RNA in the nucleus: insight into location and biological function

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.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Molecular & Integrative Physi

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2017

Author dc:creator

• Sridhar, Bharat

Contributors dc:contributor

• Zhong, Sheng
• Bagchi, Milan K.
• Prasanth, Kannanganattu V.
• Kemper, Jongsook K.

• Chromatin associated Ribonucleic acids (RNAs)
• Mapping ribonucleic acid (RNA)-genome interactions (MARGI)
• Ribonucleic acid (RNA) chromatin interactions

Statement dc:rights

• Copyright 2017 Bharat Sridhar

Language dc:language

en

Handle dc:identifier

http://hdl.handle.net/2142/98111

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/98111