A mutational analysis of the polypyrimidine tract of introns. Effects of sequence differences in pyrimidine tracts on splicing
The polypyrimidine (py) tract of introns is required for efficient spliceosome assembly and splicing of pre-mRNAs. A detailed mutational analysis of the py tract of an adenovirus 2 intron was carried out. Utilizing a “precursor in pieces” vector system, it was possible to synthesize py tract mutant...
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Veröffentlicht in: | The Journal of biological chemistry 1993-05, Vol.268 (15), p.11222-11229 |
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description | The polypyrimidine (py) tract of introns is required for efficient spliceosome assembly and splicing of pre-mRNAs. A detailed mutational analysis of the py tract of an adenovirus 2 intron was carried out. Utilizing a “precursor in pieces” vector system, it was possible to synthesize py tract mutant pre-mRNAs that were otherwise identical. The mutant pre-mRNAs that were otherwise identical. The mutant pre-mRNAs were analyzed for in vitro splicing, for formation of splicing complexes, and for binding to proteins in the HeLa nuclear extract. Chimeric pre-mRNAs that contained the yeast branch point consensus sequence (UAC-UAAC) and altered py tracts were also analyzed. Mutational analysis showed the following. First, any mutation in the py tract that affected splicing did so by interferring with complex A formation in spliceosome assembly. Second, introduction of purines into the py tract is detrimental only if the length of the tract is shortened and if there is a reduction in the number of consecutive uracil residues. Third, uracil and cytosine do not have equivalent functions in the py tract. Our results with chimeric pre-mRNAs also show that a strong py tract can partially replace a weak branch point sequence and a strong branch point sequence can partially replace a weak py tract. Finally, the one surprising finding obtained when examining protein binding was that a mutant pre-mRNA did not bind to heterogeneous nuclear ribonucleoprotein C proteins and yet spliced close to wild type level. |
doi_str_mv | 10.1016/S0021-9258(18)82114-7 |
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Effects of sequence differences in pyrimidine tracts on splicing</title><source>MEDLINE</source><source>Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals</source><source>Alma/SFX Local Collection</source><creator>Roscigno, R.F. ; Weiner, M. ; Garcia-Blanco, M.A.</creator><creatorcontrib>Roscigno, R.F. ; Weiner, M. ; Garcia-Blanco, M.A.</creatorcontrib><description>The polypyrimidine (py) tract of introns is required for efficient spliceosome assembly and splicing of pre-mRNAs. A detailed mutational analysis of the py tract of an adenovirus 2 intron was carried out. Utilizing a “precursor in pieces” vector system, it was possible to synthesize py tract mutant pre-mRNAs that were otherwise identical. The mutant pre-mRNAs that were otherwise identical. The mutant pre-mRNAs were analyzed for in vitro splicing, for formation of splicing complexes, and for binding to proteins in the HeLa nuclear extract. Chimeric pre-mRNAs that contained the yeast branch point consensus sequence (UAC-UAAC) and altered py tracts were also analyzed. Mutational analysis showed the following. First, any mutation in the py tract that affected splicing did so by interferring with complex A formation in spliceosome assembly. Second, introduction of purines into the py tract is detrimental only if the length of the tract is shortened and if there is a reduction in the number of consecutive uracil residues. Third, uracil and cytosine do not have equivalent functions in the py tract. Our results with chimeric pre-mRNAs also show that a strong py tract can partially replace a weak branch point sequence and a strong branch point sequence can partially replace a weak py tract. Finally, the one surprising finding obtained when examining protein binding was that a mutant pre-mRNA did not bind to heterogeneous nuclear ribonucleoprotein C proteins and yet spliced close to wild type level.</description><identifier>ISSN: 0021-9258</identifier><identifier>EISSN: 1083-351X</identifier><identifier>DOI: 10.1016/S0021-9258(18)82114-7</identifier><identifier>PMID: 8496178</identifier><identifier>CODEN: JBCHA3</identifier><language>eng</language><publisher>Bethesda, MD: Elsevier Inc</publisher><subject>Adenoviruses, Human - genetics ; Base Composition ; Base Sequence ; Biological and medical sciences ; Cell Nucleus - metabolism ; Cytosine ; Fundamental and applied biological sciences. Psychology ; HeLa Cells ; Humans ; Introns ; Molecular and cellular biology ; Molecular genetics ; Molecular Sequence Data ; Mutagenesis, Insertional ; Plasmids ; Pyrimidines ; RNA Precursors - genetics ; RNA Precursors - metabolism ; RNA Splicing ; Spliceosomes - metabolism ; Transcription. Transcription factor. Splicing. Rna processing ; Uracil</subject><ispartof>The Journal of biological chemistry, 1993-05, Vol.268 (15), p.11222-11229</ispartof><rights>1993 © 1993 ASBMB. Currently published by Elsevier Inc; originally published by American Society for Biochemistry and Molecular Biology.