Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Molecular Genetics and Genomics

Language

English (en)

Date of Award

1-1-2011

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Douglas Chalker

Abstract

The ciliate, Tetrahymena thermophila, like a handful of other eukaryotes, engages in massive genome reorganization known collectively as chromatin diminution. Part of this process involves large-scale DNA excision known as DNA elimination. Recent data has shown DNA elimination to be dependent on RNA interference: RNAi). Using T. thermophila, I have sought to determine the role of non-coding RNA: ncRNA) in RNAi-dependent DNA elimination through studies of DNA sequences that are to be eliminated called internal eliminated sequences: IESs) and through a conjugation-specific Dicer protein and its putative tandem dsRNA-binding motif: DSRM) protein partners. Studies of the R IES revealed the requirement of IES DNA for production of long, bidirectional ncRNA early in conjugation. This ncRNA is essential for IES excision in zygotic nuclei later in conjugation. The conjugation-specific Dicer homologue, DCL1, was shown to be required for production of a species of sRNA called scnRNAs from the long, bidirectional ncRNA from IESs. Knockouts of DCL1 displayed a loss of these scnRNAs as well as an increase in the long, bidirectional ncRNA precursors. A deficiency in these scnRNAs was sufficient to block modification of chromatin associated with IESs and prevent their rearrangement later in conjugation. Failure of DNA elimination caused DCL1 knockout cells to arrest before completion of conjugation. Further studies of the tandem DSRM-containing proteins, DRB2 and DRB1, revealed that neither are solely partners for DCL1 or any other Dicer protein but play other important roles during conjugation. Zygotic expression of DRB2 was shown to be essential for DNA elimination and completion of conjugation. Interaction with the chromo-domain containing protein, Pdd1p, by Drb2p implicates ncRNA or sRNA in later stages of conjugation after scnRNA production. Knockouts of the tandem DSRM-containing DRB1 caused higher numbers of cells to abort conjugation and therefore produce fewer progeny. Localization of this protein to the crescent micronucleus during prophase of meiosis I links DRB1 to a probable role in ensuring proper recombination during meiosis for haploid gamete production. All these studies suggest that ncRNA has many roles in conjugation-specific processes including RNAi-directed DNA elimination.

Comments

Permanent URL: http://dx.doi.org/10.7936/K7XS5SFS

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