Identification of Novel Translated Small Open Reading Frames in Escherichia coli Using Complementary Ribosome Profiling Approaches
Small proteins of
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| Veröffentlicht in: | Journal of bacteriology 2022-01, Vol.204 (1), p.1 |
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| container_title | Journal of bacteriology |
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| creator | Stringer, Anne Smith, Carol Mangano, Kyle Wade, Joseph T |
| description | Small proteins of |
| doi_str_mv | 10.1128/JB.00352-21 |
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Ribosome profiling has been used to infer the existence of small proteins by detecting the translation of the corresponding open reading frames (ORFs). Detection of translated short ORFs by ribosome profiling can be improved by treating cells with drugs that stall ribosomes at specific codons. Here, we combine the analysis of ribosome profiling data for Escherichia coli cells treated with antibiotics that stall ribosomes at either the start or stop codons. Thus, we identify ribosome-occupied start and stop codons with high sensitivity for ∼400 novel putative ORFs. The newly discovered ORFs are mostly short, with 365 encoding proteins of <51 amino acids. We validate translation of several selected short ORFs and show that many likely encode unstable proteins. Moreover, we present evidence that most of the newly identified short ORFs are not under purifying selection, suggesting that they do not impact cell fitness, although a small subset have the hallmarks of functional ORFs. IMPORTANCE Small proteins of <51 amino acids are abundant across all domains of life, but they are often overlooked because their small size makes them difficult to predict computationally and they are refractory to standard proteomic approaches. Recent studies have discovered small proteins by mapping the location of translating ribosomes on RNA using a technique known as ribosome profiling. Discovery of translated sORFs using ribosome profiling can be improved by treating cells with drugs that trap initiating ribosomes. Here, we show that combining these data with equivalent data for cells treated with a drug that stalls terminating ribosomes facilitates the discovery of small proteins. We use this approach to discover 365 putative genes that encode small proteins in Escherichia coli.</description><identifier>ISSN: 0021-9193</identifier><identifier>EISSN: 1098-5530</identifier><identifier>DOI: 10.1128/JB.00352-21</identifier><identifier>PMID: 34662240</identifier><language>eng</language><publisher>1752 N St., N.W., Washington, DC: American Society for Microbiology</publisher><subject>Amino acids ; Antibiotics ; Bacteriology ; Codons ; E coli ; Escherichia coli ; Genomics and Proteomics ; Meeting Presentation ; Open reading frames ; Proteins ; Proteomics ; Ribosomes ; Special Sections: Small Proteins, Big Questions 2021 ; Translation</subject><ispartof>Journal of bacteriology, 2022-01, Vol.204 (1), p.1</ispartof><rights>Copyright © 2022 American Society for Microbiology.</rights><rights>Copyright American Society for Microbiology Jan 2022</rights><rights>Copyright © 2022 American Society for Microbiology. 2022 American Society for Microbiology</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-a386t-25d38a701db5f472cde178c5f7bd067806db09530c42eff6f032973f4df488f23</citedby><cites>FETCH-LOGICAL-a386t-25d38a701db5f472cde178c5f7bd067806db09530c42eff6f032973f4df488f23</cites><orcidid>0000-0002-9779-3160</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/PMC8765432/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC8765432/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,727,780,784,885,27924,27925,53791,53793</link.rule.ids></links><search><contributor>Henkin, Tina M</contributor><contributor>Henkin, Tina M.