Knockout of all nematode-specific NSPC genes expressed exclusively in excretory gland cell results in transcriptomic signatures indicating affected insulin signaling

The nematode Caenorhabditis elegans is one of the best-studied model organisms in molecular biology; however, many aspects of its physiology and the functions of many genes remain poorly understood. In this study, we investigated the role of nematode-specific NSPC proteins, recently identified as primary targets of poly(A) polymerase TENT-5. Surprisingly, we found that NSPCs are exclusively expressed in the excretory gland cell, a cell with still unclear functionality. Using an optogenetic approach, we precisely ablated the excretory gland cell and observed that nematodes exhibited no transcriptomic or physiological changes in its absence. Additionally, we generated and thoroughly studied a strain with deletion of all 18 NSPC genes, which revealed that, despite previous indications, NSPCs lack antimicrobial activity. Instead, the transcriptomic analysis showed that the absence of NSPCs strongly impacts DAF-2/DAF-16 insulin signaling, suggesting that NSPCs may function as neuropeptides influencing key C. elegans signaling pathways. Although further studies are required to elucidate the physiological effects of this regulation, our findings provide new insights into this unexplored part of nematode physiology. Overall design: We implemented Illumina RNA sequencing to investigate differences in gene expression after ablation of the excreotry gland cell or deletion of nspc genes in C. elegans (either in CRISPR knockouts or RNAi silenced worms). Moreover, we checked the potential involvement of NSPCs in TENT-5 mediated polyadenylation by using Nanopore Direct RNA Sequencing .