Identification and comparative analysis of novel alternatively spliced transcripts of RhoGEF domain encoding gene in C. elegans and C. briggsae

Abstract

Y95B8A.12 gene of C. elegans encodes RhoGEF domain, which is a novel module in the Guanine nucleotide exchange factors (GEFs). Alternative splicing increases transcriptome and proteome diversification. Y95B8A.12 gene has two reported alternatively spliced transcripts by the C. elegans genome sequencing consortium. In the work presented here, we report the presence of four new spliced transcripts of Y95B8A.12 arising as a result of alternative splicing in the pre-mRNA encoded by Y95B8A.12 gene. Our methodology involved the use of various gene or exon finding programmes and several other bioinformatics tools followed by experimental validation. We have also studied alternative splicing pattern in RhoGEF domain encoding orthologues gene from C. briggsae and have obtained very similar results. These new unreported spliced transcripts, which were not detected through conventional approaches, not only point towards the extent of alternative splicing in C. elegans genes but also emphasize towards the need of analyzing genome data using a combinations of bioinformatics tools to delineate all possible gene products. Background: The guanine nucleotide exchange factors for the Rho GTPases (RhoGEFs) are a large family of proteins that share a dual structural motif designated the DH/PH domain. RhoGEFs are regulators of the Rho proteins [1] that act as molecular switches; cycling between inactive (GDP-bound) and active (GTP-bound) states. [2] The interaction of Rho with residues within the DH domain enhances the exchange of GDP for GTP and thus converts Rho into the biologically active form. Thus, the intracellular ratio of the GTP/GDP-bound forms of Rho proteins determines the activation of signal transduction pathways regulating the spatial and temporal reorganization of cytoskeletal architecture. [3] Members of the Rho subfamily of Ras-like monomeric GTPases, including Rho, Rac and Cdc42, are involved in a broad range of functions including gene transcription, cell cycle progression, cell polarity and most notably regulation of the actin cytoskeleton and cell morphology. [4] RhoGEF family is a widespread family found commonly in almost all organisms like Humans, Mouse, and Drosophila, C. elegans etc. In C. elegans this family consists of a large number of members and here the number of RhoGEF and RhoGAP regulators of Rho GTPases significantly exceeds the number of Rho family GTPases. These regulators likely provide the signaling specificity and spatial-temporal regulation required by the broadly expressed and functionally