Graduate Thesis Or Dissertation

 

Noncoding MRP RNA Function Investigated by Genetic Manipulation and Biochemical Analysis Public Deposited

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https://scholar.colorado.edu/concern/graduate_thesis_or_dissertations/rn301162r
Abstract
  • MRP RNA is an abundant, essential noncoding RNA whose functions have been proposed in yeast but are incompletely understood in humans. Mutations in the genomic locus for MRP RNA cause pleiotropic human diseases, including cartilage hair hypoplasia. MRP RNA folds into a structure similar to that of RNase P RNA and assembles with at least seven human proteins, all of which also assemble with RNase P. The thesis presents detailed investigations into the 3’ terminal profile of human MRP RNA and establishes its specific role in human pre-ribosomal RNA processing.Post-transcriptional 3’ end processing is a key component of RNA regulation. MRP RNA has been proposed to function in three distinct subcellular compartments and therefore may utilize this mode of regulation. 3’ RACE coupled with high-throughput sequencing examined the 3’ terminal sequences of human MRP RNA and other noncoding RNAs that form ribonucleoprotein (RNP) complexes. The 3’ terminal sequence of MRP RNA from HEK293T cells displayed a distinctive distribution of genomically encoded termini (including an assortment of U residues) with a portion of these selectively tagged by oligo(A) tails. This profile contrasted with the relatively homogenous 3’ terminus of an in vitro transcribed MRP RNA control and the differing 3’ terminal profiles of U3 snoRNA, RNase P RNA, and telomerase RNA (hTR), suggesting alternative modes of regulation for these distinct noncoding RNAs. To determine an essential role for MRP RNA in human cells, CRISPR-Cas9 genome editing experiments disrupted the endogenous human MRP locus to eliminate MRP RNA in the majority of cells. The resulting accumulation of uncleaved ribosomal RNA precursors, analyzed by RNA fluorescence in situ hybridization, northern blot and RNA sequencing, implicated MRP in pre-rRNA processing. Primer extension assays revealed a deficiency of 5’ ends in human ITS1 upon loss of MRP RNA. Immunopurified MRP RNP from HeLa cells cleaved the human pre-rRNA ITS1 in vitro at the same site utilized in cells, while RNP isolated from cells with CRISPR-edited MRP RNA lost this activity. Together, these experiments contribute that MRP RNA performs a vital step in human pre-ribosomal RNA processing.
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  • 2016
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  • 2019-11-16
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