Premature Stop Codon in E3 Ubiquitin Ligase of C. elegans
Research Mentor(s)
Dahlberg, Lina
Description
Misfolded proteins disrupt proteostasis and are associated with neurodegeneration. E3 ubiquitin ligases are enzymes that function in ERAD (endoplasmic reticulum associated degradation) by tagging misfolded proteins with ubiquitin, signaling for degradation by the proteasome. MARC-6 is an E3 ubiquitin ligase that we use to study protein accumulation in the neurons of Caenorhabditis elegans (C. elegans). When MARC-6 has a premature stop codon, we predicted it would fail to be properly translated due to a loss or reduction of mRNA. GLR-1 is a transmembrane protein that is synthesized in the ER(endoplasmic reticulum); we used nuIs24(pglr-1::GLR-1::GFP) to study the effect of the marc-6 mutations on protein accumulation. We genotyped the marc-6 mutants via PCR followed by a restriction digest and DNA sequencing. Western blots and fluorescence microscopy on nuIs24 and marc-6(gk121407);nuIs24 were used to quantify GLR-1 accumulation within the animals and the puncta of the ventral nerve cords, where GLR-1 is known to be localized. Protein levels showed little change in amounts of GLR-1 between the two marc-6 strains. RNA isolation and RT (reverse transcriptase)-PCR was performed on the mutant strains crossed into nuIS24 to determine the absence or reduction of the mRNA. These results suggested that the marc-6 mutants produced a loss of mRNA transcript. Although there may be little MARC-6 protein in the cell, no significant difference was observed in accumulation of GLR-1. However, further data is needed to support whether or not there is an interaction between MARC-6 and GLR-1. qPCR, behavioral nose touch assays and other experiments are the next steps in understanding the role marc-6 plays in the neurons.
Document Type
Event
Start Date
May 2018
End Date
May 2018
Department
Biology
Genre/Form
student projects, posters
Subjects – Topical (LCSH)
Protein folding; Protein engineering; Nervous system--Degeneration--Genetics
Type
Image
Rights
Copying of this document in whole or in part is allowable only for scholarly purposes. It is understood, however, that any copying or publication of this document for commercial purposes, or for financial gain, shall not be allowed without the author’s written permission.
Language
English
Format
application/pdf
Premature Stop Codon in E3 Ubiquitin Ligase of C. elegans
Misfolded proteins disrupt proteostasis and are associated with neurodegeneration. E3 ubiquitin ligases are enzymes that function in ERAD (endoplasmic reticulum associated degradation) by tagging misfolded proteins with ubiquitin, signaling for degradation by the proteasome. MARC-6 is an E3 ubiquitin ligase that we use to study protein accumulation in the neurons of Caenorhabditis elegans (C. elegans). When MARC-6 has a premature stop codon, we predicted it would fail to be properly translated due to a loss or reduction of mRNA. GLR-1 is a transmembrane protein that is synthesized in the ER(endoplasmic reticulum); we used nuIs24(pglr-1::GLR-1::GFP) to study the effect of the marc-6 mutations on protein accumulation. We genotyped the marc-6 mutants via PCR followed by a restriction digest and DNA sequencing. Western blots and fluorescence microscopy on nuIs24 and marc-6(gk121407);nuIs24 were used to quantify GLR-1 accumulation within the animals and the puncta of the ventral nerve cords, where GLR-1 is known to be localized. Protein levels showed little change in amounts of GLR-1 between the two marc-6 strains. RNA isolation and RT (reverse transcriptase)-PCR was performed on the mutant strains crossed into nuIS24 to determine the absence or reduction of the mRNA. These results suggested that the marc-6 mutants produced a loss of mRNA transcript. Although there may be little MARC-6 protein in the cell, no significant difference was observed in accumulation of GLR-1. However, further data is needed to support whether or not there is an interaction between MARC-6 and GLR-1. qPCR, behavioral nose touch assays and other experiments are the next steps in understanding the role marc-6 plays in the neurons.