Investigation of the ERK/MAP kinase long first intron and its possible role in gene regulation and germ cell development

Abstract

One distinguishing feature of eukaryotic genomes is the presence of introns within nascent transcripts. Genomic analyses of distantly related species, spanning yeast to humans, reveal conservation of intron size and position, suggesting an intron can be advantageous. Many studies have examined the relationship between short introns and gene expression; however, the effect of long introns and gene expression is not well understood. Critical developmental regulators often have an unusually long intron that is conserved in size and position and typically one of the first introns. We hypothesize that long first introns of developmental regulators affect gene expression. To investigate the relationship between a long first intron and gene expression, we have focused on the Caenorhabditis elegans gene encoding ERK/MAP kinase (mpk‐1). Like other metazoan ERK/MAP kinases, mpk‐1 harbors a long first intron in one of its isoforms. The mpk‐1b nascent transcripts include a small first exon and an 8.2 kb first intron, while mpk‐1a lacks that first exon and intron but shares other exons and introns with mpk‐1b. To visualize RNAs, we designed single molecule in situ hybridization (smFISH) probes targeting the 8.2 kb intron to visualize nascent transcripts as well as other probes targeting the shared exons to detect the mRNAs. We use confocal microscopy and MATLAB image processing to assess mpk‐1 transcription from germline stem cell self‐renewal to meiotic pachytene of differentiating cells. In parallel, we are using CRISPR to delete regions of the long mpk‐1b intron to assess its functional importance. Our plan is to compare mpk‐1 nascent and mature transcripts during germline development, both in wild type and in in mutants that lack part of most of that large intron. Our analysis will tell us if the mpk‐1b 8.2 intron is critical for the mpk‐1 regulatory program during germ cell development.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.