Lariat Intronic Rnas in the Cytoplasm of Vertebrate Cells
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Lariat intronic RNAs in the cytoplasm of vertebrate cells Gaëlle J. S. Talhouarnea,b,1 and Joseph G. Galla,2 aDepartment of Embryology, Carnegie Institution for Science, Baltimore, MD 21218; and bDepartment of Biology, Johns Hopkins University, Baltimore, MD 21218 Contributed by Joseph G. Gall, July 5, 2018 (sent for review May 22, 2018; reviewed by Lynne E. Maquat and Sandra L. Wolin) Most intronic RNAs are degraded within seconds or minutes after RBC maturation (5), some RNAs persist (6), including circular their excision from newly formed transcripts. However, stable intronic RNA or circRNA (7, 8). Intronic RNA has not yet been analyzed sequence RNAs (sisRNAs) have been described from oocytes of the in these cells. We purified RBCs from mouse blood by frac- frog Xenopus, from Drosophila embryos, and from human cell lines. In tionation on a Percoll gradient (9) and subjected the small Xenopus oocytes, sisRNAs are abundant in both the nucleus and cy- amount of recovered RNA to high-throughput sequencing. The toplasm, they occur in the form of lariats, and they are stable for days. 77 million single-end 100-bp reads that we obtained were ana- In this study we demonstrate that cytoplasmic sisRNAs are also found lyzed using a method developed by Taggart et al. (10). Of par- in human, mouse, chicken, and zebrafish cells. They exist as circular ticular interest, this method allows one to recover inverted reads (lariat) molecules, mostly 100–500 nucleotides in length, and are de- that would otherwise be thrown out by standard pipelines. We rived from many housekeeping genes. They tend to have an unusual searched for and found 524 mouse introns that were mapped by cytosine branchpoint (with the exception of those from the frog). conventional reads and also by inverted reads that contained Stable lariats are exported from the nucleus to the cytoplasm by sequences from both ends of the intron (Fig. 1A and Dataset S1). the NXF1/NXT1 system, demonstrating that their presence in the cy- Because we were looking for circular sisRNA, we disregarded toplasm is not due to passive diffusion. Lariats in the cytoplasm are not any intron with conventional reads but no inverted reads. We associated with transcripts of the genes from which they are derived. confirmed the circularity of the intronic RNA by RT-PCR and The biological significance of cytoplasmic sisRNAs remains obscure. Northern blotting of selected RNAs (Fig. 1 B and C). Two classes of circular sisRNA were detected. The most abundant circular RNA | intron | cytoplasm | vertebrate sisRNAs presumably arise from failure of the excised intronic RNA to be linearized by the debranching enzyme DBR1. An un- ntronic RNAs represent a large fraction of the unstable tran- usual feature of these stable lariats was the presence of a cytosine at Iscriptome of higher eukaryotes. These RNAs are spliced from the branchpoint rather than the more common adenine (Fig. 1D). pre-mRNA as branched circular RNAs (lariats). Within minutes Approximately 80% of the stable lariats contained cytosine at the of splicing, a dedicated debranching enzyme (DBR1) recognizes branchpoint compared with the 10% expected rate (10). On the the branchpoint and linearizes the lariat, promoting its rapid other hand, a fraction of the circular sisRNAs did not resemble degradation (1). This pathway is also necessary for processing conventional spliced lariats. Specifically, the junctions of these noncoding RNAs such as small nucleolar RNAs (2). An earlier study from our laboratory revealed the existence of unusually Significance abundant intronic RNAs in the nuclei of Xenopus oocytes (3). Many of these intronic RNAs persist for as long as 2 d and were Introns are noncoding DNA sequences interspersed among the given the name stable intronic sequence RNA, or sisRNA. In a coding sequences of genes. Shortly after transcription, the later study we found thousands of different sisRNAs in the cy- intronic sequences are spliced out of the primary RNA tran- toplasm of the Xenopus oocyte (4). We were certain of the cy- script as lariat RNAs (circular molecules with a short tail). Most toplasmic localization, because the gigantic nucleus can be of these lariats are destroyed within minutes in the cell nu- removed manually from the oocyte before the cytoplasmic RNAs cleus. We report here that many such intronic RNAs are, in fact, are isolated and sequenced. The cytoplasmic sisRNAs are stored exported to the cytoplasm, where they remain as stable cir- as circular molecules (lariats without tails). cular molecules. These cytoplasmic introns are derived from Until now, however, it has remained unknown whether cyto- hundreds of different genes of widely different functions. We plasmic circular sisRNAs are unique to frog oocytes or are more find them in cells of human, mouse, chicken, frog, and zebra- widely distributed. A major