{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/98111"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/98111","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"RNA in the nucleus: insight into location and biological function","abstract":"RNA is an important constituent of the nucleus with functions including regulation of RNA biogenesis, regulation of gene expression, and dosage compensation. Currently used technologies to study RNA chromatin interactions pull down individual RNAs and sequence the associated DNA. However, it remains challenging to identify chromatin associated RNAs and their genomic target sites. In this thesis, I will describe MARGI-seq, a method to identify chromatin associated RNAs and their genomic target sites in one unbiased experiment. This method thus challenges the one-RNA-at-a-time paradigm of currently available technologies. The technique involves ligating RNA and DNA together via a novel linker and sequencing these chimeric molecules via paired end next generation sequencing. We have developed two variations to this protocol called proximity MARGI (pxMARGI) and direct MARGI (diMARGI). pxMARGI identifies all RNA and DNA in close proximity, while diMARGI prioritizes protein mediated direct interactions. We have applied this technique to study RNA-chromatin interactions in three cell types, mouse E14 embryonic stem cell line, human H9 embryonic stem cells and human HEK 293T cells. Among the top caRNAs identified by MARGI were several nuclear body associated RNAs including SNHG1, MALAT1, NEAT1 and XIST. 80 – 95% of the DNA targets of caRNA were observed to be connected to the loci of RNA transcription, 1-2% were observed to be on the same chromosome but distal to the loci of RNA transcription and about 5 – 10 % of the DNA targets were observed to be inter-chromosomal connections. RNA attachment levels were observed to be positively correlated to H3K4me3 and H3K27Ac levels and negatively correlated to H3K9me3 levels. We also developed a new plasmid called pCRISPTET as an easily clonable system to tether RNA of interest to specific genomic loci based on the CRISPR-display technique. Gibson assembly can be used to introduce RNA of interest, while golden gate cloning can be used to clone in the PAM sequence to provide information regarding the genomic loci of interest. We used pCRISTET to study the effect of tethering lncRNA EVX1as to the EVX1/ EVX1as promoter region.","abstract_html":"RNA is an important constituent of the nucleus with functions including regulation of RNA biogenesis, regulation of gene expression, and dosage compensation. Currently used technologies to study RNA chromatin interactions pull down individual RNAs and sequence the associated DNA. However, it remains challenging to identify chromatin associated RNAs and their genomic target sites. In this thesis, I will describe MARGI-seq, a method to identify chromatin associated RNAs and their genomic target sites in one unbiased experiment. This method thus challenges the one-RNA-at-a-time paradigm of currently available technologies. The technique involves ligating RNA and DNA together via a novel linker and sequencing these chimeric molecules via paired end next generation sequencing. We have developed two variations to this protocol called proximity MARGI (pxMARGI) and direct MARGI (diMARGI). pxMARGI identifies all RNA and DNA in close proximity, while diMARGI prioritizes protein mediated direct interactions. We have applied this technique to study RNA-chromatin interactions in three cell types, mouse E14 embryonic stem cell line, human H9 embryonic stem cells and human HEK 293T cells. Among the top caRNAs identified by MARGI were several nuclear body associated RNAs including SNHG1, MALAT1, NEAT1 and XIST. 80 – 95% of the DNA targets of caRNA were observed to be connected to the loci of RNA transcription, 1-2% were observed to be on the same chromosome but distal to the loci of RNA transcription and about 5 – 10 % of the DNA targets were observed to be inter-chromosomal connections. RNA attachment levels were observed to be positively correlated to H3K4me3 and H3K27Ac levels and negatively correlated to H3K9me3 levels. We also developed a new plasmid called pCRISPTET as an easily clonable system to tether RNA of interest to specific genomic loci based on the CRISPR-display technique. Gibson assembly can be used to introduce RNA of interest, while golden gate cloning can be used to clone in the PAM sequence to provide information regarding the genomic loci of interest. We used pCRISTET to study the effect of tethering lncRNA EVX1as to the EVX1/ EVX1as promoter region.","abstract_has_math":false,"creators":["Sridhar, Bharat"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Molecular & Integrative Physi","degree_department":null,"school":null,"contributors":["Zhong, Sheng","Bagchi, Milan K.","Prasanth, Kannanganattu V.","Kemper, Jongsook K."],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2017,"date_issued":"2017-09-29T16:37:50Z","date_published":"2017-09-29T16:37:50Z","updated_at":"2026-07-22T22:24:34Z","subjects":["Chromatin associated Ribonucleic acids (RNAs)","Mapping ribonucleic acid (RNA)-genome interactions (MARGI)","Ribonucleic acid (RNA) chromatin interactions"],"languages":["en"],"rights":["Copyright 2017 Bharat Sridhar"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/2142/98111","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Zhong, Sheng","Bagchi, Milan K.","Prasanth, Kannanganattu V.","Kemper, Jongsook K."]