</rights><rights>1993 INIST-CNRS</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c496t-acc400e0916878f1e969abee964f0359b587b0b18799fbc0e776b095979981913</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27924,27925</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=4803809$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/8496178$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Roscigno, R.F.</creatorcontrib><creatorcontrib>Weiner, M.</creatorcontrib><creatorcontrib>Garcia-Blanco, M.A.</creatorcontrib><title>A mutational analysis of the polypyrimidine tract of introns. Effects of sequence differences in pyrimidine tracts on splicing</title><title>The Journal of biological chemistry</title><addtitle>J Biol Chem</addtitle><description>The polypyrimidine (py) tract of introns is required for efficient spliceosome assembly and splicing of pre-mRNAs. A detailed mutational analysis of the py tract of an adenovirus 2 intron was carried out. Utilizing a “precursor in pieces” vector system, it was possible to synthesize py tract mutant pre-mRNAs that were otherwise identical. The mutant pre-mRNAs that were otherwise identical. The mutant pre-mRNAs were analyzed for in vitro splicing, for formation of splicing complexes, and for binding to proteins in the HeLa nuclear extract. Chimeric pre-mRNAs that contained the yeast branch point consensus sequence (UAC-UAAC) and altered py tracts were also analyzed. Mutational analysis showed the following. First, any mutation in the py tract that affected splicing did so by interferring with complex A formation in spliceosome assembly. Second, introduction of purines into the py tract is detrimental only if the length of the tract is shortened and if there is a reduction in the number of consecutive uracil residues. Third, uracil and cytosine do not have equivalent functions in the py tract. Our results with chimeric pre-mRNAs also show that a strong py tract can partially replace a weak branch point sequence and a strong branch point sequence can partially replace a weak py tract. Finally, the one surprising finding obtained when examining protein binding was that a mutant pre-mRNA did not bind to heterogeneous nuclear ribonucleoprotein C proteins and yet spliced close to wild type level.</description><subject>Adenoviruses, Human - genetics</subject><subject>Base Composition</subject><subject>Base Sequence</subject><subject>Biological and medical sciences</subject><subject>Cell Nucleus - metabolism</subject><subject>Cytosine</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>HeLa Cells</subject><subject>Humans</subject><subject>Introns</subject><subject>Molecular and cellular biology</subject><subject>Molecular genetics</subject><subject>Molecular Sequence Data</subject><subject>Mutagenesis, Insertional</subject><subject>Plasmids</subject><subject>Pyrimidines</subject><subject>RNA Precursors - genetics</subject><subject>RNA Precursors - metabolism</subject><subject>RNA Splicing</subject><subject>Spliceosomes - metabolism</subject><subject>Transcription. Transcription factor. Splicing. Rna processing</subject><subject>Uracil</subject><issn>0021-9258</issn><issn>1083-351X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1993</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkM9rFDEUx4Moda3-CYUcRPQwbd78SnKSUloVCh6q4C1kMi_dyMxkTLKVvfRvb2Z3WcFLc8hL8j7fvC9fQs6AnQOD9uKOsRIKWTbiI4hPogSoC_6CrICJqqga-PWSrI7Ia_Imxt8sr1rCCTkRtWyBixV5vKTjJunk_KQHqvO2jS5Sb2laI539sJ23wY2udxPSFLRJS89NKfgpntNra9GkHR_xzwYng7R3-TEsx5hB-r8-wxON8-CMm-7fkldWDxHfHeop-Xlz_ePqa3H7_cu3q8vbwmSjqdDG1Iwhk9AKLiygbKXuMJfasqqRXSN4xzoQXErbGYactx2Tjcx3ARKqU_Jh_-8cfLYZkxpdNDgMekK_iQraVkrJRQabPWiCjzGgVXO2r8NWAVNL7mqXu1pCVSDULnfFs-7sMGDTjdgfVYegc__9oa-j0YMNejIuHrFasEow-Q9bu_v1XxdQdc6bNY6qbPO8RgGUZZmxz3sMc2YPDoOKxi2J91likuq9e8bvE-VGrGg</recordid><startdate>19930525</startdate><enddate>19930525</enddate><creator>Roscigno, R.F.</creator><creator>Weiner, M.</creator><creator>Garcia-Blanco, M.A.</creator><general>Elsevier Inc</general><general>American Society for Biochemistry and Molecular Biology</general><scope>6I.