technical problem in searching for fish. The widespread occurrence of so many stable lariat RNAs cytoplasmic sisRNAs is the difficulty of obtaining cytoplasmic in the cytoplasm suggests that they play some as-yet un- fractions completely free of nuclear contamination. For this expected role in cell metabolism. reason, we began our search for cytoplasmic sisRNAs in other species by examining mammalian red blood cells (RBCs). Author contributions: G.J.S.T. and J.G.G. designed research; G.J.S.T. and J.G.G. performed research; G.J.S.T. contributed new reagents/analytic tools; G.J.S.T. and J.G.G. analyzed Mammalian RBCs lose their nuclei during their maturation from data; and G.J.S.T. and J.G.G. wrote the paper. precursor erythroblasts and so provide a convenient source of Reviewers: L.E.M., University of Rochester; and S.L.W., National Cancer Insitute. pure cytoplasm. We found numerous circular intronic sequences The authors declare no conflict of interest. in RBCs, thereby demonstrating the existence of sisRNA in This open access article is distributed under Creative Commons Attribution-NonCommercial- mammalian somatic cells. We also found circular sisRNAs in NoDerivatives License 4.0 (CC BY-NC-ND). cultured cells and in tissues from the mouse, human, chicken, Data deposition: RNA sequencing data have been deposited in the NCBI Sequence Read and zebrafish. We discuss functions that cytoplasmic circular Archive (SRA BioProject ID PRJNA479418). sisRNAs may perform independently of their host genes. 1Present address: Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520. Results 2To whom correspondence should be addressed. Email: [email protected]. Circular SisRNAs in RBCs. Mammalian RBCs extrude their nuclei This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. before entering the circulation and hence are a source of pure 1073/pnas.1808816115/-/DCSupplemental. cytoplasm. Although extensive RNA degradation occurs during Published online August 6, 2018. E7970–E7977 | PNAS | vol. 115 | no. 34 www.pnas.org/cgi/doi/10.1073/pnas.1808816115 Downloaded by guest on October 2, 2021 Fig. 1. Typical sisRNAs found in mouse and human RBCs. (A) The two Upper tracks show conventional reads (red and purple) and inverted reads (gray) of total RNA from mature RBCs. The Lower tracks correspond to public RNA sequencing data of polyadenylated transcripts from RBC precursor cells (eryth- roblasts) (11). (B) Northern blots against two mouse stable lariats. In each case, the major band ran slower than expected for a linear molecule, consistent with the circular nature of the intronic RNA. (C) RT-PCR using conventional exonic (yellow) and intronic primers (red). Mouse RBC intronic RNAs can be amplified with the conventional (red) as well as inverted (outward facing) primers (brown), consistent with the RNAs being circular. (D) Nucleotide frequency at the branchpoint for all 374 mouse and 99 human stable lariats. circles occurred at the 3′ splice site of the intron, at the G or A however, that the designation sisRNA refers to the stability of the nucleotide, and not at the branchpoint (SI Appendix,Fig.S1A and sequences, not their location. B). These circular intronic RNAs have been previously observed in We began our search for sisRNAs in other cell types by ex- human cell culture. Presumably, they result from a postdebranching amining total RNA from mouse 3T3 cells. We treated the cells circularization event (10). In RBCs, the circularized full-length with α-amanitin to inhibit transcription and allow degradation of intronic RNAs were less abundant than stable lariats (SI Appen- transient intronic RNA. Untreated cells served as controls. Total dix,Fig.S1C). Finally, we observed that for the vast majority of extracted RNA was depleted of rRNA and treated with RNase R genes encoding a sisRNA, most reads were derived from introns, or water before high-throughput sequencing. RNase R degrades with very few exonic reads. This result suggests that linear mRNA linear RNA molecules but does not attack circular ones. This molecules are lost at some point during maturation of mammalian procedure enriches for stable circular RNA molecules but does RBCs, whereas circular RNAs are retained. not distinguish nuclear from cytoplasmic RNA. We will show Both stable lariats and circularized full-length intronic RNAs later by in situ hybridization that selected sisRNAs are, in fact, in the cytoplasm. occur in human RBCs as well. As with mouse blood, we sepa- α rated RBCs from white cells on a Percoll gradient. High-throughput In actively transcribing cells (no -amanitin), we detected ∼6,000 RNase R-resistant intronic RNAs between 65 and analysis revealed 143 circular intronic RNAs similar to the two types ∼ of mouse sisRNAs (Fig. 1 and Dataset S1). Among the 137 human 2,000 nt in length. These molecules were derived from about one third of the introns of ∼2,000 genes, with a bias for introns sisRNA host genes, 27 are common with mouse, and seven from 0.1–1.5 kb.