},{"key":"dc:creator","label":"Author","values":["Sridhar, Bharat"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2017-09-29T16:37:50Z","2017-06-16","2017-08"]},{"key":"dc:type","label":"Dc Type","values":["text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Molecular & Integrative Physi"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]},{"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":["Chromatin associated Ribonucleic acids (RNAs)","Mapping ribonucleic acid (RNA)-genome interactions (MARGI)","Ribonucleic acid (RNA) chromatin interactions"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["Copyright 2017 Bharat Sridhar"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://hdl.handle.net/2142/98111"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["RNA is an important constituent of the nucleus with functions including regulation of RNA biogenesis, regulation of gene expression, and dosage compensation. Currently used technologies to study RNA chromatin interactions pull down individual RNAs and sequence the associated DNA. However, it remains challenging to identify chromatin associated RNAs and their genomic target sites. In this thesis, I will describe MARGI-seq, a method to identify chromatin associated RNAs and their genomic target sites in one unbiased experiment. This method thus challenges the one-RNA-at-a-time paradigm of currently available technologies. The technique involves ligating RNA and DNA together via a novel linker and sequencing these chimeric molecules via paired end next generation sequencing. We have developed two variations to this protocol called proximity MARGI (pxMARGI) and direct MARGI (diMARGI). pxMARGI identifies all RNA and DNA in close proximity, while diMARGI prioritizes protein mediated direct interactions. We have applied this technique to study RNA-chromatin interactions in three cell types, mouse E14 embryonic stem cell line, human H9 embryonic stem cells and human HEK 293T cells. Among the top caRNAs identified by MARGI were several nuclear body associated RNAs including SNHG1, MALAT1, NEAT1 and XIST. 80 – 95% of the DNA targets of caRNA were observed to be connected to the loci of RNA transcription, 1-2% were observed to be on the same chromosome but distal to the loci of RNA transcription and about 5 – 10 % of the DNA targets were observed to be inter-chromosomal connections. RNA attachment levels were observed to be positively correlated to H3K4me3 and H3K27Ac levels and negatively correlated to H3K9me3 levels. We also developed a new plasmid called pCRISPTET as an easily clonable system to tether RNA of interest to specific genomic loci based on the CRISPR-display technique. Gibson assembly can be used to introduce RNA of interest, while golden gate cloning can be used to clone in the PAM sequence to provide information regarding the genomic loci of interest. We used pCRISTET to study the effect of tethering lncRNA EVX1as to the EVX1/ EVX1as promoter region.","Submission original under an indefinite embargo labeled 'Open Access'. The submission was exported from vireo on 2017-09-29 without embargo terms","The student, Bharat Sridhar, accepted the attached license on 2017-06-07 at 18:41.","The student, Bharat Sridhar, submitted this Dissertation for approval on 2017-06-07 at 19:03.","This Dissertation was approved for publication on 2017-06-16 at 12:01.","DSpace SAF Submission Ingestion Package generated from Vireo submission #11201 on 2017-09-29 at 11:26:01","Made available in DSpace on 2017-09-29T16:37:50Z (GMT). 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Currently used technologies to study RNA chromatin interactions pull down individual RNAs and sequence the associated DNA. However, it remains challenging to identify chromatin associated RNAs and their genomic target sites. In this thesis, I will describe MARGI-seq, a method to identify chromatin associated RNAs and their genomic target sites in one unbiased experiment. This method thus challenges the one-RNA-at-a-time paradigm of currently available technologies. The technique involves ligating RNA and DNA together via a novel linker and sequencing these chimeric molecules via paired end next generation sequencing. We have developed two variations to this protocol called proximity MARGI (pxMARGI) and direct MARGI (diMARGI). pxMARGI identifies all RNA and DNA in close proximity, while diMARGI prioritizes protein mediated direct interactions. We have applied this technique to study RNA-chromatin interactions in three cell types, mouse E14 embryonic stem cell line, human H9 embryonic stem cells and human HEK 293T cells. Among the top caRNAs identified by MARGI were several nuclear body associated RNAs including SNHG1, MALAT1, NEAT1 and XIST. 80 – 95% of the DNA targets of caRNA were observed to be connected to the loci of RNA transcription, 1-2% were observed to be on the same chromosome but distal to the loci of RNA transcription and about 5 – 10 % of the DNA targets were observed to be inter-chromosomal connections. RNA attachment levels were observed to be positively correlated to H3K4me3 and H3K27Ac levels and negatively correlated to H3K9me3 levels. We also developed a new plasmid called pCRISPTET as an easily clonable system to tether RNA of interest to specific genomic loci based on the CRISPR-display technique. Gibson assembly can be used to introduce RNA of interest, while golden gate cloning can be used to clone in the PAM sequence to provide information regarding the genomic loci of interest. We used pCRISTET to study the effect of tethering lncRNA EVX1as to the EVX1/ EVX1as promoter region.","Submission original under an indefinite embargo labeled 'Open Access'. The submission was exported from vireo on 2017-09-29 without embargo terms","The student, Bharat Sridhar, accepted the attached license on 2017-06-07 at 18:41.","The student, Bharat Sridhar, submitted this Dissertation for approval on 2017-06-07 at 19:03.","This Dissertation was approved for publication on 2017-06-16 at 12:01.","DSpace SAF Submission Ingestion Package generated from Vireo submission #11201 on 2017-09-29 at 11:26:01","Made available in DSpace on 2017-09-29T16:37:50Z (GMT). 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