Gene expression is the conduit by which genetic information is connected into cellular phenotypes. Recently, it was shown that gene expression in mammalian cells is governed, at least in part, by the expression of short double stranded RNA (dsRNA). This mode of gene regulation is influenced by a large group of dsRNA binding proteins that could either stabilize or trigger the degradation of dsRNA. Indeed, double stranded RNA (dsRNA) specific ribonucleases (RNases) play an important role in regulating gene expression. In most eukaryotes, members of the dsRNA specific RNase III family trigger RNA degradation and initiate cellular immune response. Disruption of human . RNase III (Dicer) deregulates fetal gene expression and promotes the development of cancer. However, very little is known about the housekeeping function of eukaryotic RNase III and the mechanism by which they distinguish between exogenous and endogenous cellular RNA species. This thesis elucidates how dsRNAs are selected for cleavage and demonstrates their contribution to RNA metabolism in yeast as model eukaryote. Initially, the reactivity determinants of yeast RNase III (Rnt1p) were identified in vitro and used to study the global impact of Rnt1p on the processing of non-coding RNA. The results indicate that Rnt1p is required for the processing of all small nucleolar RNAs (snoRNAs) involved in rRNA methylation and identify a new role of Rnt1p in the processing of intronic snoRNAs. It was shown that Rnt1p cleavage helps to coordinate the expression of some ribosomal protein genes hosting intronic snoRNAs. Direct snoRNA processing from the pre-mRNA blocks the expression of the host gene, while delayed snoRNA processing from the excised intron allows the expression of both genes. In this way, the cell can carefully calibrate the amount of snoRNA and ribosomal proteins required for ribosome biogenesis. In addition, a global analysis of snoRNA processing identified new forms of Rnt1p cleavage signals that do not exhibit a conserved sequence motif but instead use a new RNA fold to recruit the enzyme to the cleavage site. This finding led to the conclusion that Rnt1p may use a wide combination of structural motifs to identify its substrates and thus increases the theoretical number of potential degradation targets in vivo . To evaluate this possibility, a new search for snoRNA independent Rnt1p cleavage targets was performed. Interestingly, many Rnt1p cleavage signals were identified in intergenic regions devoid of known RNA transcripts. In vivo , it was shown that Rnt1p induce the termination of non-polyadenylated transcripts and functions as a surveillance mechanism for transcription read-through. This finding directly links Rnt1p to the transcription machinery and provides a new mechanism for polyadenylation independent transcription termination. Together the work described in this thesis presents an example of how eukaryotic RNase III may identify its substrates and present a case study where transcription, RNA processing and stability are linked.

Cleavage and polyadenylation are essential pre-mRNA processing reactions maturing the 3’end of almost all protein encoding eukaryotic mRNAs. Analysis of the sequences required for cleavage and polyadenylation in the human melanocortin 4 receptor (MC4R) and the human transcription factors JUNB and JUND pre-mRNAs revealed that, at least for some mammalian genes, 3’end processing of the primary transcript is independent of previously described auxiliary sequence elements located upstream or downstream of the core poly(A) sequences. The analysis of the MC4R poly(A) site, contrary to the current understanding of mammalian poly(A) sites, showed that mutations of the AUUAAA hexamer sequence had no effect on 3’end processing levels while mutations in the short DSE severely reduced cleavage efficiency. The MC4R poly(A) site uses a potent DSE and to direct maximal cleavage efficiency requires only a short upstream adenosine rich sequence. Furthermore, analysis of the endogenous A-rich human JUNB poly(A) signal validated upstream A-rich core sequences as genuine 3’end formation directing sequences in human non-canonical 3’end formation signals. The results show that a minimal human poly(A) site, similar to yeast and plants, can be defined by an adenosine rich sequence adjacent to a U/GU-rich sequence element and a cleavage site. These findings further imply that some human non-canonical poly(A) sites may be recognised via a similar DSE-dependent mechanism and may not require additional auxiliary sequence elements. Finally, results on the analysis of the EDF1 poly(A) signal show that, in a spliced environment, A-rich sequences are also 3’end formation effectors but depend on an competent upstream splicing reaction for efficient definition of the 3’end processing site.

