{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/89193"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/89193","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Single molecule pull down of double strand RNA binding proteins","abstract":"RNA molecules are transcribed as single stranded naturally, but with most of them forming in to structures composed of duplex regions, loop, bulge or mismatches. RNAs with double stranded regions, or known as double-strand RNAs (dsRNAs). The class of proteins responsible for processing dsRNAs is termed double-stranded RNA binding proteins (dsRBP). In recent decades, an increasing number of reports have shown the role of dsRBP-dsRNA interaction as core strategy in various cellular regulation pathways, including RNA interference, anti-viral immunity, mRNA transport and alternative splicing. However, little is known about the molecular mechanisms underlying the interaction between dsRBPs and dsRNA. Here we examined four human dsRBPs, ADAD2, TRBP, Staufen 1 and ADAR1 `which have various numbers of RNA binding domains expressed in mammalian cells. We applied single molecule pull-down (SiMPull) assay to investigate the intensity of various dsRNA-dsRBP interactions. Our results demonstrate that despite the highly conserved dsRNA binding domains, the dsRBPs exhibit diverse substrate specificy. While TRBP and ADAR1 have a preference for binding simple duplex RNA, ADAD2 and Staufen1 display higher affinity to imperfectly base-paired structured RNA substrates. We also demonstrate ATP-independent sliding activity of TRBP and Staufen probed by single molecule protein induced fluorescence enhancement (smPIFE), which demonstrates how single molecule approaches could be utilized to provide new insight into molecular mechanisms involved in protein-RNA interaction. Collectively, our study highlights the diverse nature of substrate specificity exhibited by dsRBPs that may be critical for their cellular function.","abstract_html":"RNA molecules are transcribed as single stranded naturally, but with most of them forming in to structures composed of duplex regions, loop, bulge or mismatches. RNAs with double stranded regions, or known as double-strand RNAs (dsRNAs). The class of proteins responsible for processing dsRNAs is termed double-stranded RNA binding proteins (dsRBP). In recent decades, an increasing number of reports have shown the role of dsRBP-dsRNA interaction as core strategy in various cellular regulation pathways, including RNA interference, anti-viral immunity, mRNA transport and alternative splicing. However, little is known about the molecular mechanisms underlying the interaction between dsRBPs and dsRNA. Here we examined four human dsRBPs, ADAD2, TRBP, Staufen 1 and ADAR1 `which have various numbers of RNA binding domains expressed in mammalian cells. We applied single molecule pull-down (SiMPull) assay to investigate the intensity of various dsRNA-dsRBP interactions. Our results demonstrate that despite the highly conserved dsRNA binding domains, the dsRBPs exhibit diverse substrate specificy. While TRBP and ADAR1 have a preference for binding simple duplex RNA, ADAD2 and Staufen1 display higher affinity to imperfectly base-paired structured RNA substrates. We also demonstrate ATP-independent sliding activity of TRBP and Staufen probed by single molecule protein induced fluorescence enhancement (smPIFE), which demonstrates how single molecule approaches could be utilized to provide new insight into molecular mechanisms involved in protein-RNA interaction. Collectively, our study highlights the diverse nature of substrate specificity exhibited by dsRBPs that may be critical for their cellular function.","abstract_has_math":false,"creators":["Wang, Xinlei"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"M.S.","degree_level":"Thesis","degree_discipline":"Bioengineering","degree_department":null,"school":null,"contributors":["Myong, Su-A"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2015,"date_issued":"2015-12","date_published":"2015-12","updated_at":"2026-07-22T22:26:32Z","subjects":["Single molecule","RNA protein interaction","double-strand RNA binding protein"],"languages":["en"],"rights":["Copyright 2015 Xinlei Wang"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/2142/89193","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Myong, Su-A"]},{"key":"dc:creator","label":"Author","values":["Wang, Xinlei"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2015-12","2016-03-02T21:06:23Z","2018-03-03T10:15:21Z","2015-11-12"]},{"key":"dc:type","label":"Dc Type","values":["text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Bioengineering"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Thesis"]},{"key":"thesis:degree_name","label":"Degree Name","values":["M.S."]