Abstract
Alternative polyadenylation (APA) is a widespread mechanism involving about half of the expressed genes, resulting in varying lengths of the 3′ untranslated region (3′UTR). Variations in length and sequence of the 3′UTR may underlie changes of post-transcriptional processing, localization, miRNA targeting and stability of mRNAs. During embryonic development a large array of mRNAs exhibit APA, with a prevalence of the longer 3′UTR versions in differentiating cells. Little is known about polyA+ site usage during differentiation of mammalian neural progenitors. Here we exploit a model of adherent neural stem (ANS) cells, which homogeneously and efficiently differentiate into GABAergic neurons. RNAseq data shows a global trend towards lengthening of the 3′UTRs during differentiation. Enriched expression of the longer 3′UTR variants of Pes1 and Gng2 was detected in the mouse brain in areas of cortical and subcortical neuronal differentiation, respectively, by two-probes fluorescent in situ hybridization (FISH). Among the coding genes upregulated during differentiation of ANS cells we found Elavl3, a neural-specific RNA-binding protein homologous to Drosophila Elav. In the insect, Elav regulates polyA+ site choice while interacting with paused Pol-II promoters. We tested the role of Elavl3 in ANS cells, by silencing Elavl3 and observed consistent changes in 3′UTR length and delayed neuronal differentiation. These results indicate that choice of the polyA+ site and lengthening of 3′UTRs is a possible additional mechanism of posttranscriptional RNA modification involved in neuronal differentiation.
Highlights
In neural progenitor cells, exit from cell cycle and initiation of neuronal differentiation is a complex process, whose fine regional regulation assures the timely generation of distinct neuronal and non-neuronal types composing the final functional networks
We tested the role of Elavl3 in adherent neural stem (ANS) cells, by silencing Elavl3 and observed consistent changes in 3 3 Untranslated Region (UTR) length and delayed neuronal differentiation. These results indicate that choice of the polyA+ site and lengthening of 3 untranslated region (3 UTR) is a possible additional mechanism of posttranscriptional RNA modification involved in neuronal differentiation
Changes in 3 UTR length due to alternative polyadenylation (APA) have been documented during embryonic brain development (Hilgers et al, 2012; Miura et al, 2013; Tallafuss et al, 2015), but a systematic examination of this phenomenon during neuronal differentiation, in particular in GABAergic neurons, is lacking
Summary
Exit from cell cycle and initiation of neuronal differentiation is a complex process, whose fine regional regulation assures the timely generation of distinct neuronal and non-neuronal types composing the final functional networks. Big progress has been made toward the understanding of molecular mechanisms directing and controlling cell cycle exit, commitment and early differentiation (Hardwick et al, 2015). Neuronal differentiation has been examined at the level of dynamics of transcriptome repertoire. The control of gene transcription at the promoter level certainly represents a key regulatory step, the complexity of the transcriptome is largely increased by additional layers of cotranscriptional and posttranscriptional regulations, including miRs-mediated silencing, competitive-endogenous RNA (ceRNA) networks, alternative splicing, non-sense mediated RNA decay (NMD) and alternative polyadenylation (APA). The various classes of regulatory molecules intersect via RNA:RNA and RNA:protein cross-regulations via complex, only partially known, mechanisms (Dai et al, 2015; Chen and Schuman, 2016; Hanan et al, 2017; Lara-Pezzi et al, 2017; Wanke et al, 2018)
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