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The RNA binding landscape of the histone stem-loop binding protein / by Lionel (Lee) Brooks 3rd.

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Author	Brooks, Lionel.
Title	The RNA binding landscape of the histone stem-loop binding protein / by Lionel (Lee) Brooks 3rd.
Date	2014
Abstract	RNA binding proteins are fundamental components of crucial biological processes in all domains of life. The development of techniques to study RNA binding protein complexes is, therefore, broadly impactful. I employed multiple genome-wide techniques to investigate the RNA binding repertoire of the histone stem-loop binding protein (SLBP) - namely, RIP-chip, RIP-seq and HITS-CLIP. These genome-wide approaches provide a comprehensive view of the ribonucleic acid components of the histone stem-loop mRNP complex. Also, the HITS-CLIP method reveals sites of molecular contact. All three techniques corroborated SLBP's specificity for histone mRNA. I developed a bioinformatic HITS-CLIP analysis pipeline that includes: (1) a clustering method for grouping genes by sequence coverage patterns, (2) a method for inference of nuclease cleavage sites to map RNP boundaries and (3) crosslink-induced mutation mapping to identify sites of molecular contact. I cross-validated my findings with crystallographic data and in vitro assays. These data mapped specific nucleotide determinants of ternary complex formation in the loop of histone stem-loop RNA. We found that SLBP and 3'hExo binding is differentially affected by mutating different uridines in the stem-loop. The different loop mutations also differentially affect crosslinking efficiency. Our SLBP HITS-CLIP data provided a gold-standard for the development of a python package for fine mapping of nuclease-resistant fragments and crosslink sites from HITS-CLIP data. I subsequently expanded the capabilities of the software to map crosslink sites from iCLIP and PAR-CLIP data. I applied the resultant "clipPyL" software package to analyze multiple public CLIP-seq datasets. These secondary data analyses proved the accuracy of the clipPyL software by reproducing previously published results. Moreover, integration of the SLBP HITS-CLIP coverage map with the UPF1 iCLIP coverage map revealed that SLBP and UPF1 bind at adjacent sites on histone mRNA. Additionally, I identified putative RNA cis-elements on the H2AFX, MYC and P53 transcripts during my secondary analysis of HuR and AGO2 CLIP-seq datasets.
Description	xvi, 140 leaves : ill. (some col.)
Format	application/pdf
College	Dartmouth Medical School. Department of Genetics.
Issuing Body	Dartmouth Dissertations are made available through the Dartmouth Digital Library.
Degree	Thesis (Ph.D.)--Dartmouth College.
Copyright	All rights reserved. No part of this book may be reproduced in any form by any electronic or mechanical means without permission in writing from the author.
DOI	http://dx.doi.org/10.1349/ddlp.1247

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