Differential 3’ processing of specific transcripts expands regulatory and protein diversity across neuronal cell types
- The Rockefeller University, United States
- University of Colorado Anschutz Medical Campus, United States
Figures
Figure 1 with 2 supplements
cTag-PAPERCLIP identifies 3’UTR isoforms expressed in specific neuronal types.
(A) Schematic of the cTag-PAPERCLIP approach. Breeding of cTag-PABP mice with Cre-expressing mice restricts expression of PABPC1-GFP to the cells of interest (B) Immunostaining of cerebella from …
Figure 1—figure supplement 1
Comparison between total uniquely mapped cTag-PAPERCLIP reads per gene and TRAP-Seq Reads Per Kilobase per Million mapped reads (RPKM) per gene from granule cells.
R: Pearson correlation coefficient. Granule cell markers are highlighted in pink and non-target cell markers are highlighted in green. The data were derived from analysis of three replicates of …
Figure 1—figure supplement 2
Comparison of marker gene ranks from non-target cell types in cTag-PAPERCLIP data and TRAP-Seq data in Purkinje cells.
We used the markers of granule cells and Bergmann glia shown in green in Figure 1E. Mann-Whitney test. p-value<0.001. The highest expressed gene has a rank of 1.
Figure 2 with 2 supplements
Differences in APA between Purkinje and granule cells.
(A) Scatterplot representing the ratio between the number of cTag-PAPERCLIP reads at the end of the proximal 3’UTR isoform and the number of cTag-PAPERCLIP reads at the end of the distal 3’UTR …
Figure 2—figure supplement 1
TRAP-Seq data for genes shown in Figure 2B and D.
TRAP-Seq data on granule and Purkinje cells from adult mice (the data represents an average of four replicates per cell type) (Mellén et al., 2012).
Figure 2—figure supplement 2
qPCR validation of 3’UTR isoform abundance differences between Purkinje and granule cells.
N = 3 for each time point for qPCR. N = 4 for cTag-PAPERCLIP on granule cells and N = 3 for cTag-PAPERCLIP on Purkinje cells. Log2(fold change) represents log2 of the ratio between the ratios of …
Figure 3 with 4 supplements
Changes in APA during granule cell development.
(A) Immunostaining showing cell type specific expression of conditionally tagged PABPC1 in P0 and P21 granule cells. EGL – external granule layer, PCL – Purkinje cell layer, IGL – internal granule …
Figure 3—figure supplement 1
The number of cTag-PAPERCLIP reads per gene is correlated with RNA sequencing reads per kilobase (RPK) per gene.
Comparison between cTag-PAPERCLIP data from granule cell precursors at P0 (average of three replicates) and RNA-seq data (average of three replicates) from the same population purified by FACS. R: …
Figure 3—figure supplement 2
RNA-seq data for genes shown in Figure 3C and E.
RNA-Seq data on FACS-sorted granule cell precursors (from P0 mice) and differentiated granule cells (from P21 mice). The data represents an average of three replicates per time point.
Figure 3—figure supplement 3
qPCR validation of 3’UTR isoform abundance differences between P0 and P21 granule cells.
N = 3 for each time point for both qPCR and cTag-PAPERCLIP. Log2(fold change) represents log2 of the ratio between the ratios of distal 3’UTR isoform abundance over total mRNA abundance in P21 vs. …
Figure 3—figure supplement 4
Distribution of log2 fold changes in ribosome-associated mRNA abundance between P0 and P21 cerebellar granule cells for groups of genes exhibiting significant differences in 3’UTR isoform expression during development.
Distribution of log2 fold-changes (logFC) in ribosome-associated mRNA abundance between P0 and P21 for genes with two 3’UTR isoforms that do not show significant changes in 3’UTR isoform expression …
Figure 4
Increase in expression of distal 3’UTR isoforms during development is cell-type specific.
(A) Overlap between genes that exhibit a significant shift towards distal 3’UTR isoform expression during granule cell development (blue circle) and genes that express significantly more of the …
Figure 5 with 2 supplements
Memo1 expression is developmentally regulated by miR-124 and APA.
(A) cTag-PAPERCLIP reads on Memo1 3’UTR in P0 and P21 granule cells. The location of miR-124 target site is indicated by the green bar. The data represents and average of three cTag-PAPERCLIP …
Figure 5—figure supplement 1
Memo1 changes APA during granule cell development and regulates granule cell precursor proliferation.
(A) Additional data showing Memo1 APA change during granule cell development. The first two tracks from above represent cTag-PAPERCLIP data from P0 and P21 granule cells. The tracks below …
Figure 5—figure supplement 2
A model for Memo1 post-transcriptional regulation during development.