SWI/SNF is a chromatin-remodeling complex and Brahma (BRM) is the ATPase subunit of SWI/SNF. BRM regulates transcription by remodeling the nucleosomes at promoter regions. BRM is also associated with RNA and affects pre-mRNA processing together with other SWI/SNF subunits. In this thesis, I will discuss the roles of BRM in both transcription and pre-mRNA processing. In Paper I, we showed that BRM, as well as other SWI/SNF subunits SNR1 and MOR, affects the alternative processing of a subset of pre-mRNAs, as shown by microarray analysis. This observation was validated by RNAi experiments both in Drosophila S2 cells and in vivo. In Paper II, we characterized the trans-splicing of transcripts derived from the mod(mdg4) gene. RNA interference (RNAi) and overexpression experiments revealed that BRM regulates the trans-splicing of mod(mdg4)-RX in an ATPase independent manner. In Paper III, we analyzed the expression of two BRM-target genes identified in Paper I, CG44250 and CG44251. RNAi and overexpression experiments showed that the expression levels of these two genes were affected by BRM in a manner that is independent of its ATPase activity. Transcriptome analysis further proved that BRM affects gene expression both in ATPase dependent and independent manners. In Paper IV, we showed that BRM is present at the 3’-end of two analyzed genes, CG5174 and CG2051. BRM facilitates the recruitment of the cleavage and polyadenylation machinery to the cleavage sites through protein-protein interactions that do not require the ATPase activity of BRM. Morevoer, BRM promotes the cleavage of the CG5174 and CG2051 pre-mRNAs. To sum up, SWI/SNF plays important roles not only in transcription but also in pre-mRNA processing. To regulate transcription, BRM can either act as an ATPase-dependent chromatin remodeler or in a manner that does not involve ATPase activity. Additionally, BRM interacts with RNA-binding proteins to regulate the processing of a subset of pre-mRNAs, and this function of BRM is independent of its chromatin remodeling activity. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p>

Abstract Failure to maintain an appropriate balance between cell death and proliferation is partly due to derangements in the regulation of apoptosis. In this work, apoptosis and the expression of apoptosis regulating proteins were studied by 3' - end labeling of fragmented apoptotic DNA (TUNEL) and immunohistochemistry in a set of 147 tissue samples consisting of 44 biopsies of normal and dysplastic bronchial epithelium, and 103 non-small cell lung carcinomas. The quantity of apoptotic cells and bodies, apoptotic index (AI%), is defined as a percentage of apoptotic cells in the entire tumor cell population. Changes in the apoptotic activity were already seen in the metaplasia-dysplasia-carcinoma sequence of the lung, where the AI% increased gradually until moderate epithelial dysplasia but started to decrease after that. Thus, the AI% for invasive NSCLC (1.20 for squamous cell carcinoma and 1.24 for adenocarcinoma) was slightly lower than in premalignant bronchial epithelium (mean 1.50), but clearly higher than in normal tissue (0.20 for normal bronchial epithelium and 0.24 for lung interstitial cells). 53% of SQCCs and 50% of ACs showed p53 positive nuclei indicative of mutated p53 protein. The immunostaining of bcl-2, bax and mcl-1 revealed diffuse, cytoplasmic staining and was present in most tissues studied. No statistically significant associations between the extent of apoptosis and the expression of p53, bcl-2, bax, or mcl-1 could be found, although . The immunostaining for caspases 3, 6 and 8 was restricted to the tumor areas, reflecting increased apoptotic activity in them. The AI% was significantly higher in NSCLCs in which the single-cell staining pattern for caspase-8 was dominant (P = 0.017), whereas the expression of caspases 3 and 6 had no association with apoptosis. The number of apoptotic cells was significantly higher in NSCLC tumors with a high number of CD3+ and CD8+ T-lymphocytes (P = 0.01) and B-cells (P = 0.05). By multivariate analysis, enhanced apoptosis in NSCLC showed a 1.9-fold risk (95% CI 1.04–3.60; P = 0.04) and p53 positivity a 2.3-fold risk (95% CI 1.30–4.10; P = 0.005) for a shortened survival. Both factors appeared as independent prognostic variables. Apoptosis is clearly enhanced in premalignant and malignant lung tissue in comparison with normal tissue. Furthermore, the expression of the apoptosis-regulating genes is different in tumor tissue from that in normal tissue, and some of the changes in their expression can be seen even in the premalignant lesions of the bronchial epithelium. The expression of caspases seen only in tumor tissue implies the activation of the apoptotic mechanisms and, thus, the lowered treshold of tumor cells to undergo apoptosis. Even in the advanced stages of the disease, the immune defense is effective and the cytotoxic action of activated CD8+ T-cells clearly involves apoptosis. Based on these results it is concluded that alterations in the apoptotic activity and changes in the expression of apoptosis-regulating genes are associated with malignant transformation and growth in lung tissue.