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["University of Illinois at Urbana-Champaign"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Single molecule","RNA protein interaction","double-strand RNA binding protein"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright 2015 Xinlei Wang"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://hdl.handle.net/2142/89193"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["RNA molecules are transcribed as single stranded naturally, but with most of them forming in to structures composed of duplex regions, loop, bulge or mismatches. RNAs with double stranded regions, or known as double-strand RNAs (dsRNAs). The class of proteins responsible for processing dsRNAs is termed double-stranded RNA binding proteins (dsRBP). In recent decades, an increasing number of reports have shown the role of dsRBP-dsRNA interaction as core strategy in various cellular regulation pathways, including RNA interference, anti-viral immunity, mRNA transport and alternative splicing. However, little is known about the molecular mechanisms underlying the interaction between dsRBPs and dsRNA. Here we examined four human dsRBPs, ADAD2, TRBP, Staufen 1 and ADAR1 `which have various numbers of RNA binding domains expressed in mammalian cells. We applied single molecule pull-down (SiMPull) assay to investigate the intensity of various dsRNA-dsRBP interactions. Our results demonstrate that despite the highly conserved dsRNA binding domains, the dsRBPs exhibit diverse substrate specificy. While TRBP and ADAR1 have a preference for binding simple duplex RNA, ADAD2 and Staufen1 display higher affinity to imperfectly base-paired structured RNA substrates. We also demonstrate ATP-independent sliding activity of TRBP and Staufen probed by single molecule protein induced fluorescence enhancement (smPIFE), which demonstrates how single molecule approaches could be utilized to provide new insight into molecular mechanisms involved in protein-RNA interaction. Collectively, our study highlights the diverse nature of substrate specificity exhibited by dsRBPs that may be critical for their cellular function.","Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2017-12-01","The student, Xinlei Wang, accepted the attached license on 2015-11-08 at 21:17.","The student, Xinlei Wang, submitted this Thesis for approval on 2015-11-08 at 21:22.","This Thesis was approved for publication on 2015-11-12 at 09:09.","DSpace SAF Submission Ingestion Package generated from Vireo submission #8767 on 2016-03-02 at 14:12:57","Made available in DSpace on 2016-03-02T21:06:23Z (GMT). No. of bitstreams: 2 WANG-THESIS-2015.pdf: 5728362 bytes, checksum: 67c53b8c9e10c006d50696de3db0a13f (MD5) LICENSE.txt: 4208 bytes, checksum: 34f4dea29d5423019a93798c118118e9 (MD5) Previous issue date: 2015-11-12","Embargo set by: Seth Robbins for item 91396 Lift date: 2018-03-02T21:07:27Z Reason: Author requested closed access (OA after 2yrs) in Vireo ETD system","Limited Restriction Lifted for Item 91396 on 2018-03-03T10:15:21Z."]},{"key":"dc:format","label":"Dc Format","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Single molecule pull down of double strand RNA binding proteins"]}]}],"canonical_facts":{"dc:contributor":["Myong, Su-A"],"dc:creator":["Wang, Xinlei"],"dc:date":["2015-12","2016-03-02T21:06:23Z","2018-03-03T10:15:21Z","2015-11-12"],"dc:description":["RNA molecules are transcribed as single stranded naturally, but with most of them forming in to structures composed of duplex regions, loop, bulge or mismatches. RNAs with double stranded regions, or known as double-strand RNAs (dsRNAs). The class of proteins responsible for processing dsRNAs is termed double-stranded RNA binding proteins (dsRBP). In recent decades, an increasing number of reports have shown the role of dsRBP-dsRNA interaction as core strategy in various cellular regulation pathways, including RNA interference, anti-viral immunity, mRNA transport and alternative splicing. However, little is known about the molecular mechanisms underlying the interaction between dsRBPs and dsRNA. Here we examined four human dsRBPs, ADAD2, TRBP, Staufen 1 and ADAR1 `which have various numbers of RNA binding domains expressed in mammalian cells. We applied single molecule pull-down (SiMPull) assay to investigate the intensity of various dsRNA-dsRBP interactions. Our results demonstrate that despite the highly conserved dsRNA binding domains, the dsRBPs exhibit diverse substrate specificy. While TRBP and ADAR1 have a preference for binding simple duplex RNA, ADAD2 and Staufen1 display higher affinity to imperfectly base-paired structured RNA substrates. We also demonstrate ATP-independent sliding activity of TRBP and Staufen probed by single molecule protein induced fluorescence enhancement (smPIFE), which demonstrates how single molecule approaches could be utilized to provide new insight into molecular mechanisms involved in protein-RNA interaction. Collectively, our study highlights the diverse nature of substrate specificity exhibited by dsRBPs that may be critical for their cellular function.","Submission published under a 24 month embargo labeled 'Closed Access', the embargo will last until 2017-12-01","The student, Xinlei Wang, accepted the attached license on 2015-11-08 at 21:17.","The student, Xinlei Wang, submitted this Thesis for approval on 2015-11-08 at 21:22.","This Thesis was approved for publication on 2015-11-12 at 09:09.","DSpace SAF Submission Ingestion Package generated from Vireo submission #8767 on 2016-03-02 at 14:12:57","Made available in DSpace on 2016-03-02T21:06:23Z (GMT). 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