Mechanism and Functional Consequences of Generating and Processing Drosophila RNase P RNA from an Intron

Bibliographic Details
Main Author:	Palsule, Geeta
Language:	English
Published:	The Ohio State University / OhioLINK 2019
Subjects:	Molecular Biology Cellular Biology
Online Access:	http://rave.ohiolink.edu/etdc/view?acc_num=osu1555628225881656

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spelling	ndltd-OhioLink-oai-etd.ohiolink.edu-osu15556282258816562021-08-03T07:10:40Z Mechanism and Functional Consequences of Generating and Processing Drosophila RNase P RNA from an Intron Palsule, Geeta Molecular Biology Cellular Biology RNase P RNA (RPR) is the catalytic component of RNase P, the housekeeping ribonucleoprotein (RNP) enzyme, which is required for the removal of the 5¢-leader frompre-tRNAs in all three domains of life. The ancestral eukaryotic RPR is a Pol III-regulated transcript generated with mature termini. In the branch of the arthropod lineage that led to the insects and crustaceans, however, a new allele arose in which RPR is embedded in an intron of a Pol II-regulated recipient gene and requires processing from the intron for maturation. In this thesis, I investigated the biogenesis mechanism for Drosophila RPR from its recipient intron. I found that intronic-RPR is processed from the excised intron to its active form by ancient nucleases that would have been present in the ancestor. Select proteins that act early in the RNase P enzyme assembly help prevent excessive nuclease trimming. Discovery of the RPR synthesis pathway casts light on events that happened ~500 million years ago when a new type of RPR gene was born and fixed in insects and crustaceans, the most successful group of animals on earth.To identify factors involved in Drosophila RPR biogenesis, I assayed RPR production following depletion of candidate nucleases using RNAi in tissue-culture cells. I discovered that post splicing and debranching, the 5¢ and the 3¢ terminal nucleotides of the precursor RPR are trimmed by the conserved nuclease Rat1/Xrn2 and the RNAexosome, respectively. These ancient nucleases are involved in processing other non- iiicoding RNAs, which suggests that the intronic RPR could have been active when it first appeared in the ancestor.In the holoenzyme, RPR associates with protein subunits, RNase P proteins (Rpps), which are required for full activity. In Drosophila there are eight predicted Rpps; Pop1, Pop4, Rpp25, Rpp20, Pop5, Rpp14, Rpp30 and Rpp21. Upon depletion of these Rpps individually, I discovered that all eight Rpps are required for pre-tRNA processing, and that a subset of Rpps (Pop1, Pop4, Rpp25 and Rpp20) are also required for RPR stability. The finding suggests that this subset of Rpps protects the RPR during nuclease processing. In keeping with this idea, I found that if RPR was generated with mature termini, and therefore did not require nuclease processing, the RNA was stable when these Rpps were depleted. These data are consistent with cryo-EM structures for eukaryotic RNase P, which show the Rpps required for stability of Drosophila RPR make the most extensive contacts with the RNA, and with the in vitro reconstitution data showing that these proteins bind the RNA early in the hierarchy of holoenzyme assembly.In conclusion, the work has identified the biogenesis mechanism for Drosophila RPR and has also provided correlative in vivo evidence for in vitro data suggesting how RPR interacts with Rpps. In future work, it will be important to determine if there is any selective advantage of intronic-RPR, despite a more complex biogenesis mechanism, by comparing Pol II-regulated RPR with ancestral Pol III-regulated RPR. This will be possible using gene editing in Drosophila. 2019-06-17 English text The Ohio State University / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=osu1555628225881656 http://rave.ohiolink.edu/etdc/view?acc_num=osu1555628225881656 restricted--full text unavailable until 2022-05-06 This thesis or dissertation is protected by copyright: some rights reserved. It is licensed for use under a Creative Commons license. Specific terms and permissions are available from this document's record in the OhioLINK ETD Center.
collection	NDLTD
language	English
sources	NDLTD
topic	Molecular Biology Cellular Biology
spellingShingle	Molecular Biology Cellular Biology Palsule, Geeta Mechanism and Functional Consequences of Generating and Processing Drosophila RNase P RNA from an Intron
author	Palsule, Geeta
author_facet	Palsule, Geeta
author_sort	Palsule, Geeta
title	Mechanism and Functional Consequences of Generating and Processing Drosophila RNase P RNA from an Intron
title_short	Mechanism and Functional Consequences of Generating and Processing Drosophila RNase P RNA from an Intron
title_full	Mechanism and Functional Consequences of Generating and Processing Drosophila RNase P RNA from an Intron
title_fullStr	Mechanism and Functional Consequences of Generating and Processing Drosophila RNase P RNA from an Intron
title_full_unstemmed	Mechanism and Functional Consequences of Generating and Processing Drosophila RNase P RNA from an Intron
title_sort	mechanism and functional consequences of generating and processing drosophila rnase p rna from an intron
publisher	The Ohio State University / OhioLINK
publishDate	2019
url	http://rave.ohiolink.edu/etdc/view?acc_num=osu1555628225881656
work_keys_str_mv	AT palsulegeeta mechanismandfunctionalconsequencesofgeneratingandprocessingdrosophilarnaseprnafromanintron
_version_	1719455514884046848

Mechanism and Functional Consequences of Generating and Processing Drosophila RNase P RNA from an Intron

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