Biology subjects

Derry, W. B.

Publications and source records attributed to Derry, W. B..

4 recordsLinked to original sources

bioRxiv2024

MRCK-1 activates non-muscle myosin for outgrowth of a unicellular tube in Caenorhabditis elegans

The formation and patterning of unicellular biological tubes is essential for metazoan development. It is well established that vascular tubes and neurons use similar guidance cues to direct their development, but the downstream mechanisms that promote the outgrowth of biological tubes are not well characterized. We show that the conserved kinase MRCK-1 and its substrate the regulatory light chain of non-muscle myosin, MLC-4, are required for outgrowth of the unicellular excretory canal in C. elegans. Structure-function analysis of MRCK-1 indicates that the kinase domain, but not the small GTPase-binding CRIB domain, is required for canal outgrowth. Expression of a phosphomimetic form of MLC-4 rescues canal truncations in mrck-1 mutants and shows enrichment at the growing canal tip. Specific depletion of MRCK-1 or MLC-4 in the canal causes severe truncations, with unlumenized projections of the basal membrane. Moreover, our work reveals a novel function for non-muscle myosin downstream of MRCK-1 in excretory canal outgrowth that may be conserved in the development of seamless tubes in other organisms.

developmental biology↗

bioRxiv2023

Regulation of the hypertonic stress response by the 3' mRNA cleavage and polyadenylation complex

Maintenance of osmotic homeostasis is one of the most aggressively defended homeostatic setpoints in physiology. One major mechanism of osmotic homeostasis involves the upregulation of proteins that catalyze the accumulation of solutes called organic osmolytes. To better understand how osmolyte accumulation proteins are regulated, we conducted forward genetic screen in C. elegans for mutants with no induction of osmolyte biosynthesis gene expression (Nio mutants). nio-3 mutants encoded a missense mutation in cpf-2/CstF64 while nio-7 mutants encoded a missense mutation in symk-1/Symplekin. Both cpf-2 and symk-1 are nuclear components of the highly conserved 3 mRNA cleavage and polyadenylation complex. cpf-2 and symk-1 block the hypertonic induction of gpdh-1 and other osmotically induced mRNAs, suggesting they act at the transcriptional level. We generated a functional auxin-inducible degron (AID) allele for symk-1 and found that acute, post-developmental degradation in the intestine and hypodermis was sufficient to cause the Nio phenotype. symk-1 and cpf-2 exhibit genetic interactions that strongly suggest they function through alterations in 3 mRNA cleavage and/or alternative polyadenylation. Consistent with this hypothesis, we find that inhibition of several other components of the mRNA cleavage complex also cause a Nio phenotype. cpf-2 and symk-1 specifically affect the osmotic stress response since heat shock-induced upregulation of a hsp-16.2::GFP reporter is normal in these mutants. Our data suggest a model in which alternative polyadenylation of one or more mRNAs is essential to regulate the hypertonic stress response.

physiology↗

bioRxiv2022

Efficient CRISPR/Cas9 mediated large insertions using long single-stranded oligonucleotide donors in C. elegans

Highly efficient generation of deletions, substitutions, and small insertions (up to [~]150 bp) into the C. elegans genome by CRISPR/Cas9 has been facilitated by use of single-stranded oligonucleotide donors as repair templates. However, efficient insertion of larger sequences such as fluorescent markers and other functional proteins remains inefficient due to lack of standardized methods for generating repair templates and labor intensive or cost prohibitive synthesis. We have optimized the simple and efficient generation of long single-stranded DNA for use as donors in CRISPR/Cas9 using a standard PCR followed by an enzymatic digest by lambda exonuclease. Comparison of long single-stranded DNA donors to previously described methods using double-stranded DNA yields orders of magnitude increased efficiency for single-stranded DNA donors. This efficiency can be leveraged to simultaneously generate multiple large insertions as well as successful edits without use of selection or co-conversion (coCRISPR) markers when necessary. Our approach complements the CRISPR/Cas9 toolkit for C. elegans to enable highly efficient insertion of longer sequences with a simple, standardized and labor-minimal protocol.

genetics↗

bioRxiv2020

Alternative polyadenylation is a determinant of oncogenic Ras function

Alternative polyadenylation of pre-mRNA has been recently shown to play important roles in development and cancer. Activating mutations in the Ras oncogene are common drivers of many human cancers but the mechanisms by which they cooperate with alternative polyadenylation are not known. By exploiting the genetics of C. elegans, we identified cfim-1/CFIm25, a subunit of the alternative polyadenylation machine, as a key determinant of hyperactive Ras function. Ablation of cfim-1 increased penetrance of multivulva phenotype in let-60/Ras gain-of-function (gf) mutant through shortening of transcripts at the 3 untranslated region, including p21 activated kinase pak-1/PAK1 and multidrug transporter mrp-5/ABCC1. Depletion of CFIm25 in human KRAS-driven cancer cells resulted in a similar shortening of 3 untranslated regions in the PAK1 and ABCC1 transcripts, which caused an epithelial-to-mesenchymal transition and increased cell migration. Exploiting the mechanisms by which alternative polyadenylation affects activated oncogene output could offer novel approaches for the treatment of Ras-driven tumors.

cancer biology↗