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, 19 (1), 8

Global Accumulation of circRNAs During Aging in Caenorhabditis Elegans


Global Accumulation of circRNAs During Aging in Caenorhabditis Elegans

Mariela Cortés-López et al. BMC Genomics.


Background: Circular RNAs (CircRNAs) are a newly appreciated class of RNAs that lack free 5' and 3' ends, are expressed by the thousands in diverse forms of life, and are mostly of enigmatic function. Ostensibly due to their resistance to exonucleases, circRNAs are known to be exceptionally stable. Previous work in Drosophila and mice have shown that circRNAs increase during aging in neural tissues.

Results: Here, we examined the global profile of circRNAs in C. elegans during aging by performing ribo-depleted total RNA-seq from the fourth larval stage (L4) through 10-day old adults. Using stringent bioinformatic criteria and experimental validation, we annotated a high-confidence set of 1166 circRNAs, including 575 newly discovered circRNAs. These circRNAs were derived from 797 genes with diverse functions, including genes involved in the determination of lifespan. A massive accumulation of circRNAs during aging was uncovered. Many hundreds of circRNAs were significantly increased among the aging time-points and increases of select circRNAs by over 40-fold during aging were quantified by RT-qPCR. The expression of 459 circRNAs was determined to be distinct from the expression of linear RNAs from the same host genes, demonstrating host gene independence of circRNA age-accumulation.

Conclusions: We attribute the global scale of circRNA age-accumulation to the high composition of post-mitotic cells in adult C. elegans, coupled with the high resistance of circRNAs to decay. These findings suggest that the exceptional stability of circRNAs might explain age-accumulation trends observed from neural tissues of other organisms, which also have a high composition of post-mitotic cells. Given the suitability of C. elegans for aging research, it is now poised as an excellent model system to determine whether there are functional consequences of circRNA accumulation during aging.

Keywords: Age-accumulation; Aging; C. elegans; Gene expression; RNA-seq; Splicing; circRNA.

