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      Sam68 binds Alu-rich introns in SMN and promotes pre-mRNA circularization

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          Abstract

          The Spinal Muscular Atrophy (SMA) gene SMN was recently duplicated ( SMN1 and SMN2) in higher primates. Furthermore, invasion of the locus by repetitive elements almost doubled its size with respect to mouse Smn, in spite of an almost identical protein-coding sequence. Herein, we found that SMN ranks among the human genes with highest density of Alus, which are evolutionary conserved in primates and often occur in inverted orientation. Inverted repeat Alus (IR Alus) negatively regulate splicing of long introns within SMN, while promoting widespread alternative circular RNA (circRNA) biogenesis. Bioinformatics analyses revealed the presence of ultra-conserved Sam68 binding sites in SMN IR Alus. Cross-link-immunoprecipitation (CLIP), mutagenesis and silencing experiments showed that Sam68 binds in proximity of intronic Alus in the SMN pre-mRNA, thus favouring circRNA biogenesis in vitro and in vivo. These findings highlight a novel layer of regulation in SMN expression, uncover the crucial impact exerted by IR Alus and reveal a role for Sam68 in SMN circRNA biogenesis.

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          DHX9 suppresses RNA processing defects originating from the Alu invasion of the human genome

          Transposable elements are viewed as ‘selfish genetic elements’, yet they contribute to gene regulation and genome evolution in diverse ways. More than half of the human genome consists of transposable elements. Alu elements belong to the short interspersed nuclear element (SINE) family of repetitive elements, and with over 1 million insertions they make up more than 10% of the human genome. Despite their abundance and the potential evolutionary advantages they confer, Alu elements can be mutagenic to the host as they can act as splice acceptors, inhibit translation of mRNAs and cause genomic instability. Alu elements are the main targets of the RNA-editing enzyme ADAR and the formation of Alu exons is suppressed by the nuclear ribonucleoprotein HNRNPC, but the broad effect of massive secondary structures formed by inverted-repeat Alu elements on RNA processing in the nucleus remains unknown. Here we show that DHX9, an abundant nuclear RNA helicase, binds specifically to inverted-repeat Alu elements that are transcribed as parts of genes. Loss of DHX9 leads to an increase in the number of circular-RNA-producing genes and amount of circular RNAs, translational repression of reporters containing inverted-repeat Alu elements, and transcriptional rewiring (the creation of mostly nonsensical novel connections between exons) of susceptible loci. Biochemical purifications of DHX9 identify the interferon-inducible isoform of ADAR (p150), but not the constitutively expressed ADAR isoform (p110), as an RNA-independent interaction partner. Co-depletion of ADAR and DHX9 augments the double-stranded RNA accumulation defects, leading to increased circular RNA production, revealing a functional link between these two enzymes. Our work uncovers an evolutionarily conserved function of DHX9. We propose that it acts as a nuclear RNA resolvase that neutralizes the immediate threat posed by transposon insertions and allows these elements to evolve as tools for the post-transcriptional regulation of gene expression.
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            A 360° view of circular RNAs: From biogenesis to functions.

            The first circular RNA (circRNA) was identified more than 40 years ago, but it was only recently appreciated that circRNAs are common outputs of many eukaryotic protein-coding genes. Some circRNAs accumulate to higher levels than their associated linear mRNAs, especially in the nervous system, and have clear regulatory functions that result in organismal phenotypes. The pre-mRNA splicing machinery generates circRNAs via backsplicing reactions, which are often facilitated by intronic repeat sequences that base pair to one another and bring the intervening splice sites into close proximity. When spliceosomal components are limiting, circRNAs can become the preferred gene output, and backsplicing reactions are further controlled by exon skipping events and the combinatorial action of RNA binding proteins. This allows circRNAs to be expressed in a tissue- and stage-specific manner. Once generated, circRNAs are highly stable transcripts that often accumulate in the cytoplasm. The functions of most circRNAs remain unknown, but some can regulate the activities of microRNAs or be translated to produce proteins. Circular RNAs can further interface with the immune system as well as control gene expression events in the nucleus, including alternative splicing decisions. Circular RNAs thus represent a large class of RNA molecules that are tightly regulated, and it is becoming increasingly clear that they likely impact many biological processes. This article is categorized under: RNA Processing > Splicing Mechanisms RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution RNA Evolution and Genomics > Computational Analyses of RNA.
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              The human SWI/SNF subunit Brm is a regulator of alternative splicing.

              The SWI/SNF (mating-type switch/sucrose nonfermenting) complex involved in chromatin remodeling on promoters has also been detected on the coding region of genes. Here we show that SWI/SNF can function as a regulator of alternative splicing. We found that the catalytic subunit Brm favors inclusion of variant exons in the mRNA of several genes, including E-cadherin, BIM, cyclin D1 and CD44. Consistent with this, Brm associates with several components of the spliceosome and with Sam68, an ERK-activated enhancer of variant exon inclusion. Examination of the CD44 gene revealed that Brm induced accumulation of RNA polymerase II (RNAPII) with a modified CTD phosphorylation pattern on regions encoding variant exons. Altogether, our data suggest that on genes regulated by SWI/SNF, Brm contributes to the crosstalk between transcription and RNA processing by decreasing RNAPII elongation rate and facilitating recruitment of the splicing machinery to variant exons with suboptimal splice sites.
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                Author and article information

                Journal
                Nucleic Acids Res
                Nucleic Acids Res
                nar
                Nucleic Acids Research
                Oxford University Press
                0305-1048
                1362-4962
                24 January 2020
                28 November 2019
                28 November 2019
                : 48
                : 2
                : 633-645
                Affiliations
                [1 ] Institute of Human Anatomy and Cell Biology, Catholic University of the Sacred Heart , Rome 00168, Italy
                [2 ] Fondazione Policlinico Universitario A . Gemelli IRCCS, Rome 00168, Italy
                [3 ] GenoSplice Technology, iPEPS-ICM, Hôpital de la Pitié Salpêtrière , Paris 75013, France
                [4 ] Laboratory of Neuroembryology, IRCCS Fondazione Santa Lucia , Rome 00143, Italy
                [5 ] Department of Biomedicine and Prevention, University of Rome Tor Vergata , Rome 00133, Italy
                Author notes
                To whom correspondence should be addressed. Tel: +39 06 3015 4915; Fax: +39 06 3015 4813; Email: claudio.sette@ 123456unicatt.it
                Author information
                http://orcid.org/0000-0003-3347-8345
                Article
                gkz1117
                10.1093/nar/gkz1117
                6954450
                31777926
                84377e9a-0175-4437-8a76-23011b18f0e5
                © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@ 123456oup.com

                History
                : 22 November 2019
                : 12 November 2019
                : 22 March 2019
                Page count
                Pages: 13
                Funding
                Funded by: Associazione Italiana per la Ricerca sul Cancro 10.13039/501100005010
                Award ID: IG18790
                Funded by: Telethon Foundation 10.13039/501100002426
                Award ID: GGP14095
                Funded by: Ministry of Education, Universities and Research 10.13039/501100003407
                Award ID: PRIN 2018
                Funded by: Ministry of Health, Italy 10.13039/501100003196
                Award ID: GR-2018-12365706
                Categories
                Gene regulation, Chromatin and Epigenetics

                Genetics
                Genetics

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