</contributor><creatorcontrib>Stringer, Anne</creatorcontrib><creatorcontrib>Smith, Carol</creatorcontrib><creatorcontrib>Mangano, Kyle</creatorcontrib><creatorcontrib>Wade, Joseph T</creatorcontrib><title>Identification of Novel Translated Small Open Reading Frames in Escherichia coli Using Complementary Ribosome Profiling Approaches</title><title>Journal of bacteriology</title><addtitle>J Bacteriol</addtitle><description>Small proteins of <51 amino acids are abundant across all domains of life, but they are often overlooked because their small size makes them difficult to predict computationally and they are refractory to standard proteomic approaches. Ribosome profiling has been used to infer the existence of small proteins by detecting the translation of the corresponding open reading frames (ORFs). Detection of translated short ORFs by ribosome profiling can be improved by treating cells with drugs that stall ribosomes at specific codons. Here, we combine the analysis of ribosome profiling data for Escherichia coli cells treated with antibiotics that stall ribosomes at either the start or stop codons. Thus, we identify ribosome-occupied start and stop codons with high sensitivity for ∼400 novel putative ORFs. The newly discovered ORFs are mostly short, with 365 encoding proteins of <51 amino acids. We validate translation of several selected short ORFs and show that many likely encode unstable proteins. Moreover, we present evidence that most of the newly identified short ORFs are not under purifying selection, suggesting that they do not impact cell fitness, although a small subset have the hallmarks of functional ORFs. IMPORTANCE Small proteins of <51 amino acids are abundant across all domains of life, but they are often overlooked because their small size makes them difficult to predict computationally and they are refractory to standard proteomic approaches. Recent studies have discovered small proteins by mapping the location of translating ribosomes on RNA using a technique known as ribosome profiling. Discovery of translated sORFs using ribosome profiling can be improved by treating cells with drugs that trap initiating ribosomes. Here, we show that combining these data with equivalent data for cells treated with a drug that stalls terminating ribosomes facilitates the discovery of small proteins. We use this approach to discover 365 putative genes that encode small proteins in Escherichia coli.</description><subject>Amino acids</subject><subject>Antibiotics</subject><subject>Bacteriology</subject><subject>Codons</subject><subject>E coli</subject><subject>Escherichia coli</subject><subject>Genomics and Proteomics</subject><subject>Meeting Presentation</subject><subject>Open reading frames</subject><subject>Proteins</subject><subject>Proteomics</subject><subject>Ribosomes</subject><subject>Special Sections: Small Proteins, Big Questions 2021</subject><subject>Translation</subject><issn>0021-9193</issn><issn>1098-5530</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2022</creationdate><recordtype>article</recordtype><recordid>eNptkUtLLDEQRoMoOj5W_oGAG0Fa8-xObwRn0HsVUfGxDuk8nEi60yY9glt_-e25I4rgqhZ16tRXFAD7GB1jTMTJ1fQYIcpJQfAamGBUi4JzitbBBCGCixrXdAts5_yCEGaMk02wRVlZEsLQBHxcGtsN3nmtBh87GB28iW82wMekuhzUYA18aFUI8La3Hby3yvjuGV4k1doMfQfPs57b5PXcK6hj8PApL4FZbPtg29Gt0ju8903MsbXwLkXnwxI46_sU1Tibd8GGUyHbvc-6A54uzh9nf4vr2z-Xs7PrQlFRDgXhhgpVIWwa7lhFtLG4Epq7qjGorAQqTYPq8XDNiHWudIiSuqKOGceEcITugNOVt180rTV6zJZUkH3y7ZhRRuXlz07n5_I5vklRlZzRpeDgU5Di68LmQb7ERerGzJKUBHMhMK9G6mhF6RRzTtZ9bcBILh8mr6by_8MkwSN9uKJVbsm37zf0Hxm5lUo</recordid><startdate>20220118</startdate><enddate>20220118</enddate><creator>Stringer, Anne</creator><creator>Smith, Carol</creator><creator>Mangano, Kyle</creator><creator>Wade, Joseph T</creator><general>American Society for Microbiology</general><scope>AAYXX</scope><scope>CITATION</scope><scope>7QL</scope><scope>7TM</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>5PM</scope><orcidid>https://orcid.org/0000-0002-9779-3160</orcidid></search><sort><creationdate>20220118</creationdate><title>Identification of Novel Translated Small Open Reading Frames in Escherichia coli Using Complementary Ribosome Profiling Approaches</title><author>Stringer, Anne ; Smith, Carol ; Mangano, Kyle ; Wade, Joseph T</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a386t-25d38a701db5f472cde178c5f7bd067806db09530c42eff6f032973f4df488f23</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2022</creationdate><topic>Amino