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Processing of microRNA primary transcripts requires heme in mammalian cells
DiGeorge syndrome critical region gene 8 (DGCR8) is the RNA-binding partner protein of the nuclease Drosha. DGCR8 and Drosha recognize and cleave primary transcripts of microRNAs (pri-miRNAs) in the maturation of canonical microRNAs (miRNAs) in animals. We previously reported that human, frog, and s...
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Published in: | Proceedings of the National Academy of Sciences - PNAS 2014-02, Vol.111 (5), p.1861-1866 |
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description | DiGeorge syndrome critical region gene 8 (DGCR8) is the RNA-binding partner protein of the nuclease Drosha. DGCR8 and Drosha recognize and cleave primary transcripts of microRNAs (pri-miRNAs) in the maturation of canonical microRNAs (miRNAs) in animals. We previously reported that human, frog, and starfish DGCR8 bind heme when expressed in Escherichia coli and that Fe (III) heme activates apoDGCR8 in reconstituted pri-miRNA processing assays. However, the physiological relevance of heme in miRNA maturation has not been clear. Here, we present a live-cell pri-miRNA processing assay that produces robust signals and faithfully indicates DGCR8 and Drosha activities. We demonstrate that all known heme-binding–deficient DGCR8 mutants are defective in pri-miRNA processing in HeLa cells. DGCR8 contains a previously uncharacterized heme-binding motif, "IPCL," that is also required for its activity. Heme availability and biosynthesis in HeLa cells positively affect pri-miRNA processing and production of mature miRNA. These results establish an essential function for heme in pri-miRNA processing in mammalian cells. Our study suggests that abnormal heme biosynthesis and degradation may contribute to diseases via miRNA-mediated gene regulation networks. |
doi_str_mv | 10.1073/pnas.1309915111 |
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DGCR8 and Drosha recognize and cleave primary transcripts of microRNAs (pri-miRNAs) in the maturation of canonical microRNAs (miRNAs) in animals. We previously reported that human, frog, and starfish DGCR8 bind heme when expressed in Escherichia coli and that Fe (III) heme activates apoDGCR8 in reconstituted pri-miRNA processing assays. However, the physiological relevance of heme in miRNA maturation has not been clear. Here, we present a live-cell pri-miRNA processing assay that produces robust signals and faithfully indicates DGCR8 and Drosha activities. We demonstrate that all known heme-binding–deficient DGCR8 mutants are defective in pri-miRNA processing in HeLa cells. DGCR8 contains a previously uncharacterized heme-binding motif, "IPCL," that is also required for its activity. Heme availability and biosynthesis in HeLa cells positively affect pri-miRNA processing and production of mature miRNA. These results establish an essential function for heme in pri-miRNA processing in mammalian cells. Our study suggests that abnormal heme biosynthesis and degradation may contribute to diseases via miRNA-mediated gene regulation networks.</description><identifier>ISSN: 0027-8424</identifier><identifier>EISSN: 1091-6490</identifier><identifier>DOI: 10.1073/pnas.1309915111</identifier><identifier>PMID: 24449907</identifier><language>eng</language><publisher>United States: National Academy of Sciences</publisher><subject>Animals ; Anura ; Asteroidea ; Base Sequence ; Biochemistry ; Biological Sciences ; Biosynthesis ; Cell lines ; E coli ; Escherichia coli ; Fluorescence ; Gene expression ; Genes, Reporter ; HeLa Cells ; Heme - biosynthesis ; Heme - metabolism ; Humans ; Iron ; Journalism ; Mammals ; Marine ; Messenger RNA ; MicroRNA ; MicroRNAs ; MicroRNAs - genetics ; MicroRNAs - metabolism ; Molecular Sequence Data ; Mutant Proteins - metabolism ; Nucleotide Motifs - genetics ; Physiology ; Plasmids ; Protein Binding ; Proteins - metabolism ; Ribonuclease III - metabolism ; RNA ; RNA Processing, Post-Transcriptional - genetics ; RNA, Messenger ; RNA-Binding Proteins ; Transfection</subject><ispartof>Proceedings of the National Academy of Sciences - PNAS, 2014-02, Vol.111 (5), p.1861-1866</ispartof><rights>copyright © 1993–2008 National Academy of Sciences of the United States of America</rights><rights>Copyright National Academy of Sciences Feb 4, 2014</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c565t-90b66e4b6384c970774deae15e86085a9f42c04eeef6cdb46f72c60da9876c953</citedby><cites>FETCH-LOGICAL-c565t-90b66e4b6384c970774deae15e86085a9f42c04eeef6cdb46f72c60da9876c953</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Uhttp://www.pnas.org/content/111/5.cover.gif</thumbnail><linktopdf>$$Uhttps://www.jstor.org/stable/pdf/23766909$$EPDF$$P50$$Gjstor$$H</linktopdf><linktohtml>$$Uhttps://www.jstor.org/stable/23766909$$EHTML$$P50$$Gjstor$$H</linktohtml><link.rule.ids>230,314,727,780,784,885,27924,27925,53791,53793,58238,58471</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/24449907$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Weitz, Sara H.</creatorcontrib><creatorcontrib>Gong, Ming</creatorcontrib><creatorcontrib>Barr, Ian</creatorcontrib><creatorcontrib>Weiss, Shimon</creatorcontrib><creatorcontrib>Guo, Feng</creatorcontrib><title>Processing of microRNA primary transcripts requires heme in mammalian cells</title><title>Proceedings of the National Academy of Sciences - PNAS</title><addtitle>Proc Natl Acad Sci U S A</addtitle><description>DiGeorge syndrome critical region gene 8 (DGCR8) is the RNA-binding partner protein of the nuclease Drosha. DGCR8 and Drosha recognize and cleave primary transcripts of microRNAs (pri-miRNAs) in the maturation of canonical microRNAs (miRNAs) in animals. We previously reported that human, frog, and starfish DGCR8 bind heme when expressed in Escherichia coli and that Fe (III) heme activates apoDGCR8 in reconstituted pri-miRNA processing assays. However, the physiological relevance of heme in miRNA maturation has not been clear. Here, we present a live-cell pri-miRNA processing assay that produces robust signals and faithfully indicates DGCR8 and Drosha activities. We demonstrate that all known heme-binding–deficient DGCR8 mutants are defective in pri-miRNA processing in HeLa cells. DGCR8 contains a previously uncharacterized heme-binding motif, "IPCL," that is also required for its activity. Heme availability and biosynthesis in HeLa cells positively affect pri-miRNA processing and production of mature miRNA. These results establish an essential function for heme in pri-miRNA processing in mammalian cells. Our study suggests that abnormal heme biosynthesis and degradation may contribute to diseases via miRNA-mediated gene regulation networks.</description><subject>Animals</subject><subject>Anura</subject><subject>Asteroidea</subject><subject>Base Sequence</subject><subject>Biochemistry</subject><subject>Biological Sciences</subject><subject>Biosynthesis</subject><subject>Cell lines</subject><subject>E coli</subject><subject>Escherichia coli</subject><subject>Fluorescence</subject><subject>Gene expression</subject><subject>Genes, Reporter</subject><subject>HeLa Cells</subject><subject>Heme - biosynthesis</subject><subject>Heme - metabolism</subject><subject>Humans</subject><subject>Iron</subject><subject>Journalism</subject><subject>Mammals</subject><subject>Marine</subject><subject>Messenger