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Vts1p is the Saccharomyces cerevisiae member of the Smaug family of post-transcriptional regulators, which is a group of sequence-specific RNA-binding proteins that regulate target mRNA expression. Vts1p is known to mediate deadenylation-dependent degradation of target transcripts through the recruitment of the Ccr4p/Pop2p/Not deadenylase complex. By conducting a functional analysis of Vts1p deletion mutants, I demonstrate that two regions within Vts1p are independently capable of downregulating the expression of an mRNA reporter. I provide both genetic and biochemical evidence that suggests residues 170-523 regulate reporter expression at the level of mRNA stability and function through a mechanism that requires the Ccr4p/Pop2p/Not deadenylase, whereas residues 1-237 repress reporter expression at the level of translation and function through a novel mechanism. In addition, I map a direct interaction between the eIF4E-binding protein, Eap1p, and the Vts1p SAM domain, which suggests a model in which residues 170-523 recruit Eap1p to mediate efficient target transcript degradation.

Vts1p is the Saccharomyces cerevisiae member of the Smaug family of post-transcriptional regulators, which is a group of sequence-specific RNA-binding proteins that regulate target mRNA expression. Vts1p is known to mediate deadenylation-dependent degradation of target transcripts through the recruitment of the Ccr4p/Pop2p/Not deadenylase complex. By conducting a functional analysis of Vts1p deletion mutants, I demonstrate that two regions within Vts1p are independently capable of downregulating the expression of an mRNA reporter. I provide both genetic and biochemical evidence that suggests residues 170-523 regulate reporter expression at the level of mRNA stability and function through a mechanism that requires the Ccr4p/Pop2p/Not deadenylase, whereas residues 1-237 repress reporter expression at the level of translation and function through a novel mechanism. In addition, I map a direct interaction between the eIF4E-binding protein, Eap1p, and the Vts1p SAM domain, which suggests a model in which residues 170-523 recruit Eap1p to mediate efficient target transcript degradation.

Thesis (Ph.D.)--Virginia Commonwealth University, 2007. / Title from title-page of electronic thesis. Prepared for: Dept. of Biochemistry. Bibliography: leaves 125-142

The localization and translation of messenger ribonucleic acids (mRNAs) play crucial roles in cellular function and diseases, and are regulated by numerous RNA-binding proteins (RBPs) and small non-coding RNAs, called trans-acting factors. Biochemical and imaging methods used to study RNA interactions with these trans-acting elements have made important discoveries in characterizing how these factors regulate gene expression and determining the RNA sequence to which they bind. However, the spatiotemporal information regarding these interactions in subcellular compartments have been difficult to determine or to quantify accurately. To image and quantify native RNA and RNA–protein interactions simultaneously in situ, we developed a proximity ligation assay that combines peptide-modified RNA imaging probes. It can detect the RNAs in live cells and the interactions at a single-interaction level. Lastly, it can produce results that are easily quantifiable. We tested the specificity and sensitivity of this technique using two models: interactions between the genomic RNA and the N protein of human respiratory syncytial virus as well as those between exogenous transcripts with or without the Human antigen R (HuR) binding site and HuR. To validate this method, its accuracy and utility have been demonstrated in three models: poly(A)+ or β-actin mRNAs binding to different cytoskeleton for localization, poly(A)+ or β-actin mRNAs interacting with HuR for stabilization, and programmed cell death 4 (PDCD4) mRNA binding to HuR or T-cell intracellular antigen (TIA1) for translational regulation.

Transposons were first discovered as "jumping genes" by Barbara McClintock, who continued to study them in maize through the 1940's and 1950's. Since then, transposons have been shown to make up a large percentage of eukaryotic genomes, including close to half of the human genome, but have been dismissed as simply "junk DNA." Recently, the importance of keeping transposons tightly regulated within the cellular environment has begun to be appreciated; the mechanisms to accomplish this have been studied and the current understanding of pathways governing transposon regulation is discussed within this dissertation. However, recent work presented within the scope of this dissertation in Drosophila melanogaster revealed a previously unknown function for condensin complexes in transposable element regulation. These studies provide a link between pathways governing chromosome pairing and transposon regulation. The potential interplay between these two pathways is intriguing and until now, largely unexplored.Aside from how transposons themselves are regulated, studies into potential roles they may play in the regulation of other protein coding genes within the cell may provide clues into the functionality of these elements within our genome. As a specific example, BRCA1 has a high density of retrotransposon sequences within its primary transcript, and studies of BRCA1 regulation presented within this dissertation has led to the development of a model for a novel gene regulatory mechanism occurring in human cells involving retrotransposons. This mechanism may provide direct relevance to cancer etiology, as retrotransposons have long been known to be misregulated in cancer.As a sum, the work presented within this dissertation extends our knowledge of how transposons are regulated and provides some of the first evidence for their functionality in gene regulatory pathways within human cells.

