An in vivo method to study post‐transcriptional regulation in germ stem cells

Abstract

Stem cells in regenerative tissues must balance factors promoting self‐renewal with those driving differentiation. Faulty regulation of this balance can lead to tumor growth or loss of regenerative ability. The tractable germline of the nematode C. elegans has allowed the identification of a wealth of key stem cell regulatory factors and remarkably, virtually all are RNA binding proteins (RBP). Despite this insight, a mechanistic understanding of how these RBPs regulate their target mRNAs is lacking. Here, we develop an in vivo method to investigate their mechanisms. Our assay probes how a given RBP affects expression of a reporter mRNA when recruited to its 3′ untranslated region (3′UTR). To perform the assay, an RBP of interest is genetically engineered to contain a small λN peptide. The λN peptide binds a specific RNA hairpin, called boxB, with high affinity. Thus, a reporter mRNA engineered to contain boxB hairpins in its 3′UTR will bind the λN tagged RBP and either increase or decrease expression, depending on the RBP regulatory function. By imaging reporter expression in germline stem cells, we can gain insight into how the RBP regulates target transcripts in its normal biological context. The simple and rapid readout of the assay allows it to be used to probe how an RBP accomplishes its RNA regulation or screen for other factors necessary for RBP function. We have applied this assay to several of the key stem cell RBP regulators to test their functions in vivo. Our results demonstrate the tractability and ease of this method to study RBPs in the C. elegans germline as well as providing mechanistic insights into how the RBPs regulate stem cell fate decisions.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.