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. 2014 Jul 29;15(7):409.
doi: 10.1186/s13059-014-0409-z.

Expanded identification and characterization of mammalian circular RNAs

Junjie U Guo, Vikram Agarwal, Huili Guo, David P Bartel

Expanded identification and characterization of mammalian circular RNAs

Junjie U Guo et al. Genome Biol. .

"VSports app下载" Abstract

Background: The recent reports of two circular RNAs (circRNAs) with strong potential to act as microRNA (miRNA) sponges suggest that circRNAs might play important roles in regulating gene expression. However, the global properties of circRNAs are not well understood. VSports手机版.

Results: We developed a computational pipeline to identify circRNAs and quantify their relative abundance from RNA-seq data. Applying this pipeline to a large set of non-poly(A)-selected RNA-seq data from the ENCODE project, we annotated 7,112 human circRNAs that were estimated to comprise at least 10% of the transcripts accumulating from their loci. Most circRNAs are expressed in only a few cell types and at low abundance, but they are no more cell-type-specific than are mRNAs with similar overall expression levels. Although most circRNAs overlap protein-coding sequences, ribosome profiling provides no evidence for their translation. We also annotated 635 mouse circRNAs, and although 20% of them are orthologous to human circRNAs, the sequence conservation of these circRNA orthologs is no higher than that of their neighboring linear exons. The previously proposed miR-7 sponge, CDR1as, is one of only two circRNAs with more miRNA sites than expected by chance, with the next best miRNA-sponge candidate deriving from a gene encoding a primate-specific zinc-finger protein, ZNF91. V体育安卓版.

Conclusions: Our results provide a new framework for future investigation of this intriguing topological isoform while raising doubts regarding a biological function of most circRNAs. V体育ios版.

