Developmentally regulated alternate 3′ end cleavage of nascent transcripts controls dynamic changes in protein expression in an adult stem cell lineage

Abstract

Alternative polyadenylation (APA) generates transcript isoforms that differ in the position of the 3′ cleavage site, resulting in the production of mRNA isoforms with different length 3′ UTRs. Although widespread, the role of APA in the biology of cells, tissues, and organisms has been controversial. We identified >500 Drosophila genes that express mRNA isoforms with a long 3′ UTR in proliferating spermatogonia but a short 3′ UTR in differentiating spermatocytes due to APA. We show that the stage-specific choice of the 3′ end cleavage site can be regulated by the arrangement of a canonical polyadenylation signal (PAS) near the distal cleavage site but a variant or no recognizable PAS near the proximal cleavage site. The emergence of transcripts with shorter 3′ UTRs in differentiating cells correlated with changes in expression of the encoded proteins, either from off in spermatogonia to on in spermatocytes or vice versa. Polysome gradient fractionation revealed >250 genes where the long 3′ UTR versus short 3′ UTR mRNA isoforms migrated differently, consistent with dramatic stage-specific changes in translation state. Thus, the developmentally regulated choice of an alternative site at which to make the 3′ end cut that terminates nascent transcripts can profoundly affect the suite of proteins expressed as cells advance through sequential steps in a differentiation lineage.

A Developmental Mechanism to Regulate Alternative Polyadenylation in an Adult Stem Cell Lineage

“…3'seq data revealed that loss of func7on of cbc or PCF11 affected APA at many loci. For roughly half of the >500 genes previously iden7fied by Berry et al (2022) as undergoing 3'UTR shortening by APA in spermatocytes compared to spermatogonia, KD of either PCF11 or cbc in spermatocytes resulted in increased usage of the distal cleavage site normally used in spermatogonia (Figure 4 C). 3'seq data was analyzed via PolyA miner (Yalamanchili et al 2020) providing as input the list of pairs of proximal PAS (pPAS) and distal PAS (dPAS) from the >500 genes most changed in spermatogonia versus spermatocytes called by Berry et al (2022).…”

“…Since transcripts from more than 70% of human genes are alterna7vely polyadenylated under one condi7on or another, APA is widespread (Gruber and Zavolan 2019). The most intriguing cases are those where the choice of 3' end cut site is developmentally regulated, leading, for example, to produc7on of transcripts with longer 3'UTRs from specific genes in neurons or shorter 3'UTRs in differen7a7ng male germ cells compared to precursor cells (Hilgers et al 2012;Miura et al 2013;Shan et al 2017;Vallejos Baier et al 2017;Grassi et al 2019;Berry et al 2022).…”

“…Developmentally regulated changes in the PAS site u7lized can have profound effects on the suite of proteins expressed as cells progress from one state to the next in a developmental sequence. For example, during spermatogenesis in flies and mice, many genes express mRNA isoforms with long 3'UTRs in prolifera7ng spermatogonia, but isoforms with shorter 3'UTRs at later stages of germ cell development (Li et al, 2016;Lee et al, 2022;Berry et al, 2022). In Drosophila melanogaster testes (Figure 1 B), over 500 genes express transcripts cleaved at a distal PAS in prolifera7ng spermatogonia but processed at a more proximal PAS in differen7a7ng spermatocytes, resul7ng in produc7on of mRNA isoforms with much shorter 3'UTRs (Figure 1 C).…”

“…The stage specific 3'UTR shortening due to APA observed in spermatocytes compared to spermatogonia (Figure 1 C) resulted in a drama7c shim in the proteins expressed, with transcripts from at least 200 genes switching from co-migra7ng with polysomes in spermatogonia to not associated with ribosomes in young spermatocytes. In addi7on, transcripts from another 50 genes were not translated in spermatogonia but associated with polysomes in spermatocytes (Berry et al, 2022). Thus, the developmental program(s) that specify whether nascent transcripts are cleaved at proximal 3'UTR sites in spermatocytes rather than the distal sites u7lized in spermatogonia can switch expression of many proteins from on in spermatogonia to off in spermatocytes or vice versa, with poten7al impacts on cell state.…”

Paralog-dependent Specialization of Paf1C subunit, Ctr9, for Sex Chromosome Gene Regulation and Male Germline Differentiation in Drosophila

“…Spermatogenesis follows a multistep differentiation program, involving changes in cell cycle dynamics, morphogenesis, organella organization, and chromatin remodelling, generating a highly polarized haploid gamete having condensed DNA and motile cilia [1][2][3]. A wide repertoire of genes takes a coordinated action during the complex processes, the expression of which is controlled by diverse transcriptional and posttranscriptional mechanisms [4][5][6][7][8][9][10]. Hence, spermatogenesis provides an attractive model for elucidating the principle of tissue/cell-specific gene regulation.…”