<p>Post-transcriptional regulation of cytoplasmic mRNAs is an efficient mechanism of regulating the amounts of active protein within a eukaryotic cell. RNA sequence elements located in the untranslated regions of mRNAs can influence transcript degradation or translation through associations with RNA-binding proteins. Tristetraprolin (TTP) is the best known member of a family of CCCH zinc finger proteins that targets adenosine-uridine rich element (ARE) binding sites in the 3’ untranslated regions (UTRs) of mRNAs, promoting transcript deadenylation through the recruitment of deadenylases. More specifically, TTP has been shown to bind AREs located in the 3’-UTRs of transcripts with known roles in the inflammatory response. The mRNA-binding region of the protein is the highly conserved CCCH tandem zinc finger (TZF) domain. The synthetic TTP TZF domain has been shown to bind with high affinity to the 13-mer sequence of UUUUAUUUAUUUU. However, the binding affinities of full-length TTP family members to the same sequence and its variants are unknown. Furthermore, the distance needed between two overlapping or neighboring UUAUUUAUU 9-mers for tandem binding events of a full-length TTP family member to a target transcript has not been explored. To address these questions, we recombinantly expressed and purified the full-length C. albicans TTP family member Zfs1. Using full-length Zfs1, tagged at the N-terminus with maltose binding protein (MBP), we determined the binding affinities of the protein to the optimal TTP binding sequence, UUAUUUAUU. Fluorescence anisotropy experiments determined that the binding affinities of MBP-Zfs1 to non-canonical AREs were influenced by ionic buffer strength, suggesting that transcript selectivity may be affected by intracellular conditions. Furthermore, electrophoretic mobility shift assays (EMSAs) revealed that separation of two core AUUUA sequences by two uridines is sufficient for tandem binding of MBP-Zfs1. Finally, we found evidence for tandem binding of MBP-Zfs1 to a 27-base RNA oligonucleotide containing only a single ARE-binding site, and showed that this was concentration and RNA length dependent; this phenomenon had not been seen previously. These data suggest that the association of the TTP TZF domain and the TZF domains of other species, to ARE-binding sites is highly conserved. Domains outside of the TZF domain may mediate transcript selectivity in changing cellular conditions, and promote protein-RNA interactions not associated with the ARE-binding TZF domain. </p><p>In summary, the evidence presented here suggests that Zfs1-mediated decay of mRNA targets may require additional interactions, in addition to ARE-TZF domain associations, to promote transcript destabilization and degradation. These studies further our understanding of post-transcriptional steps in gene regulation.</p> / Dissertation

Despite their name, a number of the cold shock proteins are expressed during normal growth, and not just during cold shock, in several species. The function of these constitutively expressed CspA paralogues is unclear. In Salmonella Typhimurium (a major worldwide cause of gastrointestinal disease) they have been linked to various stress responses and the establishment of virulence. Study of the cold shock proteins as gene regulators is therefore of great interest, and they also have potential as targets for antimicrobial development. CspE in Salmonella Typhimurium is constitutively expressed during normal growth. In order to determine its function, attempts were made to identify the interactions it forms with other cellular proteins. Initially, a proteomic investigation attempted to identify proteins which complex with CspE by in vivo cross-linking and affinity purification followed by mass spectrometry. Although no defined complex was consistently identified, the results suggested a handful of proteins which might interact with CspE in a weak or transient manner. These proteins included many from the nucleoid and ribosomal entry site, hinting at CspE’s cellular localisation. In order to investigate these transient interactions, a bacterial two-hybrid system was employed. Interactions between CspE and HupA, a nucleoid protein identified in the proteomic analysis, were probed, as were interactions between CspE and CsdA, an RNA helicase thought to function co-operatively with CspE. The twohybrid system also allowed investigation of CspE dimerisation, which has been reported in vitro but not investigated in vivo until this study. CspE appears not to interact significantly with either HupA, CsdA, or itself at 37oC. Finally in a further attempt to identify interactions of CspE, a genomic library was created to test CspE interactions by two-hybrid assay with random peptides derived from the whole Salmonella genome. The library was successfully created and screened for evidence of interaction, and revealed an association between CspE and a transcriptional repressor, DeoT. DeoT is a repressor of several genes for catabolic processes, suggesting a role for CspE in the regulation of central metabolism. The findings of this work present a number of novel discoveries and several interesting opportunities for further studies.