In the model, Memo1 acquires a longer 3’UTR during granule cell development, which subsequently leads to its downregulation due to the presence of a conserved miR-124 binding site on the long 3’UTR.
Author response image 1
Memo1 mature mRNA and pre-mRNA expression.
RT-qPCR data showing relative abundance of Memo1 mature mRNA and pre-mRNA in purified granule cells from P0 cerebella and whole P21 cerebella.N=3, Error bars: SEM. Memo1 mRNA and pre-mRNA fold …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Mus musculus) | Memo1 | NA | Entrez gene ID: 76890, Memo1 short isoform RefSeq ID: NM_133771.2 | |
| Genetic reagent (M. musculus) | Neurod1-Cre | The Jackson Laboratory | 028364 | |
| Genetic reagent (M. musculus) | Pcp2-Cre | The Jackson Laboratory | 004146 | |
| Genetic reagent (M. musculus) | Atoh1-Cre | The Jackson Laboratory | 011104 | |
| Genetic reagent (M. musculus) | Pabpc1-cTag | Hwang et al. (2017) | PMID: 28910620 | |
| Genetic reagent (M. musculus) | (Tg(Neurod1-Egfp-L10a) | Heiman et al. (2008) | PMID: 19013281 | |
| Genetic reagent (M. musculus) | (Tg(Pcp2-Egfp-L10a) | Heiman et al. (2008) | PMID: 19013281 | |
| Genetic reagent (M. musculus) | Memo1 knockout | Van Otterloo et al., 2016 | PMID: 26746790 | Memo1 ki/ki |
| Antibody | anti-GFP (for cTag-PAPERCLIP) | Heiman et al. (2008) | 19F7 and 19C8 | mouse monoclonal |
| Antibody | anti-Calb1 | Santa Cruz | sc-7691 | goat polyclonal, (1:250) |
| Antibody | anti-GFP (for IF) | Aves Labs | GFP-1020 | chicken polyclonal, (1:1000) |
| Antibody | anti-pH3 | Cell Signaling | 9701 | rabbit polyclonal, (1:100) |
| Antibody | Alexa 488, 555 and 647 secondaries | Thermo Fisher | (1:1000) | |
| Sequence-based reagent | miR-124 antagomir | Exiqon | 4102200–121 | mmu-miR-124–3 p inhibitor 3`-fluorescein labeled, (50 nM) |
| Sequence-based reagent | antagomir negative control | Exiqon | 199006–100 | Negative control A, (50 nM) |
| Sequence-based reagent | Memo1 miR-124 target blocker | Exiqon | 1999993 | Custom miRCURY LNA Power Inhibitor, (50 nM) |
| Sequence-based reagent | target blocker negative control | Exiqon | 199006–111 | Negative control A, (50 nM) |
| Commercial assay or kit | TruSeq RNA Library Prep Kit | Illumina | RS-122–2002 | For preparing RNA-seq libraries |
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All primers are listed in Supplementary file 7.
Additional files
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Source Code 1
Supplementary file 4 - source code 1.
Source code to identify genes that exhibit significantly different 3’UTR isoform expression between granule cell precursors and mature granule cells
- https://doi.org/10.7554/eLife.34042.017
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Supplementary file 1
Genes differing in 3’UTR isoform expression between Purkinje and granule cells
- https://doi.org/10.7554/eLife.34042.018
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Supplementary file 2
Genes differing in CDS length between Purkinje and granule cells
- https://doi.org/10.7554/eLife.34042.019
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Supplementary file 3
Functional gene categories enriched among genes that differ in 3’UTR isoform expression between Purkinje and granule cells
- https://doi.org/10.7554/eLife.34042.020
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Supplementary file 4
Genes differing in 3’UTR isoform expression between granule cell precursors and mature granule cells
- https://doi.org/10.7554/eLife.34042.021
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Supplementary file 5
Genes differing in CDS length between granule cell precursors and mature granule cells
- https://doi.org/10.7554/eLife.34042.022
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Supplementary file 6
Functional gene categories enriched among genes that differ in 3’UTR isoform expression between Purkinje and granule cells
- https://doi.org/10.7554/eLife.34042.023
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Supplementary file 7
List of PCR primers used in the study
- https://doi.org/10.7554/eLife.34042.024
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Supplementary file 8
Transcripts that express more of the longer 3’UTR isoform in granule cells and are downregulated compared to Purkinje cells
- https://doi.org/10.7554/eLife.34042.025
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Transparent reporting form
- https://doi.org/10.7554/eLife.34042.026
