Analysis of HeLa cell premessenger RNA splicing complexes containing the snRNP U1 by native gel electrophoresis

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dc.contributor.advisorBerget, Susan M.en_US
dc.creatorZillmann, Martinen_US
dc.date.accessioned2009-06-04T00:44:08Zen_US
dc.date.available2009-06-04T00:44:08Zen_US
dc.date.issued1988en_US
dc.description.abstractThe typical eukaryotic RNA polymerase II primary transcript is divided into regions that encode information expressed at the protein level (exons) and those which do not (introns). The latter must be removed from the transcript rapidly and with proper joining of the coding sequences during the maturation of the transcript in the nucleus. This process is termed splicing and is accompanied by the sequential addition of factors to the primary transcript resulting in the formation of a series of large ribonucleoprotein particles. The splicing reaction can be studied in vitro in HeLa cell nuclear extracts by the addition of a capped, in vitro transcribed splicing precursor RNA. A native gel electrophoresis system was developed which allowed resolution of various ribonucleoprotein complexes and the study of the splicing complex and intermediates in its formation. The HeLa cell nuclear extracts were found to immediately assemble exogenously added precursor RNAs into rapidly migrating complexes. With time, complexes migrating more slowly were observed. The complex migrating the most slowly appeared concurrently with the products of 5$\sp\prime$ junction cleavage. This complex was identified as the "active" splicing complex by the presence of reaction intermediates and the requirement for both ATP and splicing consensus sequences for its formation. Later in the reation, when large amounts of ligated RNA had been generated, rapidly migrating complexes reappeared. All of these complexes contained U snRNPs as defined by immunoprecipitation of gel-fractionated complexes. In particular, eluted active complex contained U1 snRNPs as defined by the ability of anti-U1 antiserum to immunoprecipitate this particle in the presence of competing free, unlabelled precursor RNA. Previously reported gel systems appear to resolve active complexes devoid of U1 snRNPs, a snRNP known to be required for splicing both in vivo and in vitro. Furthermore, the 5$\sp\prime$ splice junction, the region to which U1 snRNPs bind, was protected from oligonucleotide directed RNase H cleavage in eluted complexes, indicating that a 5$\sp\prime$ factor remained bound during electrophoresis. Apparently, complexes eluted from the native system retain most of the properties, other than activity, found for these complexes in whole extracts, suggesting that this gel system is an ideal tool for the study of the ribonucleoprotein complexes involved in splicing.en_US
dc.format.extent126 p.en_US
dc.format.mimetypeapplication/pdfen_US
dc.identifier.callnoThesis Biochem. 1988 Zillmannen_US
dc.identifier.citationZillmann, Martin. "Analysis of HeLa cell premessenger RNA splicing complexes containing the snRNP U1 by native gel electrophoresis." (1988) Diss., Rice University. <a href="https://hdl.handle.net/1911/16202">https://hdl.handle.net/1911/16202</a>.en_US
dc.identifier.urihttps://hdl.handle.net/1911/16202en_US
dc.language.isoengen_US
dc.rightsCopyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.en_US
dc.subjectMolecular biologyen_US
dc.titleAnalysis of HeLa cell premessenger RNA splicing complexes containing the snRNP U1 by native gel electrophoresisen_US
dc.typeThesisen_US
dc.type.materialTexten_US
thesis.degree.departmentBiochemistry and Cell Biologyen_US
thesis.degree.disciplineNatural Sciencesen_US
thesis.degree.grantorRice Universityen_US
thesis.degree.levelDoctoralen_US
thesis.degree.nameDoctor of Philosophyen_US
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