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      The emerging landscape of small nucleolar RNAs in cell biology

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          Abstract

          Small nucleolar RNAs (snoRNAs) are a large class of small noncoding RNAs present in all eukaryotes sequenced thus far. As a family, they have been well characterized as playing a central role in ribosome biogenesis, guiding either the sequence-specific chemical modification of pre-rRNA (ribosomal RNA) or its processing. However, in higher eukaryotes, numerous orphan snoRNAs were described over a decade ago, with no known target or ascribed function, suggesting the possibility of alternative cellular functionality. In recent years, thanks in great part to advances in sequencing methodologies, we have seen many examples of the diversity that exists in the snoRNA family on multiple levels. In this review, we discuss the identification of novel snoRNA members, of unexpected binding partners, as well as the clarification and extension of the snoRNA target space and the characterization of diverse new noncanonical functions, painting a new and extended picture of the snoRNA landscape. Under the deluge of novel features and functions that have recently come to light, snoRNAs emerge as a central, dynamic, and highly versatile group of small regulatory RNAs. WIREs RNA 2015, 6:381–397. doi: 10.1002/wrna.1284

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          Transcriptome-wide mapping reveals widespread dynamic-regulated pseudouridylation of ncRNA and mRNA.

          Pseudouridine is the most abundant RNA modification, yet except for a few well-studied cases, little is known about the modified positions and their function(s). Here, we develop Ψ-seq for transcriptome-wide quantitative mapping of pseudouridine. We validate Ψ-seq with spike-ins and de novo identification of previously reported positions and discover hundreds of unique sites in human and yeast mRNAs and snoRNAs. Perturbing pseudouridine synthases (PUS) uncovers which pseudouridine synthase modifies each site and their target sequence features. mRNA pseudouridinylation depends on both site-specific and snoRNA-guided pseudouridine synthases. Upon heat shock in yeast, Pus7p-mediated pseudouridylation is induced at >200 sites, and PUS7 deletion decreases the levels of otherwise pseudouridylated mRNA, suggesting a role in enhancing transcript stability. rRNA pseudouridine stoichiometries are conserved but reduced in cells from dyskeratosis congenita patients, where the PUS DKC1 is mutated. Our work identifies an enhanced, transcriptome-wide scope for pseudouridine and methods to dissect its underlying mechanisms and function. Copyright © 2014 Elsevier Inc. All rights reserved.
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            Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells

            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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              A human snoRNA with microRNA-like functions.

              Small noncoding RNAs function in concert with Argonaute (Ago) proteins to regulate gene expression at the level of transcription, mRNA stability, or translation. Ago proteins bind small RNAs and form the core of silencing complexes. Here, we report the analysis of small RNAs associated with human Ago1 and Ago2 revealed by immunoprecipitation and deep sequencing. Among the reads, we find small RNAs originating from the small nucleolar RNA (snoRNA) ACA45. Moreover, processing of ACA45 requires Dicer activity but is independent of Drosha/DGCR8. Using bioinformatic prediction algorithms and luciferase reporter assays, we uncover the mediator subunit CDC2L6 as one potential mRNA target of ACA45 small RNAs, suggesting a role for ACA45-processing products in posttranscriptional gene silencing. We further identify a number of human snoRNAs with microRNA (miRNA)-like processing signatures. We have, therefore, identified a class of small RNAs in human cells that originate from snoRNAs and can function like miRNAs.
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                Author and article information

                Journal
                Wiley Interdiscip Rev RNA
                Wiley Interdiscip Rev RNA
                wrna
                Wiley Interdisciplinary Reviews. RNA
                John Wiley & Sons, Inc. (Hoboken, USA )
                1757-7004
                1757-7012
                Jul-Aug 2015
                16 April 2015
                : 6
                : 4
                : 381-397
                Affiliations
                [1 ]Biochemistry Department, Faculty of Medicine and Health Sciences, University of Sherbrooke Sherbrooke, Canada
                [2 ]Biochemistry Department and RNA Group, Faculty of Medicine and Health Sciences, University of Sherbrooke Sherbrooke, Canada
                Author notes

                Conflict of interest: The authors declare no conflict of interest for this article.

                Article
                10.1002/wrna.1284
                4696412
                25879954
                27b9a74f-73ce-4dfa-a196-6f4884962404
                © 2015 The Authors. WIREs RNA published by John Wiley & Sons, Ltd.

                This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

                History
                : 16 January 2015
                : 18 March 2015
                : 20 March 2015
                Categories
                Advanced Reviews

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