%0 Journal Article %J Neuron %D 2019 %T Differential NOVA2-Mediated Splicing in Excitatory and Inhibitory Neurons Regulates Cortical Development and Cerebellar Function. %A Saito, Yuhki %A Yuan, Yuan %A Zucker-Scharff, Ilana %A Fak, John J %A Jereb, Saša %A Tajima, Yoko %A Licatalosi, Donny D %A Darnell, Robert B %X

RNA-binding proteins (RBPs) regulate genetic diversity, but the degree to which they do so in individual cell types in vivo is unknown. We developed NOVA2 cTag-crosslinking and immunoprecipitation (CLIP) to generate functional RBP-RNA maps from different neuronal populations in the mouse brain. Combining cell type datasets from Nova2-cTag and Nova2 conditional knockout mice revealed differential NOVA2 regulatory actions on alternative splicing (AS) on the same transcripts expressed in different neurons. This includes functional differences in transcripts expressed in cortical and cerebellar excitatory versus inhibitory neurons, where we find NOVA2 is required for, respectively, development of laminar structure, motor coordination, and synapse formation. We also find that NOVA2-regulated AS is coupled to NOVA2 regulation of intron retention in hundreds of transcripts, which can sequester the trans-acting splicing factor PTBP2. In summary, cTag-CLIP complements single-cell RNA sequencing (RNA-seq) studies by providing a means for understanding RNA regulation of functional cell diversity.

%B Neuron %V 101 %P 707-720.e5 %8 2019 Feb 20 %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/30638744?dopt=Abstract %R 10.1016/j.neuron.2018.12.019 %0 Journal Article %J Elife %D 2018 %T Differential 3' Processing of Specific Transcripts Expands Regulatory and Protein Diversity Across Neuronal Cell Types. %A Jereb, Saša %A Hwang, Hun-Way %A Van Otterloo, Eric %A Govek, Eve-Ellen %A Fak, John J %A Yuan, Yuan %A Hatten, Mary E %A Darnell, Robert B %X

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 , a transcript that shifted from expressing a short 3'UTR isoform to a longer one during granule cell differentiation. We show that 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.

%B Elife %V 7 %8 2018 Mar 26 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/29578408?dopt=Abstract %R 10.7554/eLife.34042 %0 Journal Article %J Neuron %D 2017 %T cTag-PAPERCLIP Reveals Alternative Polyadenylation Promotes Cell-Type Specific Protein Diversity and Shifts Araf Isoforms with Microglia Activation. %A Hwang, Hun-Way %A Saito, Yuhki %A Park, Christopher Y %A Blachère, Nathalie E %A Tajima, Yoko %A Fak, John J %A Zucker-Scharff, Ilana %A Darnell, Robert B %K Animals %K Antigens, Neoplasm %K Astrocytes %K Brain %K Cells, Cultured %K Female %K Humans %K Male %K Mice %K Microglia %K Nerve Tissue Proteins %K Neuro-Oncological Ventral Antigen %K Neurons %K Organ Specificity %K Polyadenylation %K Polypyrimidine Tract-Binding Protein %K Protein Isoforms %K Protein Serine-Threonine Kinases %K RNA-Binding Proteins %X

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.

%B Neuron %V 95 %P 1334-1349.e5 %8 2017 Sep 13 %G eng %N 6 %1 https://www.ncbi.nlm.nih.gov/pubmed/28910620?dopt=Abstract %R 10.1016/j.neuron.2017.08.024