ABSTRACT Cassava is currently the second most important source of carbohydrates on the African continent. In the last two decades, cassava crops have been severely affected by outbreaks of cassava mosaic disease (CMD). South African cassava mosaic virus (SACMV) has been associated with CMD outbreaks in the Mpumalanga province. Advances in post-transcriptional gene silencing (PTGS) technology have provided promising new strategies for the engineering of virus resistance in plants. Inverted repeat (IR) constructs are currently the most potent inducers of PTGS, however, these constructs are inherently unstable. The purpose of this study was to develop IR constructs with an improved stability for the efficient induction of PTGS in plants. Two mismatched inverted repeat constructs, one targeting the SACMV BC1 open reading frame, the other targeting the Maize streak virus (MSV) AC1 open reading frame, were successfully created. Sodium bisulfite was used to deaminate cytosine residues on the sense arm of the constructs. The resulting number of GT mismatches was seemingly sufficient to stabilize the linear conformation of the IR constructs, as they were efficiently propagated by E.coli DH5!, and subsequently behaved like linear DNA molecules. Furthermore, it was found that the number of mismatches on the BC1 construct (17.5%) was ideal, as the subsequent stability of the predicted RNA hairpin was not affected. Due to the higher number of mismatches on the AC1 construct (23.5%), it was found that the loop region of the RNA hairpin was marginally destabilized. Despite this, long stretches of stable dsRNA were still produced from the AC1 IR construct, and is likely to induce PTGS. Interestingly, it was observed that the mismatched IR constructs, although still replicated in E.coli, were marginally destabilized in Agrobacterium. Therefore, it was deduced that the stability of a mismatched IR construct may be influenced by the particular intracellular environment of an organism. Due to the recalcitrance of cassava to transformation, a model plant system, Nicotiana benthamiana, was used to screen constructs for toxicity, stability, and efficiency of PTGS induction. Agrobacteriummediated transformation and regeneration of N. benthamiana was optimized, and 86% transformation efficiency was achieved when using leaf disk explants. It was found that the addition of an ethylene scrubber, potassium permanganate, substantially increased the rate of regeneration by reducing the frequency of hyperhydritic plants. Transgene iv integration was confirmed by PCR amplification of the hptII gene in the T-DNA region. Transgene expression was confirmed by screening for GUS and GFP reporter genes. No toxic responses to the transgene have been observed thus far. Studies are currently underway to confirm the stability of the mismatched IR constructs in N. benthamiana. PAGE Northern blotting is being done, as the detection of siRNAs derived from the transgene will confirm that constructs are functional. In addition, infectivity assays are underway to determine the efficacy of BC1 knockdown by a stably integrated construct. Due to the enhanced stability of mismatched IR constructs, they may be an appealing alternative to currently available intron-spliced, or exact matched hairpin systems.

Dans les organites des plantes, l’édition de l’ARN consiste majoritairement en une désamination de cytidines à des sites spécifiques de l’ARNm. Trente-quatre sites d’édition ont été découverts dans les transcrits chloroplastiques d’Arabidopsis thaliana et plus de 500 dans les transcrits mitochondriaux. Depuis 2005, beaucoup de facteurs d’édition ont été trouvés. La majorité de ces protéines appartiennent à la famille des «PentatricoPeptide Repeat» (PPR). Parmi ces PPR, certaines contiennent un domaine DYW possédant de faibles similarités avec les cytidines désaminases (CDA), alors que d’autres en sont dénuées, générant un doute sur le fait qu’il ait une activité CDA. Le gène At1g47580 (DYW1) code une protéine unique chez Arabidopsis thaliana contenant «seulement» un domaine DYW. Il a été proposé que DYW1 puisse interagir avec les PPR ne contenant pas de domaine DYW, pour former un hétérodimère, capable d’éditer spécifiquement un site. En accord avec cette hypothèse, nous avons montré que DYW1 agissait sur le même site d’édition que CRR4, une PPR sans domaine DYW, et que ces protéines interagissaient in vivo. De plus, nous avons montré que DYW1 remplaçait les parties manquantes de CRR4 pour l’édition. Pour obtenir plus d’informations sur la fonction du domaine DYW, des mutations ont été introduites dans DYW1. Nous avons montré que la signature CDA dans les protéines DYW était essentielle à l’édition de l’ARN ainsi qu’à l’interaction avec les ions zinc. Les données sont en accord avec l’hypothèse d’une activité CDA dans le domaine DYW. Cependant, aucune activité CDA n’a pu être mise à jour in vitro. Il est vraisemblable qu’au moins un cofacteur doive encore être identifié. / In plant organelles, RNA editing mostly takes the form of conversions of cytidines to uridines at specific sites in mRNAs. Thirty-four editing sites have been found in Arabidopsis thaliana chloroplast transcripts and more than 500 sites in mitochondrial transcripts. Since 2005, lots of proteins have been found to act as RNA editing factors. Most of these proteins belong to the PentatricoPeptide Repeat (PPR) family. Amongst these PPR, some contain a DYW domain with weak similarity to cytidine deaminases (CDA), whilst others lack such a domain, creating doubts about whether this domain is required for editing. The gene At1g47580 (named DYW1) encodes a protein in Arabidopsis thaliana that contains “only” a DYW domain. Our initial hypothesis was that DYW1 might interact with PPR proteins that lack a DYW domain, in order to form a heterodimer, able to perform site-specific editing. In accordance with this hypothesis, we discovered that DYW1 is involved in editing the same site as CRR4, a PPR lacking a DYW domain, and that these two proteins interact together in vivo. Moreover, we showed that DYW1 replaces all the missing parts of CRR4 for editing. So, other partners need to be hypothesized for other DYW-lacking editing factors if this hypothesis is to be generalized. The highly conserved residues making up the CDA signature in DYW proteins were found to be essential for RNA editing and are also required for zinc binding, which is a known characteristic of CDAs. All the data so far are consistent with the DYW domain being (part of) a CDA activity; nevertheless, no CDA activity could be detected in vitro. It is likely that at least one required cofactor remains to be identified.