La maturation 3’ des pré-ARNm constitue une étape majeure dans la régulation post-transcriptionnelle de l’expression des gènes, indispensable à la stabilité, l’export vers le cytoplasme et la traduction des ARNm. Elle est composée de deux réactions : un clivage à l’extrémité 3’ suivie de l’addition d’une queue poly(A). Des études ont montré que la maturation en 3’ est inhibée en réponse aux dommages de l’ADN. Cependant, la cellule a mis en place des mécanismes compensatoires qui permettent à certains pré-ARNm d’être correctement maturés assurant ainsi le maintien de son intégrité. Les travaux que nous avons menés ont mis en évidence un mécanisme de résistance à l’inhibition de maturation en 3’ du pré-ARNm codant pour le suppresseur de tumeur p53. Ce mécanisme fait intervenir l’hélicase DHX36 qui déplie une structure secondaire appelée G-quadruplexe située en aval du site de clivage. Par ailleurs dans une deuxième étude, nous avons montré que la maturation en 3’ maintenue du pré-ARNm p53 en réponse aux dommages de l’ADN, est découplée du processus de transcription, contrairement au pré-ARNm TBP dont la maturation 3’ est inhibée en réponse aux dommage de l’ADN. Ce découplage a lieu grâce à un clivage co-transcriptionnelle du pré-ARNm p53 au niveau de la chromatine qui entraîne sa libération dans le nucléoplasme où il subit sa maturation en 3’. Une étude à grande échelle nous a permis de montrer que ce mécanisme de maturation en 3’ survenant dans le nucléoplasme est associé au maintien d'une maturation en 3’ efficace en réponse aux dommages de l’ADN. / The 3’-end processing of pre-mRNA, a key step in the post-transcriptional gene expression regulation, is essential for mRNA stability, export and translation. This process is a two-step reaction composed of a cleavage at the 3’-end followed by the addition of a poly(A) tail. Studies have shown that pre-mRNA 3’-end processing is inhibited in response to DNA damage. However, compensatory mechanisms exist to allow some pre-mRNA to be properly processed at their 3’-end in order to maintain cell integrity. For instance, in response to DNA damage, the 3’-end processing of the pre-mRNA coding for the tumor suppressor p53 is able to escape from its inhibition. In the present work, we have shown that the underlying mechanism involves the DHX36 helicase that unwinds a secondary structure called G-quadruplex located downstream of the cleavage site of the p53 pre-mRNA. Moreover, in a second study, we have shown that the maintained p53 pre-mRNA 3’-end processing in response to DNA damage is uncoupled from the transcription process, unlike the inhibited TBP pre-mRNA 3’-end processing. This uncoupling takes place through a co-transcriptional cleavage of p53 pre-mRNA from the chromatin and its release in the nucleoplasm where it undergoes its 3’-end processing. A genome-wide study allowed us to show that the pre-mRNA 3’-end processing occurring in the nucleoplasm is associated with a maintained 3’end processing in response to DNA damage

Poly(A) tail addition to pre-mRNAs is a highly coordinated and essential step in mRNA maturation involving multiple cis- and trans-acting factors. The trans-acting factor, poly(A) polymerase (PAP) plays an essential role in the polyadenylation of mRNA precursors. The Arabidopsis thaliana genome contains four putative PAP genes. We have found, using in silico analysis and transgenic plants expressing GUS under the control of the four PAP promoters, that each of these genes is expressed in overlapping, yet unique patterns. This gives rise to the possibility that these genes are not redundant and may be essential for plant survival. To further test this, inducible RNAi and T-DNA mutagenized plants were obtained and analyzed. Plants lacking all, or most, of each PAP gene product, due to RNAi induction, were not viable at any of the stages of plant growth tested. Furthermore, T-DNA PCR analysis determined that no plants containing a homozygous mutation, were viable. This data reveals that lack of any of the four PAP gene products has a significant effect on the plants ability of survive, thus indicating that each PAP gene is essential. Finally, transient expression experiments with each of the full length PAP cDNAs fused to GFP showed that the PAP I, PAP II and PAP IV gene products are localized throughout the nucleus and within nuclear speckles. The cellular localization of PAP III could not be determined.