RNA</subject><subject>MicroRNA</subject><subject>MicroRNAs</subject><subject>MicroRNAs - genetics</subject><subject>MicroRNAs - metabolism</subject><subject>Molecular Sequence Data</subject><subject>Mutant Proteins - metabolism</subject><subject>Nucleotide Motifs - genetics</subject><subject>Physiology</subject><subject>Plasmids</subject><subject>Protein Binding</subject><subject>Proteins - metabolism</subject><subject>Ribonuclease III - metabolism</subject><subject>RNA</subject><subject>RNA Processing, Post-Transcriptional - genetics</subject><subject>RNA, Messenger</subject><subject>RNA-Binding Proteins</subject><subject>Transfection</subject><issn>0027-8424</issn><issn>1091-6490</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2014</creationdate><recordtype>article</recordtype><recordid>eNqFkU1v1DAQhi0EokvhzAlkiQuXtDPxV3xBqiq-RAUIwdnyeietV0m8tRMk_j2JdtkWLpx88DOPZt6XsecIZwhGnO8GX85QgLWoEPEBWyFYrLS08JCtAGpTNbKWJ-xJKVsAsKqBx-ykllJaC2bFPn3NKVApcbjmqeV9DDl9-3zBdzn2Pv_iY_ZDCTnuxsIz3U4xU-E31BOPA-993_su-oEH6rrylD1qfVfo2eE9ZT_evf1--aG6-vL-4-XFVRWUVmNlYa01ybUWjQzWgDFyQ55QUaOhUd62sg4giajVYbOWujV10LDxtjE6WCVO2Zu9dzete9oEGuYtO3dY2SUf3d8_Q7xx1-mnExYbY8QseH0Q5HQ7URldH8tygh8oTcXNWdZCKCHx_-icI6KyUs7oq3_QbZryMCexUAYkoNAzdb6n5qBLydQe90ZwS6du6dTddTpPvLx_7pH_U-I9YJk86hCdctjoxfBiD2zLmPKdQBitLVjxG-MusQU</recordid><startdate>20140204</startdate><enddate>20140204</enddate><creator>Weitz, Sara H.</creator><creator>Gong, Ming</creator><creator>Barr, Ian</creator><creator>Weiss, Shimon</creator><creator>Guo, Feng</creator><general>National Academy of Sciences</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>7QG</scope><scope>7QL</scope><scope>7QP</scope><scope>7QR</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TK</scope><scope>7TM</scope><scope>7TO</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>M7N</scope><scope>P64</scope><scope>RC3</scope><scope>7X8</scope><scope>7TN</scope><scope>F1W</scope><scope>H95</scope><scope>L.G</scope><scope>5PM</scope></search><sort><creationdate>20140204</creationdate><title>Processing of microRNA primary transcripts requires heme in mammalian cells</title><author>Weitz, Sara H. ; Gong, Ming ; Barr, Ian ; Weiss, Shimon ; Guo, Feng</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c565t-90b66e4b6384c970774deae15e86085a9f42c04eeef6cdb46f72c60da9876c953</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2014</creationdate><topic>Animals</topic><topic>Anura</topic><topic>Asteroidea</topic><topic>Base Sequence</topic><topic>Biochemistry</topic><topic>Biological Sciences</topic><topic>Biosynthesis</topic><topic>Cell lines</topic><topic>E coli</topic><topic>Escherichia coli</topic><topic>Fluorescence</topic><topic>Gene expression</topic><topic>Genes, Reporter</topic><topic>HeLa Cells</topic><topic>Heme - biosynthesis</topic><topic>Heme - metabolism</topic><topic>Humans</topic><topic>Iron</topic><topic>Journalism</topic><topic>Mammals</topic><topic>Marine</topic><topic>Messenger RNA</topic><topic>MicroRNA</topic><topic>MicroRNAs</topic><topic>MicroRNAs - genetics</topic><topic>MicroRNAs - metabolism</topic><topic>Molecular Sequence Data</topic><topic>Mutant Proteins - metabolism</topic><topic>Nucleotide Motifs - genetics</topic><topic>Physiology</topic><topic>Plasmids</topic><topic>Protein Binding</topic><topic>Proteins - metabolism</topic><topic>Ribonuclease III - metabolism</topic><topic>RNA</topic><topic>RNA Processing, Post-Transcriptional - genetics</topic><topic>RNA, Messenger</topic><topic>RNA-Binding Proteins</topic><topic>Transfection</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Weitz, Sara H.