Identification of a Functional Mammalian Spliceosome Containing Unspliced Pre-mRNA
Functional 60S spliceosomes were assembled under conditions that block the first step of the mRNA splicing reaction. This block was imposed by carrying out the splicing reaction in nuclear extracts preincubated in 2.5 mM EDTA. Preparative amounts of the spliceosomes were isolated by gel filtration c...
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| Veröffentlicht in: | Proceedings of the National Academy of Sciences - PNAS 1988-10, Vol.85 (19), p.7216-7220 |
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| container_title | Proceedings of the National Academy of Sciences - PNAS |
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| creator | Abmayr, Susan M. Reed, Robin Maniatis, Tom |
| description | Functional 60S spliceosomes were assembled under conditions that block the first step of the mRNA splicing reaction. This block was imposed by carrying out the splicing reaction in nuclear extracts preincubated in 2.5 mM EDTA. Preparative amounts of the spliceosomes were isolated by gel filtration chromatography and shown to be functional by in vitro complementation assays. The unspliced pre-mRNA in the complex was converted to spliced products when incubated in cytoplasmic S100 extracts or in heat-treated or micrococcal nuclease-treated nuclear extracts. The latter result, in conjunction with the size of the complex, suggests that the spliceosome contains all of the small nuclear ribonucleoproteins (snRNPs) required for both steps of the splicing reaction. Biochemical characterization of the 5′ cleavage reaction revealed that ATP and MgCl2 are required for this step in the splicing pathway. The presence of U1 snRNP in the blocked complex was demonstrated by quantitative immunoprecipitation of this complex by an anti-U1 snRNP monoclonal antibody. |
| doi_str_mv | 10.1073/pnas.85.19.7216 |
| format | Article |
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This block was imposed by carrying out the splicing reaction in nuclear extracts preincubated in 2.5 mM EDTA. Preparative amounts of the spliceosomes were isolated by gel filtration chromatography and shown to be functional by in vitro complementation assays. The unspliced pre-mRNA in the complex was converted to spliced products when incubated in cytoplasmic S100 extracts or in heat-treated or micrococcal nuclease-treated nuclear extracts. The latter result, in conjunction with the size of the complex, suggests that the spliceosome contains all of the small nuclear ribonucleoproteins (snRNPs) required for both steps of the splicing reaction. Biochemical characterization of the 5′ cleavage reaction revealed that ATP and MgCl2 are required for this step in the splicing pathway. The presence of U1 snRNP in the blocked complex was demonstrated by quantitative immunoprecipitation of this complex by an anti-U1 snRNP monoclonal antibody.</description><identifier>ISSN: 0027-8424</identifier><identifier>EISSN: 1091-6490</identifier><identifier>DOI: 10.1073/pnas.85.19.7216</identifier><identifier>PMID: 2971971</identifier><language>eng</language><publisher>United States: National Academy of Sciences of the United States of America</publisher><subject>Animals ; Biochemistry ; Chromatography, Gel ; Complementation ; Edetic Acid - pharmacology ; Electrophoresis, Polyacrylamide Gel ; Gel chromatography ; Gels ; Humans ; Messenger RNA ; Rabbits ; Ribonucleoproteins - analysis ; Ribonucleoproteins, Small Nuclear ; RNA ; RNA Precursors - analysis ; RNA Splicing ; Small nuclear ribonucleoproteins ; Spliceosomes ; Splicing ; Yeasts</subject><ispartof>Proceedings of the National Academy of Sciences - PNAS, 1988-10, Vol.85 (19), p.7216-7220</ispartof><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c4196-d5dfa6286a9d5ba2992a21ed9585b33175fdbf6df254a94ba378ad57354acbf33</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Uhttp://www.pnas.org/content/85/19.cover.gif</thumbnail><linktopdf>$$Uhttps://www.jstor.org/stable/pdf/32101$$EPDF$$P50$$Gjstor$$H</linktopdf><linktohtml>$$Uhttps://www.jstor.org/stable/32101$$EHTML$$P50$$Gjstor$$H</linktohtml><link.rule.ids>314,776,780,799,27901,27902,57992,58225</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/2971971$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Abmayr, Susan M.