Biochemical and genetic analysis of RNA processing and decay

L'expression des gènes est le conduit par lequel l'information génétique est traduite dans les phénotypes cellulaires. Récemment, il a été démontré que le programme de l'expression des gènes dans les cellules de mammifères est régi, au moins en partie par l'expression d'ARN double brin court (ARNdb). Ce mode de régulation des gènes est influencé par un grand groupe de protéines de liaison à l'ARN double brin qui peuvent soit stabiliser ou déclencher la dégradation de l'ARN double brin. En effet, les ribonucléases (RNases) spécifiques à l'ARN double brin jouent un rôle important dans l'expression des gènes. Dans la plupart des eucaryotes, les membres de la famille des RNase Ill spécifiques ~ l'ARNdb déclenchent la dégradation de l'ARN et initient la réponse immune de la cellule. Un défaut dans l'activité de la RNase Ill (DICER) inhibe l'expression des gènes et favorise le développement du cancer. D'autre part, la surexpression de la RNase Ill bloque l'infection virale. Cependant, très peu est connu sur la fonction de gestion domestique des RNases Ill chez les eucaryotes et le mécanisme par lequel ils font la distinction entre les espèces d'ARN cellulaire et l'infection virale. Cette thèse pave la voie sur la manière dont les ARNdbs sont choisis pour être clivés et démontre leur contribution dans le mécanisme de l'ARN en utilisant la levure comme modèle d'étude. Initialement, les déterminants de réactivité de la RNase Ill chez la levure (Rnt1 p) ont été identifiés in vitro et utilisés pour étudier l'impact global de Rnt1 p sur la maturation des ARNs noncodants. Les résultats indiquent que Rnt1 p est nécessaire pour la maturation de tous les petits ARN nucléolaires (snoRNAs) impliqués dans la méthylation de l'ARNr et ils identifient un nouveau rôle de Rnt1 p dans la maturation des snoRNAs introniques. Il a été démontré que le clivage de Rnt1 p contribue à coordonner l'expression de certaines protéines ribosomales et des snoRNA contenus dans leurs introns. La maturation du snoRNA à partir' de l'ARN prémessager bloque l'expression du gène hôte, alors qu'en retardant la maturation du snoRNA, celle-ci se séroule sur l'intron excisé ce qui permet l'expression des deux gènes. De cette façon, la cellule peut coordonner soigneusement la quantité de protéines ribosomales et de snoRNAs requises pour la biogénèse des ribosomes. En outre, l'analyse globale de la maturation des snoRNAs a identifié de nouveaux signaux de clivage de Rnt1 p qui ne présentent pas un motif de séquence conservé.Abstract: 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

Yeast RNase III triggers polyadenylation-independent transcription termination

Transcription termination of messenger RNA (mRNA) is normally achieved by polyadenylation followed by Rat1p-dependent 5'-3' exoribonuleolytic degradation of the downstream transcript. Here we show that the yeast ortholog of the dsRNA-specific ribonuclease III (Rnt1p) may trigger Rat1p-dependent termination of RNA transcripts that fail to terminate near polyadenylation signals. Rnt1p cleavage sites were found downstream of several genes, and the deletion of RNT1 resulted in transcription readthrough. Inactivation of Rat1p impaired Rnt1p-dependent termination and resulted in the accumulation of 3' end cleavage products. These results support a model for transcription termination in which cotranscriptional cleavage by Rnt1p provides access for exoribonucleases in the absence of polyadenylation signals.This work was supported by a grant from the Canadian Institute of Health Research. S. A. is a Chercheur Boursier National of the Fonds de la Recherche en Santé du Québec. F.R. holds a New Investigator Award from the Canadian Institute of Health Research. P-É.J. holds a post-doctoral award from the IRCM training program in cancer research funded by the CIHR. J.-R.L is a research fellow of the Terry Fox Foundation through an award from the National Cancer Institute of Canada

Biochemical and genetic analysis of RNA processing and decay

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

Genome-Wide Prediction and Analysis of Yeast RNase III-Dependent snoRNA Processing Signals