</creatorcontrib><creatorcontrib>Gong, Ming</creatorcontrib><creatorcontrib>Barr, Ian</creatorcontrib><creatorcontrib>Weiss, Shimon</creatorcontrib><creatorcontrib>Guo, Feng</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Animal Behavior Abstracts</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Chemoreception Abstracts</collection><collection>Ecology Abstracts</collection><collection>Entomology Abstracts (Full archive)</collection><collection>Immunology Abstracts</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Oncogenes and Growth Factors Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><collection>Oceanic Abstracts</collection><collection>ASFA: Aquatic Sciences and Fisheries Abstracts</collection><collection>Aquatic Science & Fisheries Abstracts (ASFA) 1: Biological Sciences & Living Resources</collection><collection>Aquatic Science & Fisheries Abstracts (ASFA) Professional</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Proceedings of the National Academy of Sciences - PNAS</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Weitz, Sara H.</au><au>Gong, Ming</au><au>Barr, Ian</au><au>Weiss, Shimon</au><au>Guo, Feng</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Processing of microRNA primary transcripts requires heme in mammalian cells</atitle><jtitle>Proceedings of the National Academy of Sciences - PNAS</jtitle><addtitle>Proc Natl Acad Sci U S A</addtitle><date>2014-02-04</date><risdate>2014</risdate><volume>111</volume><issue>5</issue><spage>1861</spage><epage>1866</epage><pages>1861-1866</pages><issn>0027-8424</issn><eissn>1091-6490</eissn><abstract>DiGeorge syndrome critical region gene 8 (DGCR8) is the RNA-binding partner protein of the nuclease Drosha. DGCR8 and Drosha recognize and cleave primary transcripts of microRNAs (pri-miRNAs) in the maturation of canonical microRNAs (miRNAs) in animals. We previously reported that human, frog, and starfish DGCR8 bind heme when expressed in Escherichia coli and that Fe (III) heme activates apoDGCR8 in reconstituted pri-miRNA processing assays. However, the physiological relevance of heme in miRNA maturation has not been clear. Here, we present a live-cell pri-miRNA processing assay that produces robust signals and faithfully indicates DGCR8 and Drosha activities. We demonstrate that all known heme-binding–deficient DGCR8 mutants are defective in pri-miRNA processing in HeLa cells. DGCR8 contains a previously uncharacterized heme-binding motif, "IPCL," that is also required for its activity. Heme availability and biosynthesis in HeLa cells positively affect pri-miRNA processing and production of mature miRNA. These results establish an essential function for heme in pri-miRNA processing in mammalian cells. Our study suggests that abnormal heme biosynthesis and degradation may contribute to diseases via miRNA-mediated gene regulation networks.</abstract><cop>United States</cop><pub>National Academy of Sciences</pub><pmid>24449907</pmid><doi>10.1073/pnas.1309915111</doi><tpages>6</tpages><oa>free_for_read</oa></addata></record> |
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subjects | Animals Anura Asteroidea Base Sequence Biochemistry Biological Sciences Biosynthesis Cell lines E coli Escherichia coli Fluorescence Gene expression Genes, Reporter HeLa Cells Heme - biosynthesis Heme - metabolism Humans Iron Journalism Mammals Marine Messenger RNA MicroRNA MicroRNAs MicroRNAs - genetics MicroRNAs - metabolism Molecular Sequence Data Mutant Proteins - metabolism Nucleotide Motifs - genetics Physiology Plasmids Protein Binding Proteins - metabolism Ribonuclease III - metabolism RNA RNA Processing, Post-Transcriptional - genetics RNA, Messenger RNA-Binding Proteins Transfection |
title | Processing of microRNA primary transcripts requires heme in mammalian cells |
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