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RIPPLiT and ChimeraTie: High throughput tools for understanding higher order RNP structures

Metkar, Mihir
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Student Authors
Faculty Advisor
Melissa J. MooreJob Dekker
Academic Program
Interdisciplinary Graduate Program
UMass Chan Affiliations
Document Type
Doctoral Dissertation
Publication Date
2018-07-30
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Abstract

Even after their discovery more than 60 years ago, little is known about how messenger RNAs (mRNAs) are packaged inside the cells. To ensure efficient and accurate delivery of the intended message to its proper destination, it is important to package the informational molecule in a way that protects it from premature degradation but also proper decoding at the destination. However, very little is known about the this fundamentally important step of mRNA packaging inside eukaryotic cells. To this end, we developed a novel approach, RIPPLiT (RNA ImmunoPrecipitation and Proximity Ligation in Tandem), to capture the 3D architecture of the ribonucleoprotein particles (RNPs) of interest transcriptome-wide. To begin with, we applied RIPPLiT to the exon-junction complex (EJC), a set of proteins stably bound to a spliced RNA. EJCs have been shown to interact with other proteins like SR- and SR-like to form megadalton sized complexes and help protect large regions of mRNAs. Thus, we hypothesized that these RNPs would provide an ideal system to elucidate the higher order organization of mRNPs.

Preliminary analysis of data obtained from RIPPLiT consisted of “chimeric reads”, reads with multiple RNA fragments ligated together, which could not be analyzed with any of the existing bioinformatics tools. Thus, we developed a new bioinformatics suite, ChimeraTie, to map, analyze and visualize chimeric reads. Performing polymer analysis on chimeric reads obtained for hundreds of mRNAs, we were able to predict that mRNPs are linearly and densely packed into flexible rod-like structures before they undergo translation.

In this thesis, along with the detailed biological conclusion, I have also provided a step-wise manual to perform RIPPLiT experiment and analyze the ensuing data using ChimeraTie.

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DOI
10.13028/jdwh-kp41
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Licensed under a Creative Commons license