Applying a high-throughput fluorescence polarization assay for the discovery of chemical probes blocking La:RNA interactions in vitro and in cells
The RNA-binding protein La is overexpressed in a number of tumor tissues and is thought to support tumorigenesis by binding to and facilitating the expression of mRNAs encoding tumor-promoting and anti-apoptotic factors. Hence, small molecules able to block the binding of La to specific RNAs could h...
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| creator | Sommer, Gunhild Fedarovich, Alena Kota, Venkatesh Rodriguez, Reycel Smith, Charles D Heise, Tilman |
| description | The RNA-binding protein La is overexpressed in a number of tumor tissues and is thought to support tumorigenesis by binding to and facilitating the expression of mRNAs encoding tumor-promoting and anti-apoptotic factors. Hence, small molecules able to block the binding of La to specific RNAs could have a therapeutic impact by reducing the expression of tumor-promoting and anti-apoptotic factors. Toward this novel therapeutic strategy, we aimed to develop a high-throughput fluorescence polarization assay to screen small compound libraries for molecules blocking the binding of La to an RNA element derived from cyclin D1 mRNA. Herein, we make use of a robust fluorescence polarization assay and the validation of primary hits by electrophoretic mobility shift assays. We showed recently that La protects cells against cisplatin treatment by stimulating the protein synthesis of the anti-apoptotic factor Bcl2. Here, we show by RNA immunoprecipitation experiments that one small compound specifically impairs the association of La with Bcl2 mRNA in cells and sensitizes cells for cipslatin-induced cell death. In summary, we report the application of a high-throughput fluorescence polarization assay to identify small compounds that impair the binding of La to target RNAs in vitro and in cells. |
| doi_str_mv | 10.1371/journal.pone.0173246 |
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Hence, small molecules able to block the binding of La to specific RNAs could have a therapeutic impact by reducing the expression of tumor-promoting and anti-apoptotic factors. Toward this novel therapeutic strategy, we aimed to develop a high-throughput fluorescence polarization assay to screen small compound libraries for molecules blocking the binding of La to an RNA element derived from cyclin D1 mRNA. Herein, we make use of a robust fluorescence polarization assay and the validation of primary hits by electrophoretic mobility shift assays. We showed recently that La protects cells against cisplatin treatment by stimulating the protein synthesis of the anti-apoptotic factor Bcl2. Here, we show by RNA immunoprecipitation experiments that one small compound specifically impairs the association of La with Bcl2 mRNA in cells and sensitizes cells for cipslatin-induced cell death. 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This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2017 Sommer et al 2017 Sommer et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c725t-d5e48417e428d0952755a2f05429c52c24ee1bc63504097a67029e2327e3d0fa3</citedby><cites>FETCH-LOGICAL-c725t-d5e48417e428d0952755a2f05429c52c24ee1bc63504097a67029e2327e3d0fa3</cites><orcidid>0000-0002-3248-3129</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/PMC5349447/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5349447/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,2096,2915,23845,27901,27902,53766,53768,79342,79343</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/28291789$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Sommer, Gunhild</creatorcontrib><creatorcontrib>Fedarovich, Alena</creatorcontrib><creatorcontrib>Kota, Venkatesh</creatorcontrib><creatorcontrib>Rodriguez, Reycel</creatorcontrib><creatorcontrib>Smith, Charles D</creatorcontrib><creatorcontrib>Heise, Tilman</creatorcontrib><title>Applying a high-throughput fluorescence polarization assay for the discovery of chemical probes blocking La:RNA interactions in vitro and in cells</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>The RNA-binding protein La is overexpressed in a number of tumor tissues and is thought to support tumorigenesis by binding to and facilitating the expression of mRNAs encoding tumor-promoting and anti-apoptotic factors. 