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. 2017 Jun;206(2):757-774.
doi: 10.1534/genetics.116.196774. Epub 2017 Mar 27.

Alternative Polyadenylation Directs Tissue-Specific miRNA Targeting in Caenorhabditis elegans Somatic Tissues

Stephen M Blazie  1   2 , Heather C Geissel  1   2 , Henry Wilky  3 , Rajan Joshi  4 , Jason Newbern  1   3 , Marco Mangone  5   2   3
Affiliations

Alternative Polyadenylation Directs Tissue-Specific miRNA Targeting in Caenorhabditis elegans Somatic Tissues

Stephen M Blazie et al. Genetics. 2017 Jun.

Abstract

mRNA expression dynamics promote and maintain the identity of somatic tissues in living organisms; however, their impact in post-transcriptional gene regulation in these processes is not fully understood. Here, we applied the PAT-Seq approach to systematically isolate, sequence, and map tissue-specific mRNA from five highly studied Caenorhabditis elegans somatic tissues: GABAergic and NMDA neurons, arcade and intestinal valve cells, seam cells, and hypodermal tissues, and studied their mRNA expression dynamics. The integration of these datasets with previously profiled transcriptomes of intestine, pharynx, and body muscle tissues, precisely assigns tissue-specific expression dynamics for 60% of all annotated C. elegans protein-coding genes, providing an important resource for the scientific community. The mapping of 15,956 unique high-quality tissue-specific polyA sites in all eight somatic tissues reveals extensive tissue-specific 3'untranslated region (3'UTR) isoform switching through alternative polyadenylation (APA) . Almost all ubiquitously transcribed genes use APA and harbor miRNA targets in their 3'UTRs, which are commonly lost in a tissue-specific manner, suggesting widespread usage of post-transcriptional gene regulation modulated through APA to fine tune tissue-specific protein expression. Within this pool, the human disease gene C. elegans orthologs rack-1 and tct-1 use APA to switch to shorter 3'UTR isoforms in order to evade miRNA regulation in the body muscle tissue, resulting in increased protein expression needed for proper body muscle function. Our results highlight a major positive regulatory role for APA, allowing genes to counteract miRNA regulation on a tissue-specific basis.

Keywords: C. elegans; alternative polyadenylation; miRNA; transcriptome.