</creatorcontrib><creatorcontrib>Reed, Robin</creatorcontrib><creatorcontrib>Maniatis, Tom</creatorcontrib><title>Identification of a Functional Mammalian Spliceosome Containing Unspliced Pre-mRNA</title><title>Proceedings of the National Academy of Sciences - PNAS</title><addtitle>Proc Natl Acad Sci U S A</addtitle><description>Functional 60S spliceosomes were assembled under conditions that block the first step of the mRNA splicing reaction. This block was imposed by carrying out the splicing reaction in nuclear extracts preincubated in 2.5 mM EDTA. Preparative amounts of the spliceosomes were isolated by gel filtration chromatography and shown to be functional by in vitro complementation assays. The unspliced pre-mRNA in the complex was converted to spliced products when incubated in cytoplasmic S100 extracts or in heat-treated or micrococcal nuclease-treated nuclear extracts. The latter result, in conjunction with the size of the complex, suggests that the spliceosome contains all of the small nuclear ribonucleoproteins (snRNPs) required for both steps of the splicing reaction. Biochemical characterization of the 5′ cleavage reaction revealed that ATP and MgCl2 are required for this step in the splicing pathway. The presence of U1 snRNP in the blocked complex was demonstrated by quantitative immunoprecipitation of this complex by an anti-U1 snRNP monoclonal antibody.</description><subject>Animals</subject><subject>Biochemistry</subject><subject>Chromatography, Gel</subject><subject>Complementation</subject><subject>Edetic Acid - pharmacology</subject><subject>Electrophoresis, Polyacrylamide Gel</subject><subject>Gel chromatography</subject><subject>Gels</subject><subject>Humans</subject><subject>Messenger RNA</subject><subject>Rabbits</subject><subject>Ribonucleoproteins - analysis</subject><subject>Ribonucleoproteins, Small Nuclear</subject><subject>RNA</subject><subject>RNA Precursors - analysis</subject><subject>RNA Splicing</subject><subject>Small nuclear ribonucleoproteins</subject><subject>Spliceosomes</subject><subject>Splicing</subject><subject>Yeasts</subject><issn>0027-8424</issn><issn>1091-6490</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1988</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkEtLxDAUhYMoOj7WgqB0pauOebbNchh8gS98rMNtk0ikTcamBf33ts4ouFG4cLmc7xwuB6F9gqcE5-x04SFOCzElcppTkq2hCcGSpBmXeB1NMKZ5WnDKt9B2jK8YYykKvIk2qczJMBP0cKWN75x1FXQu-CTYBJLz3lfjBXVyA00DtQOfPC5qV5kQQ2OSefAdOO_8S_Ls45egk_vWpM3D7WwXbVioo9lb7R30fH72NL9Mr-8uruaz67TiRGapFtpCRosMpBYlUCkpUGL08KEoGSO5sLq0mbZUcJC8BJYXoEXOhrMqLWM76HiZu2jDW29ipxoXK1PX4E3oo8oLng0p5F-QCMxZQfkAni7Bqg0xtsaqResaaD8UwWqsW411q0IoItVY9-A4XEX3ZWP0D7_qd9CPVvpo_FZ_BZz8CSjb13Vn3ruBPFiSr7EL7Q_KKMGEfQIyCpxe</recordid><startdate>198810</startdate><enddate>198810</enddate><creator>Abmayr, Susan M.