acids</topic><topic>Antibiotics</topic><topic>Bacteriology</topic><topic>Codons</topic><topic>E coli</topic><topic>Escherichia coli</topic><topic>Genomics and Proteomics</topic><topic>Meeting Presentation</topic><topic>Open reading frames</topic><topic>Proteins</topic><topic>Proteomics</topic><topic>Ribosomes</topic><topic>Special Sections: Small Proteins, Big Questions 2021</topic><topic>Translation</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Stringer, Anne</creatorcontrib><creatorcontrib>Smith, Carol</creatorcontrib><creatorcontrib>Mangano, Kyle</creatorcontrib><creatorcontrib>Wade, Joseph T</creatorcontrib><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Nucleic Acids 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>PubMed Central (Full Participant titles)</collection><jtitle>Journal of bacteriology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Stringer, Anne</au><au>Smith, Carol</au><au>Mangano, Kyle</au><au>Wade, Joseph T</au><au>Henkin, Tina M</au><au>Henkin, Tina M.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Identification of Novel Translated Small Open Reading Frames in Escherichia coli Using Complementary Ribosome Profiling Approaches</atitle><jtitle>Journal of bacteriology</jtitle><stitle>J Bacteriol</stitle><date>2022-01-18</date><risdate>2022</risdate><volume>204</volume><issue>1</issue><spage>1</spage><pages>1-</pages><issn>0021-9193</issn><eissn>1098-5530</eissn><abstract>Small proteins of <51 amino acids are abundant across all domains of life, but they are often overlooked because their small size makes them difficult to predict computationally and they are refractory to standard proteomic approaches. Ribosome profiling has been used to infer the existence of small proteins by detecting the translation of the corresponding open reading frames (ORFs). Detection of translated short ORFs by ribosome profiling can be improved by treating cells with drugs that stall ribosomes at specific codons. Here, we combine the analysis of ribosome profiling data for Escherichia coli cells treated with antibiotics that stall ribosomes at either the start or stop codons. Thus, we identify ribosome-occupied start and stop codons with high sensitivity for ∼400 novel putative ORFs. The newly discovered ORFs are mostly short, with 365 encoding proteins of <51 amino acids. We validate translation of several selected short ORFs and show that many likely encode unstable proteins. Moreover, we present evidence that most of the newly identified short ORFs are not under purifying selection, suggesting that they do not impact cell fitness, although a small subset have the hallmarks of functional ORFs. IMPORTANCE Small proteins of <51 amino acids are abundant across all domains of life, but they are often overlooked because their small size makes them difficult to predict computationally and they are refractory to standard proteomic approaches. Recent studies have discovered small proteins by mapping the location of translating ribosomes on RNA using a technique known as ribosome profiling. Discovery of translated sORFs using ribosome profiling can be improved by treating cells with drugs that trap initiating ribosomes. Here, we show that combining these data with equivalent data for cells treated with a drug that stalls terminating ribosomes facilitates the discovery of small proteins. We use this approach to discover 365 putative genes that encode small proteins in Escherichia coli.</abstract><cop>1752 N St., N.W., Washington, DC</cop><pub>American Society for Microbiology</pub><pmid>34662240</pmid><doi>10.1128/JB.00352-21</doi><tpages>13</tpages><orcidid>https://orcid.org/0000-0002-9779-3160</orcidid><oa>free_for_read</oa></addata></record> |
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| subjects | Amino acids Antibiotics Bacteriology Codons E coli Escherichia coli Genomics and Proteomics Meeting Presentation Open reading frames Proteins Proteomics Ribosomes Special Sections: Small Proteins, Big Questions 2021 Translation |
| title | Identification of Novel Translated Small Open Reading Frames in Escherichia coli Using Complementary Ribosome Profiling Approaches |
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