Collawn, J. | Search bioRxiv

bioRxiv2023

IRE1-mediated degradation of pre-miR-301a promotes apoptosis through upregulation of GADD45A

The unfolded protein response is a survival signaling pathway that is induced during various types of ER stress. Here we focus on the IRE1 pathway to determine IRE1s role in miRNA regulation during ER stress. During induction of ER stress in human bronchial epithelial cells, we utilized next generation sequencing to demonstrate that pre-miR-301a and pre-miR-106b, were significantly increased in the presence of an IRE1 inhibitor. Conversely, using nuclear-cytosolic fractionation on ER stressed cells, we found that these three pre-miRNAs were decreased in the nuclear fractions without the IRE1 inhibitor. We also found that miR-301a-3p targets the proapoptotic UPR factor, growth arrest and DNA-damage-inducible alpha (GADD45A). Inhibiting miR-301a-3p levels or blocking its predicted miRNA binding site in GADD45As 3 UTR with a target protector increased GADD45A mRNA expression. An elevation of XBP1s expression had no effect on GADD45A mRNA expression. We also demonstrated that the introduction of a target protector for the miR-301a-3p binding site in GADD45A mRNA during ER stress promoted cell death in the airway epithelial cells. These results indicated that IRE1s endonuclease activity is a two-edged sword that splices XBP1 mRNA for survival and degrades pre-miR-301a to elevate the mRNA expression of a pro-apoptotic gene, GADD45A.

cell biology↗

bioRxiv2021

An RNAi screen in C. elegans for genes that play a role in secretion and cleavage of VAPB MSP domain.

VAPB (VPR-1 in C. elegans) is a type-II ER transmembrane protein whose N-terminal Major Sperm Protein domain (MSPd) is cleaved and secreted. Mutations in the MSPd of human VAPB cause impaired secretion and are associated with Amyotrophic Lateral Sclerosis (ALS). In C. elegans, the secreted MSPd signals non-cell-autonomously to regulate striated muscle mitochondrial morphology and gonad development. As VAPB/VPR-1 does not have a signal peptide and its MSPd extends into the cytosol, it is unclear how the protein is proteolytically cleaved and secreted. To identify genes that are involved in VPR-1 cleavage and unconventional secretion, we performed an RNA interference (RNAi) screen in C. elegans. Worms null for vpr-1 are sterile and have striated muscle mitochondrial abnormalities. These defects can be rescued by vpr-1 expression in the neurons, germline, or intestine, implying that these three tissues share a common machinery to cleave and secrete the MSPd. Examination of shared gene expression in these tissues revealed a list of 422 genes, which we targeted with RNAi. vpr-1 null worms expressing vpr-1 from intestine were used in the screen, and the brood size of these worms after RNAi knockdown was scored. Disruption of factors involved in VPR-1 MSPd processing and/or secretion should revert fertility phenotypes in these worms. We identified many genes that induce compromised fertility when knocked down in these but not wild type worms, including a V-SNARE, several proteasome components, stress response molecules, and mitochondrial genes. Our screen thus identified many potential players involved in MSPd processing and/or secretion. SummaryThe MSP domain (MSPd) of a type-II ER transmembrane protein called VAPB is cleaved and secreted to function as a non-cell-autonomous signal. The topology of VAPB positions MSPd in the cytosol. It is thus unclear how MSPd is cleaved from VAPB and released extracellularly. Using C. elegans, we screened 422 genes by RNAi to identify potential candidates regulating MSPd cleaving and secretion. We identified the Golgi v-SNARE YKT-6 and several components of the 20S and 19S proteasome that may mediate MSPd trafficking and cleaving, respectively. These results have promising implications in advancing our understanding of MSPd signaling.

cell biology↗