Differential 3' Processing of Specific Transcripts Expands Regulatory and Protein Diversity Across Neuronal Cell Types.

Saša Jereb,Yuan Yuan,Robert B Darnell,Eric Van Otterloo,John J Fak,Mary E Hatten,Eve-Ellen Govek,Hun-Way Hwang

doi:10.7554/elife.34042

Abstract

Alternative polyadenylation (APA) regulates mRNA translation, stability, and protein localization. However, it is unclear to what extent APA regulates these processes uniquely in specific cell types. Using a new technique, cTag-PAPERCLIP, we discovered significant differences in APA between the principal types of mouse cerebellar neurons, the Purkinje and granule cells, as well as between proliferating and differentiated granule cells. Transcripts that differed in APA in these comparisons were enriched in key neuronal functions and many differed in coding sequence in addition to 3'UTR length. We characterize Memo1, a transcript that shifted from expressing a short 3'UTR isoform to a longer one during granule cell differentiation. We show that Memo1 regulates granule cell precursor proliferation and that its long 3'UTR isoform is targeted by miR-124, contributing to its downregulation during development. Our findings provide insight into roles for APA in specific cell types and establish a platform for further functional studies.

Highlights

Alternative polyadenylation (APA) is a process by which different ends to an mRNA transcript are determined
We recently developed cTag-PAPERCLIP (conditionally-tagged poly (A) binding protein-mediated mRNA 3’ end retrieval by crosslinking immunoprecipitation). cTagPAPERCLIP – which is based on PAPERCLIP (Hwang et al, 2016) and CLIP (Licatalosi et al, 2008; Ule et al, 2003) – enables purification and sequencing of 30 untranslated region (3’UTR) ends of polyadenylated transcripts via their interaction with poly-A binding protein cytoplasmic 1 (PABPC1), a protein that binds with high specificity to mRNA poly(A) tails
Examples of genes that showed greater expression of the long 3’UTR isoform in differentiated granule cells compared to proliferating granule cell precursors and Purkinje cells include (Figure 4B) Mbd2, which codes for methyl CpG-binding domain protein two and has been shown to regulate the proliferation of olfactory receptor neuron precursors (Macdonald et al, 2010), Tmem57, which codes for macoilin, and has been shown to regulate neuronal excitability (ArellanoCarbajal et al, 2011), and Memo1, which we describe in more detail below

Summary

Introduction

Alternative polyadenylation (APA) is a process by which different ends to an mRNA transcript are determined. New approaches, like translating ribosome affinity purification (TRAP), have been developed that enable sequencing of mRNA from specific neurons in a cell type-specific manner (Mellen et al, 2012; Sanz et al, 2013), but they lack the resolution to precisely identify 3’UTR ends. Purifying 3’UTR ends via PABPC1 immuno-precipitation exhibited less internal priming to A-rich regions other than poly-A tails compared to 3’UTR end sequencing techniques based exclusively on oligo-dT priming (Hwang et al, 2016) Another major strength of the CLIP approach is that by covalently crosslinking RNA to protein via ultraviolet light, this method captures direct RNA-protein interactions in situ, allowing stringent immunopurification of physiological interactions from non-specific interactions, which is especially important when purifying mRNA from rare cell populations. Our findings demonstrate the potential of single cell-type resolution of APA regulation to reveal quantitative as well as qualitative control of functional diversity in different neuronal populations

Results

Discussion

Materials and methods

Funding Funder National Institutes of Health