</creator><creator>Reed, Robin</creator><creator>Maniatis, Tom</creator><general>National Academy of Sciences of the United States of America</general><general>National Acad Sciences</general><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>7TM</scope><scope>7X8</scope></search><sort><creationdate>198810</creationdate><title>Identification of a Functional Mammalian Spliceosome Containing Unspliced Pre-mRNA</title><author>Abmayr, Susan M. ; Reed, Robin ; Maniatis, Tom</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4196-d5dfa6286a9d5ba2992a21ed9585b33175fdbf6df254a94ba378ad57354acbf33</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1988</creationdate><topic>Animals</topic><topic>Biochemistry</topic><topic>Chromatography, Gel</topic><topic>Complementation</topic><topic>Edetic Acid - pharmacology</topic><topic>Electrophoresis, Polyacrylamide Gel</topic><topic>Gel chromatography</topic><topic>Gels</topic><topic>Humans</topic><topic>Messenger RNA</topic><topic>Rabbits</topic><topic>Ribonucleoproteins - analysis</topic><topic>Ribonucleoproteins, Small Nuclear</topic><topic>RNA</topic><topic>RNA Precursors - analysis</topic><topic>RNA Splicing</topic><topic>Small nuclear ribonucleoproteins</topic><topic>Spliceosomes</topic><topic>Splicing</topic><topic>Yeasts</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Abmayr, Susan M.</creatorcontrib><creatorcontrib>Reed, Robin</creatorcontrib><creatorcontrib>Maniatis, Tom</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Nucleic Acids Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Proceedings of the National Academy of Sciences - PNAS</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Abmayr, Susan M.</au><au>Reed, Robin</au><au>Maniatis, Tom</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Identification of a Functional Mammalian Spliceosome Containing Unspliced Pre-mRNA</atitle><jtitle>Proceedings of the National Academy of Sciences - PNAS</jtitle><addtitle>Proc Natl Acad Sci U S A</addtitle><date>1988-10</date><risdate>1988</risdate><volume>85</volume><issue>19</issue><spage>7216</spage><epage>7220</epage><pages>7216-7220</pages><issn>0027-8424</issn><eissn>1091-6490</eissn><abstract>Functional 60S spliceosomes were assembled under conditions that block the first step of the mRNA splicing reaction. This block was imposed by carrying out the splicing reaction in nuclear extracts preincubated in 2.5 mM EDTA. Preparative amounts of the spliceosomes were isolated by gel filtration chromatography and shown to be functional by in vitro complementation assays. The unspliced pre-mRNA in the complex was converted to spliced products when incubated in cytoplasmic S100 extracts or in heat-treated or micrococcal nuclease-treated nuclear extracts. The latter result, in conjunction with the size of the complex, suggests that the spliceosome contains all of the small nuclear ribonucleoproteins (snRNPs) required for both steps of the splicing reaction. Biochemical characterization of the 5′ cleavage reaction revealed that ATP and MgCl2 are required for this step in the splicing pathway. The presence of U1 snRNP in the blocked complex was demonstrated by quantitative immunoprecipitation of this complex by an anti-U1 snRNP monoclonal antibody.</abstract><cop>United States</cop><pub>National Academy of Sciences of the United States of America</pub><pmid>2971971</pmid><doi>10.1073/pnas.85.19.7216</doi><tpages>5</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | Proceedings of the National Academy of Sciences - PNAS, 1988-10, Vol.85 (19), p.7216-7220 |
| issn | 0027-8424 1091-6490 |
| language | eng |
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| source | Jstor Complete Legacy; MEDLINE; PubMed Central; Alma/SFX Local Collection; Free Full-Text Journals in Chemistry |
| subjects | Animals Biochemistry Chromatography, Gel Complementation Edetic Acid - pharmacology Electrophoresis, Polyacrylamide Gel Gel chromatography Gels Humans Messenger RNA Rabbits Ribonucleoproteins - analysis Ribonucleoproteins, Small Nuclear RNA RNA Precursors - analysis RNA Splicing Small nuclear ribonucleoproteins Spliceosomes Splicing Yeasts |
| title | Identification of a Functional Mammalian Spliceosome Containing Unspliced Pre-mRNA |
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