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Blocking of an intronic splicing silencer completely rescues IKBKAP exon 20 splicing in familial dysautonomia patient cells
Abstract Familial dysautonomia (FD) is a severe genetic disorder causing sensory and autonomic dysfunction. It is predominantly caused by a c.2204+6T>C mutation in the IKBKAP gene. This mutation decreases the 5′ splice site strength of IKBKAP exon 20 leading to exon 20 skipping and decreased amou...
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Published in: | Nucleic acids research 2018-09, Vol.46 (15), p.7938-7952 |
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container_title | Nucleic acids research |
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creator | Bruun, Gitte H Bang, Jeanne M V Christensen, Lise L Brøner, Sabrina Petersen, Ulrika S S Guerra, Barbara Grønning, Alexander G B Doktor, Thomas K Andresen, Brage S |
description | Abstract
Familial dysautonomia (FD) is a severe genetic disorder causing sensory and autonomic dysfunction. It is predominantly caused by a c.2204+6T>C mutation in the IKBKAP gene. This mutation decreases the 5′ splice site strength of IKBKAP exon 20 leading to exon 20 skipping and decreased amounts of full-length IKAP protein. We identified a binding site for the splicing regulatory protein hnRNP A1 downstream of the IKBKAP exon 20 5′-splice site. We show that hnRNP A1 binds to this splicing regulatory element (SRE) and that two previously described inhibitory SREs inside IKBKAP exon 20 are also bound by hnRNP A1. Knockdown of hnRNP A1 in FD patient fibroblasts increases IKBKAP exon 20 inclusion demonstrating that hnRNP A1 is a negative regulator of IKBKAP exon 20 splicing. Furthermore, by mutating the SREs in an IKBKAP minigene we show that all three SREs cause hnRNP A1-mediated exon repression. We designed splice switching oligonucleotides (SSO) that blocks the intronic hnRNP A1 binding site, and demonstrate that this completely rescues splicing of IKBKAP exon 20 in FD patient fibroblasts and increases the amounts of IKAP protein. We propose that this may be developed into a potential new specific treatment of FD. |
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Familial dysautonomia (FD) is a severe genetic disorder causing sensory and autonomic dysfunction. It is predominantly caused by a c.2204+6T>C mutation in the IKBKAP gene. This mutation decreases the 5′ splice site strength of IKBKAP exon 20 leading to exon 20 skipping and decreased amounts of full-length IKAP protein. We identified a binding site for the splicing regulatory protein hnRNP A1 downstream of the IKBKAP exon 20 5′-splice site. We show that hnRNP A1 binds to this splicing regulatory element (SRE) and that two previously described inhibitory SREs inside IKBKAP exon 20 are also bound by hnRNP A1. Knockdown of hnRNP A1 in FD patient fibroblasts increases IKBKAP exon 20 inclusion demonstrating that hnRNP A1 is a negative regulator of IKBKAP exon 20 splicing. Furthermore, by mutating the SREs in an IKBKAP minigene we show that all three SREs cause hnRNP A1-mediated exon repression. We designed splice switching oligonucleotides (SSO) that blocks the intronic hnRNP A1 binding site, and demonstrate that this completely rescues splicing of IKBKAP exon 20 in FD patient fibroblasts and increases the amounts of IKAP protein. We propose that this may be developed into a potential new specific treatment of FD.</description><identifier>ISSN: 0305-1048</identifier><identifier>EISSN: 1362-4962</identifier><identifier>DOI: 10.1093/nar/gky395</identifier><identifier>PMID: 29762696</identifier><language>eng</language><publisher>England: Oxford University Press</publisher><subject>Base Sequence ; Binding Sites - genetics ; Carrier Proteins - genetics ; Carrier Proteins - metabolism ; Cell Line ; Cells, Cultured ; Exons - genetics ; Fibroblasts - metabolism ; Heterogeneous Nuclear Ribonucleoprotein A1 - genetics ; Heterogeneous Nuclear Ribonucleoprotein A1 - metabolism ; Humans ; Introns - genetics ; Mutation ; Oligonucleotides - genetics ; Oligonucleotides - metabolism ; Regulatory Sequences, Nucleic Acid - genetics ; RNA and RNA-protein complexes ; RNA Splicing</subject><ispartof>Nucleic acids research, 2018-09, Vol.46 (15), p.7938-7952</ispartof><rights>The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research. 