The role of mRNA translation mechanisms in germ cell and embryonic development in C. elegans
Author
Umphlett, Hannah Bassett
Abstract
Reproductive cells and embryos use mRNA regulation as a primary means of gene expression. mRNA translational mechanisms rely on the eIF4 translation factors. These include eIF4E, which binds mRNA 7-methylguanosine caps, and eIF4G, which binds eIF4E and guides the mRNA to the ribosome for translation. Germ cells and embryos have the unusual capacity to use both cap-dependent and cap-independent translation mechanisms. Both use eIF4G (called “IFG-1” in C. elegans), but only cap-dependent uses eIF4G. Previous research in the model organism C. elegans has indicated that these mechanisms function at different times and for different mRNAs during germ cell development into sperm and oocytes. Germ cell development is also highly temperature dependent. Each of these aspects in turn impact the fertility of the nematode.
Genetic engineering by means of the CRISPR-Cas9 system allowed us to introduce N-terminal tags into the ifg-1 gene. We noticed early that some of these tags had deleterious effects on fertility, specifically on embryo hatching, while others were fully tolerated. This study addresses the viability of IFG-1 tagged with short peptides including V5, 3xMyc, and a short internal deletion of V5, along with the longer fluorophore mCherry. Embryonic lethality was compensated by balancer chromosomes in strains where tags were not tolerated. Indeed, addition of 3xMyc and the full mCherry protein (3xMyc::mCherry) fused to IFG-1 was viable. However, replacement of the 3xMyc tag with the short peptide V5 caused full embryonic lethality at all temperatures tested (15C, 20C, 25C). To determine if the V5 per se was toxic, we used CRISPR-Cas9 to make an internal deletion in V5. Remarkably, deletion of just 3 amino acids within V5 (dV5) restored nearly wild type levels of fertility (egg laying) at all temperatures. Remarkably, the viability of those embryos was observed only at 25C. To exclude the possibility of a recombination event in the dV5::ifg-1 gene that restored a functionally wild type ifg-1 gene, we conducted genomic PCR in the strains, even those mothers used in the egg laying/hatching experiment at 25C. Correct predicted genotypes were observed for all strains at the ifg-1 locus by PCR.
Our results indicate that the N-terminus of the IFG-1 is very sensitive to structural or sequence changes that affect the protein synthesis required for embryo hatching. This portion of the protein is upstream of the portion that binds to eIF4E—the cap binding protein. Cap-dependent mRNA translation is known to be critical for cells in growing or differentiating stages. We are attempting to evaluate the importance of the balance between cap-dependent and -independent mechanisms in the developmental processes associated with embryo hatching.
Date
2023-05-03
Citation:
APA:
Umphlett, Hannah Bassett.
(May 2023).
The role of mRNA translation mechanisms in germ cell and embryonic development in C. elegans
(Honors Thesis, East Carolina University). Retrieved from the Scholarship.
(http://hdl.handle.net/10342/12986.)
MLA:
Umphlett, Hannah Bassett.
The role of mRNA translation mechanisms in germ cell and embryonic development in C. elegans.
Honors Thesis. East Carolina University,
May 2023. The Scholarship.
http://hdl.handle.net/10342/12986.
May 21, 2024.
Chicago:
Umphlett, Hannah Bassett,
“The role of mRNA translation mechanisms in germ cell and embryonic development in C. elegans”
(Honors Thesis., East Carolina University,
May 2023).
AMA:
Umphlett, Hannah Bassett.
The role of mRNA translation mechanisms in germ cell and embryonic development in C. elegans
[Honors Thesis]. Greenville, NC: East Carolina University;
May 2023.
Collections
Publisher
East Carolina University