Plant polyadenylation factors have proven difficult to purify and characterize, owing to the presence of excessive nuclease activity in plant nuclear extracts, thereby precluding the identification of polyadenylation signal-dependent processing and polyadenylation in crude extracts. As an alternative approach to identifying such factors, a screen was conducted for activities that inhibit the non-specific activity of plant poly(A) polymerases (PAP). One such factor (termed here as Putative Polyadenylation Factor B, or PPF-B) was identified in a screen of DEAE-Sepharose column fractions using a partially purified preparation of a plant nuclear poly(A) polymerase. This factor was purified to near homogeneity. Surprisingly, in addition to being an effective inhibitor of the nuclear PAP, PPF-B inhibited the activity of a chloroplast PAP. In contrast, this factor stimulated the activity of the yeast PAP. Direct assays of ATPase, proteinase, and nuclease activities indicated that inhibition of PAP activity was not due to depletion of substrates or degradation of products of the PAP reaction. The major polypeptide component of PPF-B proved to be a novel linker histone (RSP), which copurified with inhibitory activity by affinity chromatography on DNA-cellulose. The association of inhibitory activity with a linker histone and the spectrum of inhibitory activity, raise interesting possibilities regarding the role of PPF-B in nuclear RNA metabolism. These include a link between DNA damage and polyadenylation, as well as a role for limiting the polyadenylation of stable RNAs in the nucleus and nucleolus. The Arabidopsis genome possesses genes encoding probable homologs of most of the polyadenylation subunits that have been identified in mammals and yeast. Two of these reside on chromosome III and V and have the potential to encode a protein that is related to the yeast and mammalian Fip1 subunit (AtFip1-III and AtFip1-V). These genes are universally expressed in Arabidopsis tissues. AtFip1-V stimulates the non-specific activity of at least one Arabidopsis nuclear PAP, binds RNA, and interacts with other polyadenylation homologs AtCstF77 and AtCPSF30. These studies suggest that AtFip1- V is an authentic polyadenylation factor that coordinates other subunits and plays a role in regulating the activityof PAP in plants.

Thesis (Ph.D. in Molecular Biology) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 126-149). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Philosophiae Doctor - PhD (Biochemistry) / Human Retinoblastoma Binding Protein 6 (RBBP6) is a 200 kDa protein that has been implicated in a number of crucial cellular processes. It forms part of the mRNA 3'-end processing complex in both humans and yeast, and it contains an RS-like domain and interacts with core splicing proteins, suggesting multiple roles in mRNA processing. Through its RING finger domain it has been implicated in catalysing ubiquitination of the tumour suppressor p53, the oncogene Y-Box Binding Protein 1 (YB-1) and the DNA replication-associated protein zBTB38. It is one of only a few proteins known to bind to both p53 and pRb. At the N-terminus of the protein is the DWNN domain, an ubiquitin-like domain which is found only in this protein family. Four protein isoforms of RBBP6 have been identified in humans, all of which contain the DWNN domain: isoform 1 contains 1972 residues, isoform 2 contains 1758 residues and isoform 4 contains 952 residues. Isoform 3, which contains the first 101 residues of the full length protein (isoform 1), including the DWNN domain, followed by an unique 17-amino acid tail, is reported to be expressed independently of the other isoforms and to be down-regulated in a number of cancers.

Durant ce processus central qu’est la biogenèse des ARNm, la formation de la queue polyA est une étape clé impliquant de nombreuses activités enzymatiques et complexe protéiques. CF IA (Facteur de Clivage 1A) est un complexe macromoléculaire essentiel pour les deux étapes de clivage et de polyadénylation durant la formation de la queue poly(A) à l’extrémité 3’ de l’ARNm de levure. Constitué par les protéines RNA14, RNA15, Pcf11 et CLP1 dans une stœchiométrie supposée 2:2:1:1. Cependant, contrairement au complexe CPF (Facteur de Clivage et de Polyadénylation) qui porte les activités de clivage et de polyadénylation, aucune activité enzymatique n’a pu être associé au CF IA, suggérant un rôle d’architecture via d’une part la liaison à l’ARN et à d’autres complexes d’autre part. Dans ce travail, j’ai pu combiner les données obtenues par différentes approches biophysiques pour apporter des précisions sur l’organisation structurale au sein du CF IA mais également étudier l’importance biologique de certains motifs spécifiques. / During this major process which is mRNA biogenesis, the formation of the polyA tail is a key step involving numerous enzymatic activities and protein complex. CF IA (Cleavage Factor IA) is a macromolecular complex essential for both cleavage and polyadenylation steps during the formation of the 3'-end poly(A) tail of the yeast mRNA. Composed by RNA14, RNA15, Pcf11 and CLP1 yeast proteins in an assumed stochiometry of 2:2:1:1. However, unlike CPF (Cleavage and Polyadenylation Factor) complex hosting the both cleavage and polyadenylation activities, no enzymatic activity has been associated to CF IA, suggesting a scaffolding and/or positioning activity through the binding on the one hand to the RNA and on the other hand to other complexes. In this work, I was able to cross-use different biophysical technics to get insights on the structural organization within the CF IA as well as studying the biological importance of some specifics sequences.