2018</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c408t-42da6894cc4d3b564ff2239281b88fcad8f6def470f267937186cf18367c508d3</citedby><cites>FETCH-LOGICAL-c408t-42da6894cc4d3b564ff2239281b88fcad8f6def470f267937186cf18367c508d3</cites><orcidid>0000-0001-7488-3035</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6125618/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6125618/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,885,1604,27924,27925,53791,53793</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/29762696$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Bruun, Gitte H</creatorcontrib><creatorcontrib>Bang, Jeanne M V</creatorcontrib><creatorcontrib>Christensen, Lise L</creatorcontrib><creatorcontrib>Brøner, Sabrina</creatorcontrib><creatorcontrib>Petersen, Ulrika S S</creatorcontrib><creatorcontrib>Guerra, Barbara</creatorcontrib><creatorcontrib>Grønning, Alexander G B</creatorcontrib><creatorcontrib>Doktor, Thomas K</creatorcontrib><creatorcontrib>Andresen, Brage S</creatorcontrib><title>Blocking of an intronic splicing silencer completely rescues IKBKAP exon 20 splicing in familial dysautonomia patient cells</title><title>Nucleic acids research</title><addtitle>Nucleic Acids Res</addtitle><description>Abstract
Familial dysautonomia (FD) is a severe genetic disorder causing sensory and autonomic dysfunction. It is predominantly caused by a c.2204+6T>C mutation in the IKBKAP gene. This mutation decreases the 5′ splice site strength of IKBKAP exon 20 leading to exon 20 skipping and decreased amounts of full-length IKAP protein. We identified a binding site for the splicing regulatory protein hnRNP A1 downstream of the IKBKAP exon 20 5′-splice site. We show that hnRNP A1 binds to this splicing regulatory element (SRE) and that two previously described inhibitory SREs inside IKBKAP exon 20 are also bound by hnRNP A1. Knockdown of hnRNP A1 in FD patient fibroblasts increases IKBKAP exon 20 inclusion demonstrating that hnRNP A1 is a negative regulator of IKBKAP exon 20 splicing. Furthermore, by mutating the SREs in an IKBKAP minigene we show that all three SREs cause hnRNP A1-mediated exon repression. We designed splice switching oligonucleotides (SSO) that blocks the intronic hnRNP A1 binding site, and demonstrate that this completely rescues splicing of IKBKAP exon 20 in FD patient fibroblasts and increases the amounts of IKAP protein. We propose that this may be developed into a potential new specific treatment of FD.</description><subject>Base Sequence</subject><subject>Binding Sites - genetics</subject><subject>Carrier Proteins - genetics</subject><subject>Carrier Proteins - metabolism</subject><subject>Cell Line</subject><subject>Cells, Cultured</subject><subject>Exons - genetics</subject><subject>Fibroblasts - metabolism</subject><subject>Heterogeneous Nuclear Ribonucleoprotein A1 - genetics</subject><subject>Heterogeneous Nuclear Ribonucleoprotein A1 - metabolism</subject><subject>Humans</subject><subject>Introns - genetics</subject><subject>Mutation</subject><subject>Oligonucleotides - genetics</subject><subject>Oligonucleotides - metabolism</subject><subject>Regulatory Sequences, Nucleic Acid - genetics</subject><subject>RNA and RNA-protein complexes</subject><subject>RNA Splicing</subject><issn>0305-1048</issn><issn>1362-4962</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2018</creationdate><recordtype>article</recordtype><sourceid>TOX</sourceid><recordid>eNp9kU1rFTEUhoNY2uvVjT9AshFEmDZfk0k2QlvUlhbaha5Dbia5xmaSMZkRL_3zzeX2w266Cpw85z3vOS8A7zE6xEjSo6jz0fpmQ2X7Ciww5aRhkpPXYIEoahuMmDgAb0r5jRBmuGX74IDIjhMu-QLcnoRkbnxcw-SgjtDHKafoDSxj8GZbLz7YaGyGJg1jsJMNG5htMbMt8Pzi5OL4Gtp_KUKCnnp8hE4PPngdYL8pep5STIPXcNSTt3GCxoZQ3oI9p0Ox7-7fJfj57euP07Pm8ur7-enxZWMYElPDSK-5kMwY1tNVy5lzhFBJBF4J4YzuheO9daxDjvBO0g4LbhwWlHemRaKnS_BlpzvOq8H2phrIOqgx-0HnjUraq-c_0f9S6_RXcUxaXoWW4NO9QE5_6t6TGnzZrqCjTXNRBFU7sh4bV_TzDjU5lZKtexyDkdqmpWpaapdWhT_8b-wRfYinAh93QJrHl4TuAFXqoKA</recordid><startdate>20180906</startdate><enddate>20180906</enddate><creator>Bruun, Gitte H</creator><creator>Bang, Jeanne M V</creator><creator>Christensen, Lise L</creator><creator>Brøner, Sabrina</creator><creator>Petersen, Ulrika S S</creator><creator>Guerra, Barbara</creator><creator>Grønning, Alexander G B</creator><creator>Doktor, Thomas K</creator><creator>Andresen, Brage S</creator><general>Oxford University Press</general><scope>TOX</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><scope>5PM</scope><orcidid>https://orcid.org/0000-0001-7488-3035</orcidid></search><sort><creationdate>20180906</creationdate><title>Blocking of an intronic splicing silencer completely rescues IKBKAP exon 20 splicing in familial dysautonomia patient cells</title><author>Bruun, Gitte H ; Bang, Jeanne M V ; Christensen, Lise L ; Brøner, Sabrina ; Petersen, Ulrika S S ; Guerra, Barbara ; Grønning, Alexander G B ; Doktor, Thomas K ; Andresen, Brage S</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c408t-42da6894cc4d3b564ff2239281b88fcad8f6def470f267937186cf18367c508d3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2018</creationdate><topic>Base Sequence</topic><topic>Binding Sites - genetics</topic><topic>Carrier Proteins - genetics</topic><topic>Carrier Proteins - metabolism</topic><topic>Cell Line</topic><topic>Cells, Cultured</topic><topic>Exons - genetics</topic><topic>Fibroblasts - metabolism</topic><topic>Heterogeneous Nuclear Ribonucleoprotein A1 - genetics</topic><topic>Heterogeneous Nuclear Ribonucleoprotein A1 - metabolism</topic><topic>Humans</topic><topic>Introns - genetics</topic><topic>Mutation</topic><topic>Oligonucleotides - genetics</topic><topic>Oligonucleotides - metabolism</topic><topic>Regulatory Sequences, Nucleic Acid - genetics</topic><topic>RNA and RNA-protein complexes</topic><topic>RNA Splicing</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Bruun, Gitte H</creatorcontrib><creatorcontrib>Bang, Jeanne M V</creatorcontrib><creatorcontrib>Christensen, Lise L</creatorcontrib><creatorcontrib>Brøner, Sabrina</creatorcontrib><creatorcontrib>Petersen, Ulrika S S</creatorcontrib><creatorcontrib>Guerra, Barbara</creatorcontrib><creatorcontrib>Grønning, Alexander G B</creatorcontrib><creatorcontrib>Doktor, Thomas K</creatorcontrib><creatorcontrib>Andresen, Brage S</creatorcontrib><collection>Oxford University Press Open Access</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Nucleic acids research</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Bruun, Gitte H</au><au>Bang, Jeanne M V</au><au>Christensen, Lise L</au><au>Brøner, Sabrina</au><au>Petersen, Ulrika S S</au><au>Guerra, Barbara</au><au>Grønning, Alexander G B</au><au>Doktor, Thomas K</au><au>Andresen, Brage S</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Blocking of an intronic splicing silencer completely rescues IKBKAP exon 20 splicing in familial dysautonomia patient cells</atitle><jtitle>Nucleic acids research</jtitle><addtitle>Nucleic Acids Res</addtitle><date>2018-09-06</date><risdate>2018</risdate><volume>46</volume><issue>15</issue><spage>7938</spage><epage>7952</epage><pages>7938-7952</pages><issn>0305-1048</issn><eissn>1362-4962</eissn><abstract>Abstract
Familial dysautonomia (FD) is a severe genetic disorder causing sensory and autonomic dysfunction. It is predominantly caused by a c.2204+6T>C mutation in the IKBKAP gene. This mutation decreases the 5′ splice site strength of IKBKAP exon 20 leading to exon 20 skipping and decreased amounts of full-length IKAP protein. We identified a binding site for the splicing regulatory protein hnRNP A1 downstream of the IKBKAP exon 20 5′-splice site. We show that hnRNP A1 binds to this splicing regulatory element (SRE) and that two previously described inhibitory SREs inside IKBKAP exon 20 are also bound by hnRNP A1. Knockdown of hnRNP A1 in FD patient fibroblasts increases IKBKAP exon 20 inclusion demonstrating that hnRNP A1 is a negative regulator of IKBKAP exon 20 splicing. Furthermore, by mutating the SREs in an IKBKAP minigene we show that all three SREs cause hnRNP A1-mediated exon repression. We designed splice switching oligonucleotides (SSO) that blocks the intronic hnRNP A1 binding site, and demonstrate that this completely rescues splicing of IKBKAP exon 20 in FD patient fibroblasts and increases the amounts of IKAP protein. We propose that this may be developed into a potential new specific treatment of FD.</abstract><cop>England</cop><pub>Oxford University Press</pub><pmid>29762696</pmid><doi>10.1093/nar/gky395</doi><tpages>15</tpages><orcidid>https://orcid.org/0000-0001-7488-3035</orcidid><oa>free_for_read</oa></addata></record> |
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subjects | Base Sequence Binding Sites - genetics Carrier Proteins - genetics Carrier Proteins - metabolism Cell Line Cells, Cultured Exons - genetics Fibroblasts - metabolism Heterogeneous Nuclear Ribonucleoprotein A1 - genetics Heterogeneous Nuclear Ribonucleoprotein A1 - metabolism Humans Introns - genetics Mutation Oligonucleotides - genetics Oligonucleotides - metabolism Regulatory Sequences, Nucleic Acid - genetics RNA and RNA-protein complexes RNA Splicing |
title | Blocking of an intronic splicing silencer completely rescues IKBKAP exon 20 splicing in familial dysautonomia patient cells |
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