Date of Award

5-2015

Document Type

Honors Thesis

Degree Name

Bachelor of Science

Department

Biology

Advisor/Committee Chair

Rupa Choudhary

Committee Member

Ben Szaro

Committee Member

Richard Zitomer

Abstract

The amphibian Xenopus laevis has the ability to regenerate axons of its optic nerve even after metamorphosis. From previous studies done in our lab, we found that Suppressor of Cytokine Signaling 2 (SOCS2) could be involved in the complex pathway of genes regulating nervous system development and regeneration. 3’ and 5’ Rapid Amplification of cDNA Ends (RACE) revealed that the 5’ untranslated region (UTR) of SOCS2 contains two splice forms. One splice form contained a previously unidentified 68 base pair exon, which will be referred to as ‘Exon 2’, which we hypothesized is involved in post-transcriptional regulation of SOCS2. I performed in situ hybridization on retina sections of the regenerating and non-regenerating eye 12 days after optic nerve crush to test whether Exon 2 is specifically expressed during optic nerve regeneration. I observed that expression of the splice form containing Exon 2 increased in the retina. This suggests that Exon 2 does play a role in the regulation of expression of SOCS2 during regeneration. RT-PCR and qPCR were performed to study the expression differences of the two splice forms at 3 days and 7 days after optic nerve crush. These data, when combined with the in situ hybridization data, suggest that the 5’UTR of SOCS2 is differentially expressed relative to the stages of regeneration. The form of the SOCS2 5’UTR that contains Exon 2 is expressed more in the intermediate to late stages of optic nerve regeneration, whereas the form that lacks this exon is associated with the early stages. Based on these observations, I hypothesized that the 5’UTR with Exon 2 or without it could be functioning as an internal ribosome entry site (IRES), to facilitate translation of SOCS2 protein under stress conditions where cap-dependent translation is suppressed. To test this in vivo, I have created a bicistronic fluorescent protein reporter plasmid that contains the SOCS2 5’ UTR sequences with and without Exon 2. In vitro transcribed mRNA from two control constructs was injected into X. laevis embryos. Assaying for expression indicated that this method can be used to determine IRES activity. mRNA from the experimental constructs containing the SOCS2 5’UTR will be injected into embryos for confirmation or denial of my hypothesis. From my study I hope to better understand the regulatory mechanisms of the SOCS2 5’UTR during axon regeneration in X. laevis.

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