Examination of RAL Proteins in C. elegans
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Date
2020-05
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The Ohio State University
Abstract
Introduction: Ras-related proteins (RAL proteins) are part of the Ras superfamily and these proteins are relevant in many cancers such as breast, colorectal, and prostate. In humans, there are two RAL genes, RALA and RALB. While the structures of RALA and RALB proteins are similar, and they compensate for each other in many cases, they have some specific, distinct functions. In particular, RALA is overexpressed in triple-negative/basal-like breast cancer, while RALB is under expressed (Data not yet published). Reasons and mechanisms for the functional differences are currently unknown, and further research is vital to understanding these proteins. Caenorhabditis elegans, the nematode worm, has a single ortholog to the human proteins, RAL-1. Rescuing nematodes that lack RAL-1 with RALA or RALB can be used to observe differences between the two proteins, which could lead to better understanding of these proteins.
Methods: The promoter, cDNA, and 3' UTR were cloned from DNA, and cDNA from human were used to generate worm-specific expression constructs for each protein (RAL-1, RALA, and RALB) using PCR. These were added together into a plasmid using a Gibson Assembly. The constructs were then inserted into C. elegans using the MosSCI method; the construct is inserted into a transposable element in the genome, which allows for the expression of a single copy of the gene. This created 3 different strains. The capability of each strain to rescue a C. elegans ral-1 mutant was tested. Production of lateral alae, paraquat resistance, and gonad structure were tested. Chimeric constructs containing the RAL-1 N-terminus and the RALA/RALB strain are being developed using PCR to determine regions of the proteins that lead to functional differences.
Results: No significant differences occurred in lifespan or paraquat resistance in RAL-1, RALA, or RALB, suggesting that the human RAL proteins are able to compensate for RAL-1 interactions with reactive oxygen species (ROS).RALB fully rescues lateral alae production, a structure in the cuticle dependent on exosome secretion, whereas RALA rescues incompletely. The gonad structure is similar between RALA and RALB, where egg yolk accumulates, and the animals are sterile. The rachis width is more narrow in RALB, whereas RALA width is more comparable to wild-type (WT). Comparison of RAL-1, RALA, and RALB in C. elegans suggests in vivo differences.
Conclusions: RALA and RALB both rescue to viability. RALB is unable to rescue the rachis, whereas RALA rescues the rachis. RALB rescues the lateral alae, but RALA does not. This suggests that this assay can be used to distinguish functions of RALA and RALB. RALA and RALB may also be used to observe the effects of RAL-1 on fertility in C. elegans.
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Keywords
Molecular Genetics, Cancer, Cell signaling, Cell Biology