Discovery of Replicating Circular RNAs by RNA-Seq and Computational Algorithms

PLoS Pathogens, Dec 2014

Replicating circular RNAs are independent plant pathogens known as viroids, or act to modulate the pathogenesis of plant and animal viruses as their satellite RNAs. The rate of discovery of these subviral pathogens was low over the past 40 years because the classical approaches are technical demanding and time-consuming. We previously described an approach for homology-independent discovery of replicating circular RNAs by analysing the total small RNA populations from samples of diseased tissues with a computational program known as progressive filtering of overlapping small RNAs (PFOR). However, PFOR written in PERL language is extremely slow and is unable to discover those subviral pathogens that do not trigger in vivo accumulation of extensively overlapping small RNAs. Moreover, PFOR is yet to identify a new viroid capable of initiating independent infection. Here we report the development of PFOR2 that adopted parallel programming in the C++ language and was 3 to 8 times faster than PFOR. A new computational program was further developed and incorporated into PFOR2 to allow the identification of circular RNAs by deep sequencing of long RNAs instead of small RNAs. PFOR2 analysis of the small RNA libraries from grapevine and apple plants led to the discovery of Grapevine latent viroid (GLVd) and Apple hammerhead viroid-like RNA (AHVd-like RNA), respectively. GLVd was proposed as a new species in the genus Apscaviroid, because it contained the typical structural elements found in this group of viroids and initiated independent infection in grapevine seedlings. AHVd-like RNA encoded a biologically active hammerhead ribozyme in both polarities, and was not specifically associated with any of the viruses found in apple plants. We propose that these computational algorithms have the potential to discover novel circular RNAs in plants, invertebrates and vertebrates regardless of whether they replicate and/or induce the in vivo accumulation of small RNAs.

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Discovery of Replicating Circular RNAs by RNA-Seq and Computational Algorithms

et al. (2014) Discovery of Replicating Circular RNAs by RNA-Seq and Computational Algorithms. PLoS Pathog 10(12): e1004553. doi:10.1371/journal.ppat.1004553 Discovery of Replicating Circular RNAs by RNA-Seq and Computational Algorithms Zhixiang Zhang 0 Shuishui Qi 0 Nan Tang 0 Xinxin Zhang 0 Shanshan Chen 0 Pengfei Zhu 0 Lin Ma 0 Jinping Cheng 0 Yun Xu 0 Meiguang Lu 0 Hongqing Wang 0 Shou-Wei Ding 0 Shifang Li 0 Qingfa Wu 0 Biao Ding, The Ohio State University, United States of America 0 1 School of Life Sciences, University of Science and Technology of China , Hefei, Anhui , China , 2 State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences , Beijing , China , 3 Department of Computer Science and Technology, University of Science and Technology of China , Hefei, Anhui , China , 4 Department of Fruit Science, College of Agronomy and Biotechnology, China Agricultural University , Beijing , China , 5 Department of Plant Pathology and Microbiology, University of California, Riverside, Riverside, California, United States of America, 6 The CAS Key Laboratory of Innate Immunity and Chronic Disease, University of Science and Technology of China , Hefei, Anhui , China Replicating circular RNAs are independent plant pathogens known as viroids, or act to modulate the pathogenesis of plant and animal viruses as their satellite RNAs. The rate of discovery of these subviral pathogens was low over the past 40 years because the classical approaches are technical demanding and time-consuming. We previously described an approach for homology-independent discovery of replicating circular RNAs by analysing the total small RNA populations from samples of diseased tissues with a computational program known as progressive filtering of overlapping small RNAs (PFOR). However, PFOR written in PERL language is extremely slow and is unable to discover those subviral pathogens that do not trigger in vivo accumulation of extensively overlapping small RNAs. Moreover, PFOR is yet to identify a new viroid capable of initiating independent infection. Here we report the development of PFOR2 that adopted parallel programming in the C++ language and was 3 to 8 times faster than PFOR. A new computational program was further developed and incorporated into PFOR2 to allow the identification of circular RNAs by deep sequencing of long RNAs instead of small RNAs. PFOR2 analysis of the small RNA libraries from grapevine and apple plants led to the discovery of Grapevine latent viroid (GLVd) and Apple hammerhead viroid-like RNA (AHVd-like RNA), respectively. GLVd was proposed as a new species in the genus Apscaviroid, because it contained the typical structural elements found in this group of viroids and initiated independent infection in grapevine seedlings. AHVd-like RNA encoded a biologically active hammerhead ribozyme in both polarities, and was not specifically associated with any of the viruses found in apple plants. We propose that these computational algorithms have the potential to discover novel circular RNAs in plants, invertebrates and vertebrates regardless of whether they replicate and/or induce the in vivo accumulation of small RNAs. - Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. Programs and supporting data sets are available from the website (http://staff.ustc.edu.cn/,wuqf/resources.html). The small RNA data sets of grapevine and apple are available from NCBI SRA database under the accession number SRR890616 and SRR890731. Funding: This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant XDB11040400), the Ministry of Science and Technology of China (Grants 2014CB138405, 2011CBA01103), the National Natural Science Foundation of China (Grants 31272011, 31471747), the Special Fund for Agro-scientific Research in the Public Interest (201203067), the Doctoral Fund of the Ministry of Education of China (20123402110013) and the earmarked fund for the China Agriculture Research System (CARS-31-2-03). SD is supported by a grant of US Department of Agriculture Research Service (6659-22000-025). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Viroids and a group of satellite RNAs (satRNAs) have singlestranded circular RNA genomes that range in size from 220 to 457 nucleotides (nt) [14]. These subviral pathogenic RNAs lack protein-coding capabilities and thus depend on either hostencoded DNA-dependent RNA polymerase (viroids) or helper virus-encoded RNA-dependent RNA polymerase (circular satRNAs) for replication [5,6]. Viroids and circular satRNAs have been proven to be excellent biological models for studying non-coding RNAs (...truncated)


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Zhixiang Zhang, Shuishui Qi, Nan Tang, Xinxin Zhang, Shanshan Chen, Pengfei Zhu, Lin Ma, Jinping Cheng, Yun Xu, Meiguang Lu, Hongqing Wang, Shou-Wei Ding, Shifang Li, Qingfa Wu. Discovery of Replicating Circular RNAs by RNA-Seq and Computational Algorithms, PLoS Pathogens, 2014, Volume 10, Issue 12, DOI: 10.1371/journal.ppat.1004553