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In summary, we report the application of a high-throughput fluorescence polarization assay to identify small compounds that impair the binding of La to target RNAs in vitro and in cells.</description><subject>Analysis</subject><subject>Apoptosis</subject><subject>Assaying</subject><subject>Binding</subject><subject>Biochemical assays</subject><subject>Biochemistry</subject><subject>Biology and life sciences</subject><subject>Blocking</subject><subject>Cancer</subject><subject>Cell death</subject><subject>Cell Line</subject><subject>Cisplatin</subject><subject>Cyclin D1</subject><subject>DNA probes</subject><subject>Electrophoretic mobility</subject><subject>Electrophoretic Mobility Shift Assay</subject><subject>Fluorescence</subject><subject>Fluorescence polarization</subject><subject>Fluorescence Polarization - methods</subject><subject>Fluorescent indicators</subject><subject>Hepatitis</subject><subject>High-Throughput Screening Assays - methods</subject><subject>Humans</subject><subject>Immunoprecipitation</subject><subject>In Vitro Techniques</subject><subject>Messenger RNA</subject><subject>Molecular biology</subject><subject>Molecular Probes - 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Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Sommer, Gunhild</au><au>Fedarovich, Alena</au><au>Kota, Venkatesh</au><au>Rodriguez, Reycel</au><au>Smith, Charles D</au><au>Heise, Tilman</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Applying a high-throughput fluorescence polarization assay for the discovery of chemical probes blocking La:RNA interactions in vitro and in cells</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2017-03-14</date><risdate>2017</risdate><volume>12</volume><issue>3</issue><spage>e0173246</spage><epage>e0173246</epage><pages>e0173246-e0173246</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>The RNA-binding protein La is overexpressed in a number of tumor tissues and is thought to support tumorigenesis by binding to and facilitating the expression of mRNAs encoding tumor-promoting and anti-apoptotic factors. Hence, small molecules able to block the binding of La to specific RNAs could have a therapeutic impact by reducing the expression of tumor-promoting and anti-apoptotic factors. Toward this novel therapeutic strategy, we aimed to develop a high-throughput fluorescence polarization assay to screen small compound libraries for molecules blocking the binding of La to an RNA element derived from cyclin D1 mRNA. Herein, we make use of a robust fluorescence polarization assay and the validation of primary hits by electrophoretic mobility shift assays. We showed recently that La protects cells against cisplatin treatment by stimulating the protein synthesis of the anti-apoptotic factor Bcl2. Here, we show by RNA immunoprecipitation experiments that one small compound specifically impairs the association of La with Bcl2 mRNA in cells and sensitizes cells for cipslatin-induced cell death. In summary, we report the application of a high-throughput fluorescence polarization assay to identify small compounds that impair the binding of La to target RNAs in vitro and in cells.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>28291789</pmid><doi>10.1371/journal.pone.0173246</doi><tpages>e0173246</tpages><orcidid>https://orcid.org/0000-0002-3248-3129</orcidid><oa>free_for_read</oa></addata></record> |
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| subjects | Analysis Apoptosis Assaying Binding Biochemical assays Biochemistry Biology and life sciences Blocking Cancer Cell death Cell Line Cisplatin Cyclin D1 DNA probes Electrophoretic mobility Electrophoretic Mobility Shift Assay Fluorescence Fluorescence polarization Fluorescence Polarization - methods Fluorescent indicators Hepatitis High-Throughput Screening Assays - methods Humans Immunoprecipitation In Vitro Techniques Messenger RNA Molecular biology Molecular Probes - chemistry Molecular Probes - pharmacology mRNA Phosphorylation Polarization Protein Binding Protein biosynthesis Protein synthesis Proteins Research and Analysis Methods Ribonucleic acid RNA RNA - antagonists & inhibitors RNA - metabolism RNA polymerase RNA probes RNA-binding protein RNA-Binding Proteins - antagonists & inhibitors RNA-Binding Proteins - metabolism Tissues Tumorigenesis Viral infections Viruses |
| title | Applying a high-throughput fluorescence polarization assay for the discovery of chemical probes blocking La:RNA interactions in vitro and in cells |
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