cTag-PAPERCLIP Reveals Alternative Polyadenylation Promotes Cell-Type Specific Protein Diversity and Shifts Araf Isoforms with Microglia Activation
Open accessAbstract
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.
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The Cx3cr1-CreM line was deployed in Hwang et al. (2017), who found it to drive microglial-specific expression of a reporter.
“Notably, this Cre-line was also deployed in a recent study by Hwang et al. (2017), who found it to drive microglial-specific expression of a reporter.”
From Targeting Microglia Using Cx3cr1-Cre Lines: Revisiting the Specificity (2019) · cites this paper as Hwang et al. (2017)
The Results section (page unavailable) reports that Cx3cr1-Cre; Pabpc1cTag mice showed highly restricted PABP-GFP expression matching cell-type markers, supporting the reporter-expression finding. But the paper calls the line Cx3cr1-Cre, not Cx3cr1-CreM, and STAR Methods (page unavailable) notes occasional GFP expression in neurons; our copy therefore does not establish the exact line designation or absolute microglial specificity. (LLM, conf=0.82)
Experimental — accuracy not yet measuredHwang et al. (2017) briefly mentioned that ∼10% of their animals displayed significant leakage into neurons, but the data were not provided.
“However, the authors also briefly mentioned that ∼ 10% of their animals displayed significant leakage into neurons, but the data were not provided (Hwang et al., 2017).”
From Targeting Microglia Using Cx3cr1-Cre Lines: Revisiting the Specificity (2019) · cites this paper as Hwang et al., 2017
STAR METHODS (Experimental Model and Subject Details; page unavailable) reports occasional GFP-expression leakiness in neuronal lineages in fewer than 10% of tested Cx3cr1-Cre; Pabpc1cTag mice. This supports the central observation, but the paper does not call the leakage significant or give a figure documenting those cases; its stated rate is <10%, rather than approximately 10%. (LLM, conf=0.87)
Experimental — accuracy not yet measuredIn Hwang et al. (2017), the GFP reporter line was created by the researchers themselves.
“Moreover, in the same study, the GFP reporter line was created by the researchers themselves.”
From Targeting Microglia Using Cx3cr1-Cre Lines: Revisiting the Specificity (2019) · cites this paper as Hwang et al., 2017
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- Authors
- Hwang, H.-W., Saito, Y., Park, C. Y., Blachère, N. E., Tajima, Y., Fak, J. J., Zucker-Scharff, I., & Darnell, R. B.
- Affiliations
- Howard Hughes Medical Institute (US)
- Corresponding author
- Hwang, Hun-Way
- Darnell, Robert B.
- Venue
- Neuron
- Publisher
- Elsevier BV
- Published
- 2017-09
- Volume
- 95
- Issue
- 6
- Pages
- 1334-1349.e5
- ISSN
- 0896-6273 (ISSN-L)
- PMC
- PMC5637551
- OpenAlex
- W2755381547
- Cited by
- 74
- References
- 77
- Citations by year
- 2026: 7 · 2025: 4 · 2024: 7 · 2023: 3 · 2022: 7 · 2021: 11 · 2020: 8 · 2019: 14 · 2018: 11 · 2017: 2
- FWCI
- 4.02
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- Locations
- Neuron (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Institutes of Health (NS034389, NS081706, NS097404)
- Simons Foundation (SFARI 240432)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
- Keywords
- alternative polyadenylation, single-cell RNA sequencing, microglial activation, cell type-specific transcriptome, NOVA2, PTBP2, RNA-binding proteins, brain cell types, excitatory neurons, inhibitory neurons, astrocytes, inflammatory cytokines
- MeSH terms
- Neuro-Oncological Ventral Antigen, Animals, Antigens, Neoplasm, Astrocytes, Brain, Cells, Cultured, Female, Humans, Male, Nerve Tissue Proteins, Neurons, Organ Specificity, RNA-Binding Proteins, Protein Serine-Threonine Kinases, Microglia, Protein Isoforms, Polyadenylation, Polypyrimidine Tract-Binding Protein, Mice
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