took advantage of the evolutionary conservation between these two proteins and swapped the last third residues of Slfn13 with those of Slfn11. This chimera was readily expressed indicating that the regulation was post-translational. Furthermore, this indicates that the last third of Slfn13 is responsible for the instability of this protein. Since the Slfn11 portion in the chimera does not have anti-viral activity, we also investigated the activity of Slfn13/11 Chimera against HIV-1 and flaviviruses. Our findings indicate that the Slfn13/1l chimera lacks anti-viral activity. However, we cannot ascertain the presence or absence of Slfn13 through normal methods. We believe that localization plays a role in the expression of this protein as the c-terminal of region of Slfn11 in the Slfn13/11 contains a predicted nuclear localization sequence. This led us to probe which of the last third of the c-terminal is responsible for the instability of this protein and how we could exploit this feature for molecular biology purposes. In summary, because the Slfn13/11 Chimera is 80 percent identical to Slfn13 and it is known that c-terminal has no activity; we can say that Slfn13 also lacks anti-viral activity.

Language

en

Provenance

Received from ProQuest

File Size

61 pages

File Format

application/pdf

Rights Holder

Jordan Winfield

Download

Included in

Virology Commons

Share

COinS