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Fig. 1
Fig. 1
Genomic features of C. elegans circRNAs. a Schematic showing a circRNA generated by backsplicing of exons, and the mapping of reads to the back-spliced junction. b Distribution of circRNAs in the C. elegans genome. Data was mapped from 12 total RNA-seq libraries of N2 worms, including L4 larvae (L4), Day 1 (D-1), Day 7 (D-7), and Day 10 (D-10). CDS, protein coding sequence. c Forward and reverse splicing patterns for the haf-4 gene. Linear spliced read count (green) and back-splicing read count (brown) are shown. Numbers correspond to the number of spliced reads detected in the D-10 datasets. Only reads corresponding to the junctions included in circRNAs are shown. The gene haf-4 generates a single circRNA that extends across 8 exons. d afd-1 generates 8 circRNAs. e Bar plot showing the number of expressed circRNAs per gene. f Number of exons contained within exonic circRNAs. g Ranked position of circRNA first exon for circRNAs containing more than 1 exon. h Presence of Reverse Complementary Matches (RCM) in introns flanking circRNA exons is greater than non-circRNA generating exon controls. Number above bars correspond to # of loci. *, P < 0.0001 on Kruskal-Wallis test with Dunn’s post-hoc test for multiple comparisons
Fig. 2
Fig. 2
Experimental validation of circRNAs. a RT-PCR strategy to detect circRNAs exclusively using outward facing primer sets. Sanger sequencing of PCR products confirmed 10/10 circRNAs tested (Additional file 4: Table S2). b RT-qPCR experiments on RNase R treated mixed age adult worms. Equal amounts of mock-treated and RNase R treated RNA were used for cDNA preparation prior to qPCR. Note the enrichment of circRNAs with RNase R treatment, whereas linear cdc-42 mRNA is not enriched. c RNA-seq track visualized using Integrated Genomics Viewer from D-7 worms showing read pileup at the crh-1 gene. Note the increased read number overlapping the circularized exon. cel_circ_0000438 and cel_circ_0000439 differ by 6 nucleotides in length at the 5′ end of the exon. d Northern blot using a probe overlapping the circularized exon of crh-1 (see panel c) detects bands corresponding to circRNA and mRNA from mixed age adult RNA. Relative circRNA to mRNA abundance is enriched in RNase R treated and polyA- samples compared to polyA+ samples. e Northern blot performed using a probe that detects afd-1 circRNA and mRNA. Red arrows denote circRNA bands. Black arrows denote likely linear RNA bands
Fig. 3
Fig. 3
Global circRNA accumulation during aging. a Principal component analysis of circRNA Transcripts Per Million reads (TPM) shows clear clustering of young (L4, D-1) versus old ages (D-7, D-10). b Plot of circRNA TPM fold changes in aging time-point pairwise comparisons. Red line represents 1.5-fold increase and blue line represents 1.5-fold decrease. c CircRNA TPM compared among the four aging time-points: L4 larvae (L4), Day 1 (D-1), Day 7 (D-7) and Day 10 (D-10). P values reflect non-parametrical Kruskal-Wallis with Nemenyi post-hoc test for multiple comparisons. d Pairwise comparisons of age-increased and decreased circRNAs among the aging time-points (>1.5 FC, P < 0.05, FDR < 0.2). e Histogram showing number of circRNAs specifically expressed at a single time-point (6 or more reads among 3 biological replicates)
Fig. 4
Fig. 4
Validation of circRNA age-accumulation. a RNA-seq quantification of select circRNAs during aging (TPM fold-change with L4 set at 1). Total number of reads across all libraries for each circRNA is noted above graph. Labels display circRNA names with host gene in brackets. b RT-qPCR data for the same selected circRNAs as in A). Data is normalized to cdc-42 mRNA. Note the greater magnitude of age-related expression changes reported by RT-qPCR versus RNA-seq for all circRNAs. Note that for cel_circ_0001331, there was a significant reduction (P < 0.05) between D-1 and D-10 (Additional file 5: Table S3). Error bars represent SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001, two-tailed t-test compared to L4
Fig. 5
Fig. 5
Comparison of circRNA and linear RNA expression during aging. a Scatterplot showing pairwise comparisons between aging time-points for linear RNA levels (a-d) and circRNAs (e-h). Log2 linear RNA Fragments Per Kilobase per Million reads (FPKM) value scatterplots are shown for (a) D-7 vs L4, (b) D-10 vs L4, (c) D-7 vs D-1, and (d) D-10 vs D-1. Log2 circRNA TPM scatterplots are shown for (e) D-7 vs L4, (f) D-10 vs L4, (g) D-7 vs D-1, and (h) D-10 vs D-1. For circRNA TPM analysis, significant changes have a fold-change >1.5, P < 0.05, FDR < 0.2. Significant changes for linear RNA were computed using CuffDiff (Fold change >1.5, P < 0.05; see Methods). Red data points show age-upregulated transcripts, whereas blue data points show age-downregulated transcripts
Fig. 6
Fig. 6
Age-accumulation of circRNAs is independent of host gene expression. Density plots for CircTest-derived circRNA read counts fold-change versus linear read count RNA fold-change. Log2 fold-changes of circRNAs versus log2 fold-changes of linear RNAs from host genes are shown. a D-1 vs L4, (b) D-7 vs L4, (c) D-10 vs L4, (d) D-7 vs L4, and (e) D-10 vs D-1, (f) D-10 vs D-7. Scale bar inset in panel A represents circRNA number and applies to all the density plots. For old versus young time-point comparisons, it is evident that upregulation of circRNAs is largely independent of linear RNA expression from the same gene (upward shift in plots). Pearson correlation values are shown in the upper right corner, indicating weak correlation between the circular and linear ratios in all comparisons. Plots include circRNAs with a minimum of 6 reads for each time-point under comparison. g Pairwise comparisons of CircTest-derived counts showing significant host gene independent changes (Significant expression changes: P < 0.05, FDR < 0.2)

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