In Saccharomyces cerevisiae, the maturation of both pre-rRNA and pre-small nucleolar RNAs (pre-snoRNAs) involves common factors, thereby providing a potential mechanism for the coregulation of snoRNA and rRNA synthesis. In this study, we examined the global impact of the double-stranded-RNA-specific RNase Rnt1p, which is required for pre-rRNA processing, on the maturation of all known snoRNAs. In silico searches for Rnt1p cleavage signals, and genome-wide analysis of the Rnt1p-dependent expression profile, identified seven new Rnt1p substrates. Interestingly, two of the newly identified Rnt1p-dependent snoRNAs, snR39 and snR59, are located in the introns of the ribosomal protein genes RPL7A and RPL7B. In vitro and in vivo experiments indicated that snR39 is normally processed from the lariat of RPL7A, suggesting that the expressions of RPL7A and snR39 are linked. In contrast, snR59 is produced by a direct cleavage of the RPL7B pre-mRNA, indicating that a single pre-mRNA transcript cannot be spliced to produce a mature RPL7B mRNA and processed by Rnt1p to produce a mature snR59 simultaneously. The results presented here reveal a new role of yeast RNase III in the processing of intron-encoded snoRNAs that permits independent regulation of the host mRNA and its associated snoRNA

The RNA catabolic enzymes Rex4p, Rnt1p, and Dbr1p show genetic interaction with trans-acting factors involved in processing of ITS1 in Saccharomyces cerevisiae pre-rRNA

Eukaryotes have two types of ribosomes containing either 5.8S(L) or 5.8S(S) rRNA that are produced by alternative pre-rRNA processing. The exact processing pathway for the minor 5.8S(L) rRNA species is poorly documented. We have previously shown that the trans-acting factor Rrp5p and the RNA exonuclease Rex4p genetically interact to influence the ratio between the two forms of 5.8S rRNA in the yeast Saccharomyces cerevisiae. Here we report a further analysis of ITS1 processing in various yeast mutants that reveals genetic interactions between, on the one hand, Rrp5p and RNase MRP, the endonuclease required for 5.8S(S) rRNA synthesis, and, on the other, Rex4p, the RNase III homolog Rnt1p, and the debranching enzyme Dbr1p. Yeast cells carrying a temperature-sensitive mutation in RNase MRP (rrp2-1) exhibit a pre-rRNA processing phenotype very similar to that of the previously studied rrp5-33 mutant: ITS2 processing precedes ITS1 processing, 5.8S(L) rRNA becomes the major species, and ITS1 is processed at the recently reported novel site A4 located midway between sites A2 and A3. As in the rrp5-Δ3 mutant, all of these phenotypical processing features disappear upon inactivation of the REX4 gene. Moreover, inactivation of the DBR1 gene in rrp2-1, or the RNT1 gene in rrp5-Δ3 mutant cells also negates the effects of the original mutation on pre-rRNA processing. These data link a total of three RNA catabolic enzymes, Rex4p, Rnt1p, and Dbr1p, to ITS1 processing and the relative production of 5.8S(S) and 5.8S(L) rRNA. A possible model for the indirect involvement of the three enzymes in yeast pre-rRNA processing is discussed

Dataset Figure_5: Whi5-GFP intensity versus size and time in a synchronous G1 population

Whi5-GFP intensity as a function of time for the different FOV of elutriated cells. This dataset contains: 1. Raw TIF_images_NADH – These are autofluorescence images taken at each time point for the purposes of calculating cell size. Time points were every 10 minutes except at 30 minutes which had to be discarded due to poor focus. 2. Raw TIF_images_WHI5 – These raw image files correspond to images of Whi5-GFP excited at 1000 nm fpr all 11 time points of different FOV obtained at 3 different z positions (1-3) (0, -0.5 um, + 0.5 um). Whi5-GFP intensity values for each nucleus were taken for the z-position that gave the highest intensity for each nucleus. 3. NADHDATA_with_time plot.xlsx is the analysis of the raw images of auto-fluorescence exciting at 750 nm. The only relevant information for the Figure is in Colume C sheet 1. It is the cell area in total pixels. 4. Whi5Data_BestFocusPlanes_All_FOV.xlsx is the analysis of the Whi5-GFP images for Whi5-GFP intensity vs time and size for each time point (which corresponds to a different FOV)

Dataset Figure_4: Nuclear Whi5-GFP intensity versus time from repeated imaging of the same individual cells

Whi5-GFP intensity as a function of time for the same FOVs. Only small daughter cells in the asynchronous population at time point 0 were quantified as a function of time. The folder 'Image Files' contains raw image files for Whi5-GFP for FOV1,3,4,5 & 6 at each of the 6 time points (0, 20, 40, 60, 80, 100 min). The folder 'Excel_analysis_output' contains the output files for all five FOV (1,3,4,5,6) for 1. fovX_t0 or time 100_whi5_nadh_*.xlsx – at time 0 and time 100 minutes. These files correspond to the analysis of the background auto-fluorescence excited at 750 nm used to calculate size. The only relevant information from these analyses is the Cyto size (fL) column in tab 3 of each file 2. fovX_tX_whi5_*.xlsx Analysis of all time points and all FOV for Whi5-GFP intensities