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

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.

The Future of Cross-Linking and Immunoprecipitation (CLIP)

“…cTag-PAPERCLIP data obtained from cTag-PABP mouse models are very consistent with data generated in wild-type mouse through a variety of methods in multiple occasions (Hwang et al, 2017; Jereb et al, 2018; Saito et al, 2018). Therefore, it is evident that cTag-CLIP approach in general does not interfere with normal RBP biology and hence provides a powerful means to dissecting cell-type specific biology.…”

“…The latent GFP-tagged allele is “off” by default, leaving the native allele unaffected, and only turns “on” in the presence of Cre recombinase (Figure 2A) (Hwang et al, 2017). Importantly, the GFP-tagged allele is expressed by homologous recombination into the endogenous locus, such that the quantitative, qualitative (alternate isoforms), spatial and temporal expression of GFP-tagged RBP is minimally disruptive to the biology of the system, and can be precisely controlled through Cre expression to achieve cell-type specificity.…”

“…First, since cTag mice are created using a “knock-in” approach, the proper cellular expression of RBPs and the RNA-RBP stoichiometry is likely to be maintained, both of which are difficult to achieve with either traditional or even bacterial artificial chromosome (BAC) transgenes (Gerfen et al, 2013; He et al, 2012) (Figure 2A). Second, the utilization of a mix of high affinity GFP antibodies (Heiman et al, 2014) allows cTag-CLIP analysis to be performed with a standardized and optimized high stringency protocol for different RBPs (Hwang et al, 2017). The standardization eliminates the need for RBP-specific antibodies and the subsequent experimental optimization to achieve high stringency immunoprecipitation for individual RBPs.…”

“…We validated this approach using conditionally tagged PABPC1, the poly(A)-binding protein (PABP), to generate the cTag-PABP mouse and to establish the cTag-PAPERCLIP technique (Hwang et al, 2017). cTag-PAPERCLIP utilizes PABP as a biological filter to specifically clone distal mRNA 3′ ends to generate comprehensive mRNA alternative polyadenylation (APA) maps.…”

Resolving CNS mRNA Heterogeneity: Examining mRNA Alternative Polyadenylation at a Cell-Type-Specific Level

“…However, even though the PAPERCLIP methodology is ideal for high-performance identification of mRNA 3'ends, and thus APA, the near-universal expression of PABP limits the resolution of PAPERCLIP data in tissues constituted of many heterogeneous cell types, such as in the CNS. In this issue of Neuron, Hwang and colleagues report a new version of the PAPERCLIP methodology that can be used in a cell type-specific manner (Hwang et al, 2017). For this purpose, they generated a knock-in mouse line in which PABP is tagged with GFP in a conditional Cre-dependent fashion.…”