</scope><scope>AAFTH</scope><scope>IQODW</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>7TM</scope><scope>7U9</scope><scope>H94</scope></search><sort><creationdate>19930525</creationdate><title>A mutational analysis of the polypyrimidine tract of introns. Effects of sequence differences in pyrimidine tracts on splicing</title><author>Roscigno, R.F. ; Weiner, M. ; Garcia-Blanco, M.A.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c496t-acc400e0916878f1e969abee964f0359b587b0b18799fbc0e776b095979981913</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1993</creationdate><topic>Adenoviruses, Human - genetics</topic><topic>Base Composition</topic><topic>Base Sequence</topic><topic>Biological and medical sciences</topic><topic>Cell Nucleus - metabolism</topic><topic>Cytosine</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>HeLa Cells</topic><topic>Humans</topic><topic>Introns</topic><topic>Molecular and cellular biology</topic><topic>Molecular genetics</topic><topic>Molecular Sequence Data</topic><topic>Mutagenesis, Insertional</topic><topic>Plasmids</topic><topic>Pyrimidines</topic><topic>RNA Precursors - genetics</topic><topic>RNA Precursors - metabolism</topic><topic>RNA Splicing</topic><topic>Spliceosomes - metabolism</topic><topic>Transcription. Transcription factor. Splicing. Rna processing</topic><topic>Uracil</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Roscigno, R.F.</creatorcontrib><creatorcontrib>Weiner, M.</creatorcontrib><creatorcontrib>Garcia-Blanco, M.A.</creatorcontrib><collection>ScienceDirect Open Access Titles</collection><collection>Elsevier:ScienceDirect:Open Access</collection><collection>Pascal-Francis</collection><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>Virology and AIDS Abstracts</collection><collection>AIDS and Cancer Research Abstracts</collection><jtitle>The Journal of biological chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Roscigno, R.F.</au><au>Weiner, M.</au><au>Garcia-Blanco, M.A.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>A mutational analysis of the polypyrimidine tract of introns. Effects of sequence differences in pyrimidine tracts on splicing</atitle><jtitle>The Journal of biological chemistry</jtitle><addtitle>J Biol Chem</addtitle><date>1993-05-25</date><risdate>1993</risdate><volume>268</volume><issue>15</issue><spage>11222</spage><epage>11229</epage><pages>11222-11229</pages><issn>0021-9258</issn><eissn>1083-351X</eissn><coden>JBCHA3</coden><abstract>The polypyrimidine (py) tract of introns is required for efficient spliceosome assembly and splicing of pre-mRNAs. A detailed mutational analysis of the py tract of an adenovirus 2 intron was carried out. Utilizing a “precursor in pieces” vector system, it was possible to synthesize py tract mutant pre-mRNAs that were otherwise identical. The mutant pre-mRNAs that were otherwise identical. The mutant pre-mRNAs were analyzed for in vitro splicing, for formation of splicing complexes, and for binding to proteins in the HeLa nuclear extract. Chimeric pre-mRNAs that contained the yeast branch point consensus sequence (UAC-UAAC) and altered py tracts were also analyzed. Mutational analysis showed the following. First, any mutation in the py tract that affected splicing did so by interferring with complex A formation in spliceosome assembly. Second, introduction of purines into the py tract is detrimental only if the length of the tract is shortened and if there is a reduction in the number of consecutive uracil residues. Third, uracil and cytosine do not have equivalent functions in the py tract. Our results with chimeric pre-mRNAs also show that a strong py tract can partially replace a weak branch point sequence and a strong branch point sequence can partially replace a weak py tract. Finally, the one surprising finding obtained when examining protein binding was that a mutant pre-mRNA did not bind to heterogeneous nuclear ribonucleoprotein C proteins and yet spliced close to wild type level.</abstract><cop>Bethesda, MD</cop><pub>Elsevier Inc</pub><pmid>8496178</pmid><doi>10.1016/S0021-9258(18)82114-7</doi><tpages>8</tpages><oa>free_for_read</oa></addata></record> |
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subjects | Adenoviruses, Human - genetics Base Composition Base Sequence Biological and medical sciences Cell Nucleus - metabolism Cytosine Fundamental and applied biological sciences. Psychology HeLa Cells Humans Introns Molecular and cellular biology Molecular genetics Molecular Sequence Data Mutagenesis, Insertional Plasmids Pyrimidines RNA Precursors - genetics RNA Precursors - metabolism RNA Splicing Spliceosomes - metabolism Transcription. Transcription factor. Splicing. Rna processing Uracil |
title | A mutational analysis of the polypyrimidine tract of introns. Effects of sequence differences in pyrimidine tracts on splicing |
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