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Figures

Figure 1
Figure 1
Global identification of human circRNAs. (A) Schematic illustration of the alternative-splicing isoforms generated from linear splicing (left) and back splicing (right). Two-part alignments identified junction-spanning reads indicative of circRNAs (bottom left). Exons are colored, and donor (GU) and acceptor (AG) signals at splice sites are indicated. (B) The computational pipeline developed to identify and quantify circRNAs from long-read RNA-seq data. (C) Enrichment of donor GT and acceptor AG splicing signals in genomic windows flanking candidate circular junctions supported by ≥5 junction-spanning reads in the CD34 sample. Similar results were obtained from all other cell types. (D) Distribution of circular fractions for circRNA candidates in (C), grouped based on whether their circular junctions were flanked by splicing signals of the major or minor spliceosome (GT-AG- and AT-AC-flanking, respectively). (E) Distributions of exon numbers for circRNAs, mRNAs, and other annotated ncRNAs. (F) Annotations of genomic regions mapping to inferred circRNA exons. CDS, coding sequence; lincRNA, long intervening ncRNA; UTR, untranslated region. (G) Splicing within circRNAs of the CD34 sample. Mapped locations of the mates of junction-spanning reads were compared to the genomic annotations 200 nucleotides downstream and upstream of back-spliced acceptors and donors, respectively. Because the fragment size for the paired-end sequencing averaged 200 nucleotides, these genomic annotations resembled those expected if the introns within the circRNAs were retained.
Figure 2
Figure 2
Trans-splicing rarely contributed to back-spliced junctions. (A) Schematic illustration of the analysis of paired-end reads used to distinguish trans-spliced products from circRNAs. Depending on the insert size, mate reads of trans-spliced but not back-spliced junction-spanning reads could potentially map to adjacent linear exons. Based on the insert sizes of the ENCODE paired-end RNA-seq libraries, we only considered circRNAs that were <400 nucleotides. (B) Distances of all mapped mate reads from the acceptors (left) and donors (right). Two possible trans-spliced events are indicated. (C) The identified trans-spliced event from the ANKRD28 locus. (D) Circular fractions of 598 circRNAs detected in non-poly(A)-selected RNA-seq data from U2OS cells, analyzed using non-poly(A)-selected RNA-seq data (Ribo-Zero) and poly(A)-selected RNA-seq data (poly(A)+).
Figure 3
Figure 3
Expression of human circRNAs. (A) Levels of circRNAs in CD34+ hematopoietic progenitor cells. The expression level was estimated for each circRNA (using its circular fraction and the FPKM of the corresponding gene, which included both circular and linear isoforms) and the cumulative distribution of levels is plotted. For comparison, the levels of mRNAs with FPKM ≥0.1 are also plotted. (B) Fractions of mRNA-mapping reads estimated to derive from circRNAs. Reads derived from each circRNA were estimated as the product of the circular fraction, the gene FPKM and the length of the circRNA exonic sequence. The fraction was estimated for each sample, and the distribution of fractions is plotted. (C) Numbers of circRNAs identified in each biological sample. The number of circRNAs was tallied for each sample, and the distribution of values is plotted. (D) Numbers of samples in which ≥10% circular fraction was observed. The number of samples with ≥10% circular fraction was tallied for each circRNA, and the distribution of values is plotted. (E) Cumulative distribution of cell-type-specificity scores of circRNAs compared to mRNAs with similar overall expression levels (linear controls). (F) Unsupervised hierarchical clustering of the circular fractions of 1,299 circRNAs for which the availability of both the donor and the acceptor sites were each supported by ≥5 reads in all 39 samples.
Figure 4
Figure 4
Conservation between human and mouse circRNAs. (A) Analysis of enrichment in circRNAs from human orthologs of mouse genes for which circRNAs were found. Only the mouse genes that had one-to-one human orthologs were considered. (B) Extent to which mouse circRNAs align with human circRNA loci. (C) An example of conserved circRNAs, which derives from human PHF21A and mouse Phf21a loci. (D) Relationship between average circular fractions observed for circRNAs conserved in human and mouse (n = 130). Spearman’s rank correlation coefficient is shown. (E) Sequence conservation for the conserved circRNAs, compared with that of their neighboring exons. Distributions are of average mammalian phyloP scores for each of the three codon positions in circular exons and their neighboring linear exons. No significant difference was observed at any of the three positions (P > 0.1, paired Mann-Whitney test).
Figure 5
Figure 5
No evidence for translation of human circRNAs. (A) Numbers of RNA-seq and RPF reads that spanned the linear junction at the donor end, the circular junction, and the linear junction at the acceptor end of 224 circRNAs that contained RPF reads corresponding to both linear junctions in U2OS cells. (B) Circular fractions of 224 of the circRNAs of (A), calculated using either RNA-seq or RPF reads.
Figure 6
Figure 6
A search for additional circRNAs with the expected properties of miRNA sponges. (A) Frequency of AGO2-crosslinking clusters observed in circRNAs compared with that of clusters observed in their neighboring exons (left). See Figure 4E for color keys. For comparison, the analysis was repeated for a negative control, IGF2BP1 (right). No significant difference was observed between circular exons and their neighboring exons (P > 0.1, paired Mann-Whitney test). (B) Numbers of AGO2-crosslinking clusters assigned to individual miRNA families. The number of crosslinking clusters was tallied for each circRNA-miRNA pair, and the distribution of values is plotted. The outlying CDR1as-miR-7 pair is indicated. (C) Numbers of 7- and 8-nucleotide sites for individual miRNA families found within each circRNA. The number of sites was tallied for each circRNA-miRNA pair, and the distribution of values is plotted. The black curve indicates the averaged results when repeating the analysis 1,000 times using different permutations of the site sequences. The two outlying pairs are indicated. (D) Numbers of miRNA target sites in CDR1as and top-ranking ZNF circRNAs. (E) Part of the ZNF91 locus containing the circRNA. miR-23 and miR-296 seed matches are indicated.
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