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      Advances in analyzing RNA diversity in eukaryotic transcriptomes: peering through the Omics lens

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          Abstract

          Alternative splicing, polyadenylation, and chemical modifications of RNA generate astonishing complexity within eukaryotic transcriptomes. The last decade has brought numerous advances in sequencing technologies that allow biologists to investigate these phenomena with greater depth and accuracy while reducing time and cost. A commensurate development in biochemical techniques for the enrichment and analysis of different RNA variants has accompanied the advancement of global sequencing analysis platforms. Here, we present a detailed overview of the latest biochemical methods, along with bioinformatics pipelines that have aided in identifying different RNA variants. We also highlight the ongoing developments and challenges associated with RNA variant detection and quantification, including sample heterogeneity and isolation, as well as ‘Omics’ big data handling.

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          An integrated semiconductor device enabling non-optical genome sequencing.

          Jonathan Rothberg, Wolfgang Hinz, Todd M. Rearick … (2011)
          The seminal importance of DNA sequencing to the life sciences, biotechnology and medicine has driven the search for more scalable and lower-cost solutions. Here we describe a DNA sequencing technology in which scalable, low-cost semiconductor manufacturing techniques are used to make an integrated circuit able to directly perform non-optical DNA sequencing of genomes. Sequence data are obtained by directly sensing the ions produced by template-directed DNA polymerase synthesis using all-natural nucleotides on this massively parallel semiconductor-sensing device or ion chip. The ion chip contains ion-sensitive, field-effect transistor-based sensors in perfect register with 1.2 million wells, which provide confinement and allow parallel, simultaneous detection of independent sequencing reactions. Use of the most widely used technology for constructing integrated circuits, the complementary metal-oxide semiconductor (CMOS) process, allows for low-cost, large-scale production and scaling of the device to higher densities and larger array sizes. We show the performance of the system by sequencing three bacterial genomes, its robustness and scalability by producing ion chips with up to 10 times as many sensors and sequencing a human genome.
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            The evolutionary landscape of alternative splicing in vertebrate species.

            Nuno Barbosa-Morais, Manuel Irimia, Qun Pan … (2012)
            How species with similar repertoires of protein-coding genes differ so markedly at the phenotypic level is poorly understood. By comparing organ transcriptomes from vertebrate species spanning ~350 million years of evolution, we observed significant differences in alternative splicing complexity between vertebrate lineages, with the highest complexity in primates. Within 6 million years, the splicing profiles of physiologically equivalent organs diverged such that they are more strongly related to the identity of a species than they are to organ type. Most vertebrate species-specific splicing patterns are cis-directed. However, a subset of pronounced splicing changes are predicted to remodel protein interactions involving trans-acting regulators. These events likely further contributed to the diversification of splicing and other transcriptomic changes that underlie phenotypic differences among vertebrate species.
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              Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells

              Thomas Carlile, Maria Rojas-Duran, Boris Zinshteyn … (2014)
              Post-transcriptional modification of RNA nucleosides occurs in all living organisms. Pseudouridine, the most abundant modified nucleoside in non-coding RNAs 1 , enhances the function of transfer RNA and ribosomal RNA by stabilizing RNA structure 2–8 . mRNAs were not known to contain pseudouridine, but artificial pseudouridylation dramatically affects mRNA function – it changes the genetic code by facilitating non-canonical base pairing in the ribosome decoding center 9,10 . However, without evidence of naturally occurring mRNA pseudouridylation, its physiological was unclear. Here we present a comprehensive analysis of pseudouridylation in yeast and human RNAs using Pseudo-seq, a genome-wide, single-nucleotide-resolution method for pseudouridine identification. Pseudo-seq accurately identifies known modification sites as well as 100 novel sites in non-coding RNAs, and reveals hundreds of pseudouridylated sites in mRNAs. Genetic analysis allowed us to assign most of the new modification sites to one of seven conserved pseudouridine synthases, Pus1–4, 6, 7 and 9. Notably, the majority of pseudouridines in mRNA are regulated in response to environmental signals, such as nutrient deprivation in yeast and serum starvation in human cells. These results suggest a mechanism for the rapid and regulated rewiring of the genetic code through inducible mRNA modifications. Our findings reveal unanticipated roles for pseudouridylation and provide a resource for identifying the targets of pseudouridine synthases implicated in human disease 11–13 .
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                Author and article information

                Journal
                Journal ID (nlm-ta): F1000Res
                Journal ID (iso-abbrev): F1000Res
                Journal ID (pmc): F1000Research
                Title: F1000Research
                Publisher: F1000Research (London, UK )
                ISSN (Electronic): 2046-1402
                Publication date (Electronic): 14 November 2016
                Publication date Collection: 2016
                Volume: 5
                Electronic Location Identifier: 2668
                Affiliations
                [1 ]Department of Biochemistry, University of Illinois at Urbana-Champaign, Illinois, USA
                [2 ]Institute of Genomic Biology, University of Illinois at Urbana-Champaign, Illinois, USA
                [3 ]College of Medicine, University of Illinois at Urbana-Champaign, Illinois, USA
                Author notes

                Competing interests: The authors declare that they have no competing interests.

                Author information
                http://orcid.org/0000-0002-1011-0006
                Article
                DOI: 10.12688/f1000research.9511.1
                PMC ID: 5112568
                SO-VID: 99fd1ce8-4fcf-4179-a13c-9dadc5345fb2
                Copyright © Copyright: © 2016 Bangru S and Kalsotra A
                License:

                This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                Date accepted : 8 November 2016
                Funding
                Funded by: National Institutes of Health
                Award ID: R01HL126845
                Funded by: March of Dimes
                Award ID: 5-FY14-112
                AK is supported by grants from the US National Institutes of Health (R01HL126845), the March of Dimes (5-FY14-112), and the Center for Advanced Study at the University of Illinois.
                The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Subject: Review
                Subject: Articles
                Subject: Bioinformatics
                Subject: Biomacromolecule-Ligand Interactions
                Subject: Cell Growth & Division
                Subject: Chemical Biology of the Cell
                Subject: Control of Gene Expression
                Subject: Developmental Molecular Mechanisms
                Subject: Genomics
                Subject: Macromolecular Chemistry
                Subject: Morphogenesis & Cell Biology
                Subject: Pattern Formation
                Subject: Protein Chemistry & Proteomics

                Keywords: rna variants,alternative splicing,next generation sequencing,mrna modifications,computational pipelines,post-transcriptional gene regulation
                Data availability:
                Keywords: rna variants, alternative splicing, next generation sequencing, mrna modifications, computational pipelines, post-transcriptional gene regulation

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