cTag-PAPERCLIP Reveals Alternative Polyadenylation Promotes Cell-Type Specific Protein Diversity and Shifts Araf Isoforms with Microglia Activation

Neuron. 2017 Sep 13;95(6):1334-1349.e5. doi: 10.1016/j.neuron.2017.08.024.

Abstract

Alternative polyadenylation (APA) is increasingly recognized to regulate gene expression across different cell types, but obtaining APA maps from individual cell types typically requires prior purification, a stressful procedure that can itself alter cellular states. Here, we describe a new platform, cTag-PAPERCLIP, that generates APA profiles from single cell populations in intact tissues; cTag-PAPERCLIP requires no tissue dissociation and preserves transcripts in native states. Applying cTag-PAPERCLIP to profile four major cell types in the mouse brain revealed common APA preferences between excitatory and inhibitory neurons distinct from astrocytes and microglia, regulated in part by neuron-specific RNA-binding proteins NOVA2 and PTBP2. We further identified a role of APA in switching Araf protein isoforms during microglia activation, impacting production of downstream inflammatory cytokines. Our results demonstrate the broad applicability of cTag-PAPERCLIP and a previously undiscovered role of APA in contributing to protein diversity between different cell types and cellular states within the brain.

Keywords: ARAF; NOVA; PTB; alternative polyadenylation; crosslinking immunoprecipitation; microglia; neuron; single cell type.

MeSH terms

  • Animals
  • Antigens, Neoplasm / physiology
  • Astrocytes / metabolism
  • Brain / cytology*
  • Brain / metabolism
  • Cells, Cultured
  • Female
  • Humans
  • Male
  • Mice
  • Microglia / cytology
  • Microglia / metabolism*
  • Nerve Tissue Proteins / physiology
  • Neurons / metabolism*
  • Organ Specificity
  • Polyadenylation*
  • Polypyrimidine Tract-Binding Protein / physiology
  • Protein Isoforms / metabolism
  • Protein-Serine-Threonine Kinases / metabolism*
  • RNA-Binding Proteins / physiology

Substances

  • Antigens, Neoplasm
  • Nerve Tissue Proteins
  • Nova2 protein, mouse
  • Protein Isoforms
  • Ptbp2 protein, mouse
  • RNA-Binding Proteins
  • Polypyrimidine Tract-Binding Protein
  • Protein-Serine-Threonine Kinases