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Figures

Figure 1
Figure 1
PAT-Seq to study the transcriptomes of AIV cells, NMDA-type neurons, GABAergic neurons, seam cells, and hypodermis. (A) Diagram with the anatomical location of tissues we have profiled in this study. Red labels mark tissues that our group has previously profiled (Blazie et al. 2015), and that have been remapped in this study and used in the analysis. (B) Overview of the PAT-Seq approach. The tissue-specific expression of the Poly(A)-Pull cassette containing GFP fused to 3×FLAG-tagged pab-1 is achieved through usage of selected tissue-specific promoters. Transgenic C. elegans lines that express this construct are then prepared and grown in liquid culture, followed by crosslinking, lysis and immunoprecipitation of 3×FLAG-tagged PAB-1 complexes. The tissue-specific mRNA extracted from the IP is then used to prepare cDNA libraries for next generation sequencing and transcriptome mapping, which is stored in the UTRome.org database (Mangone et al. 2008). (C) Examples of fluorescent images of transgenic lines expressing the Poly(A)-Pull cassette in the tissue profiled in this study. Yellow arrows mark small cells expressing the construct.
Figure 2
Figure 2
Distribution of gene expression among eight somatic tissues profiled with PAT-Seq. (A) Venn diagram displaying the portions of genes expressed between four tissue groups we have profiled in this study and the germline transcriptome from Ortiz et al. (2014). Tissues from our study were grouped by muscle (pharynx and body muscle), neuronal (GABAergic and NMDA-type neurons), epithelial (intestine and AIV cells), and epidermal (hypodermis and seam cells) groups. (B) Venn diagram of the epithelial-unique (Top left), epidermal-unique (Top right), neuronal-unique (Bottom left), and muscle-unique genes (Bottom right) identified by PAT-Seq. Epithelial tissue (AIV cells and intestine) and the epidermal tissue (seam cells and hypodermis) transcriptomes were highly similar (**P < 0.01, hypergeometric distribution).
Figure 3
Figure 3
PolyA Cluster mapping. (A) We have bioinformatically assembled ∼25,000 high-quality polyA clusters distributed in eight somatic C. elegans tissues from our RNA-seq data. These clusters map the 3′ends of protein coding genes. Genes in these tissues use APA at a ∼31% rate; >83% of these mapped polyA clusters map 3′UTR isoforms previously detected by Mangone et al. (2010) and Jan et al. (2011) (B) Tissue-specific 3′UTR isoform preferences in 91 protein coding genes detected with only two 3′UTR isoforms in this study. Most of the genes in this analysis use tissue-specific APA. Instances where a 3′UTR was not mapped in a tissue are indicated in gray (n/a). (C) Example of polyA clusters prepared for the gene rpl-12. The distal 3′UTR isoform is absent in both muscle tissues (dotted red box).
Figure 4
Figure 4
Commonly transcribed genes are enriched in APA and miRNA targets. (A) Histogram comparing the length distribution of 3′UTRs of tissue-restricted (red) and commonly transcribed genes (blue) with the C. elegans 3′UTRome (dotted black). Commonly transcribed genes have longer 3′UTRs, on average. (B) Portion of commonly transcribed or tissue-restricted genes with at least one PicTar or mirANDA predicted miRNA target in their 3′UTRs. Most commonly transcribed genes have at least one predicted miRNA target. (C) Pie charts displaying the proportion of tissue-restricted genes (left chart) or commonly transcribed genes (right chart) with >1 3′UTR isoform (APA). Nearly all commonly transcribed genes are prepared with at least two 3′UTR isoforms.
Figure 5
Figure 5
Tissue-specific APA events allow rack-1 and tct-1 to escape miRNA mediated gene repression in body muscle. (A) Illustration (not to scale) of all 3′UTR isoforms identified in our study for tct-1 (orange) and rack-1 (blue) with the location of PicTar predicted miRNAs targets. We detected their short 3′UTR isoforms in the body muscle (bm) and the long isoforms exclusively in the intestine (int). (B) pAPAreg: A dual-color reporting system to study post-transcriptional gene regulation in vivo. The system uses the Gateway multisite technology. When expressed in C. elegans using tissue-specific promoters, the operon cassette (SE) is cleaved, and both fluorochromes are expressed in the same molar ratio in a given tissue. After the trans-splicing of the spliceable element (SE), the mCherry fluorochrome is translated independently of GFP-PEST, which is instead subject to post-transcriptional repression via miRNAs that target (purple asterisk) in the 3′UTR placed downstream of it. Deletion of PAS1 allows expression of the long 3′UTR isoform containing the miRNA target in the body muscle where it is not normally expressed. (C) rack-1 escapes miR-50 and miR-85 regulation in the body muscle through APA. Left: Representative mCherry and GFP fluorescent images of transgenic lines expressing pAPAreg in the body muscle using the myo-3 promoter and the rack-1 3′UTR with (i) wild-type sequence (WT), (ii) deleted PAS1 (ΔPAS1), (iii) deleted PAS1 and miR-50 target (ΔPAS1;ΔmiR-50), or (iv) deleted PAS1 and miR-85 target (ΔPAS1;ΔmiR-85). Right: Quantification of GFP fluorescence intensity relative to mCherry for each rack-1 transgenic C. elegans line pictured in left panel using Image-J software (n = 34, ****P < 0.0001, paired T-test). (D) tct-1 escapes miR-50 regulation in the body muscle through APA. Left: Representative mCherry and GFP fluorescent images of transgenic C. elegans lines expressing pAPAreg in the body muscle using the myo-3 promoter and the tct-1 3′UTR with (i) wild-type sequence (wt), (ii) deleted PAS1 (ΔPAS1), or (iii) deleted PAS1 and miR-50 target (ΔPAS1;ΔmiR-50). Right: Quantification of GFP fluorescence intensity relative to mCherry for each tct-1 transgenic C. elegans line pictured in left panel using Image-J software (n = 27, ****P < 0.0001, paired T-test). (E) RNAi mediated knockdown of rack-1 and tct-1 results in partial embryonic lethality, (n = 10). C. elegans animals fed rack-1 or tct-1 RNAi exhibit uncoordinated locomotion. Shown are the results from larval C. elegans animals that bypassed embryonic lethality (n = 10). (F) Model depicting the manner in which APA allows rack-1 and tct-1 to escape regulation by the miR-50 miRNA in a tissue-specific manner.
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