Thesis advisor: Michelle M. Meyer / Ribosomes play a vital role in all cellular life translating the genetic code into functional proteins. This pivotal function is derived from its structure. The large and small subunits of the ribosome consist of 3 ribosomal RNA strands and over 50 individual ribosomal proteins that come together in a highly coordinated manner. There are striking differences between eukaryotic and prokaryotic ribosomes and many of the most potent antibacterial drugs target bacterial ribosomes (e.g. tetracycline and kanamycin). Bacteria spend a large amount of energy and nutrients on the production and maintenance of these molecular machines: during exponential growth as much as 40% of dry bacterial mass is ribosomes (Harvey 1970). Because of this, bacteria have evolved an elegant negative feedback mechanism for the regulation of their ribosomal proteins, known as autoregulation. When excess ribosomal protein is produced, unneeded for ribosome assembly, the protein binds a structured portion of its own mRNA transcript to prevent further expression of that operon. Autoregulation facilitates a quick response to changing environmental conditions and ensures economical use of nutrients. My thesis has investigated the autoregulatory function of ribosomal protein S15 in diverse bacterial phyla. In many bacterial species, when there is excess S15 the protein interacts with an RNA structure formed in the 5’-UTR of its own mRNA transcript that enables autoregulation of the S15-encoding operon, rpsO. For many ribosomal proteins (ex. L1, L20, S2) there is striking homology and often mimicry between the recognition motifs within the rRNA and the regulatory mRNA structure. However, this is not the case for S15-three different regulatory RNA structures have been previously described in E. coli, G. stearothermophilus, and T. thermophilus (Portier 1990, Scott 2001, Serganov 2003). These RNAs share little to no structural homology to one another, nor the rRNA, and they are narrowly distributed to their respective bacterial phyla, Gammaproteobacteria, Firmicutes, and Thermales. It is unknown which regulatory RNA structures control the expression of S15 outside of these phyla. Additionally, previous work has shown the S15 homolog from G. stearothermophilus is unable to regulate expression using the mRNA from E. coli. These observations formulate the crux of the question this thesis work endeavors to answer: What drove the evolution of such diverse regulatory RNA structures in these different bacteria? In Chapter II, “Discover and Validate Novel Regulatory Structures for Ribosomal Protein S15in Diverse Bacterial Phyla”, I present evidence for the in silico identification of three novel regulatory RNA structures for S15 and present experimental evidence that one of these novel structures is distinct from those previously described. In Chapter III, “Co-evolution of Ribosomal Protein S15 with Diverse Regulatory RNA Structures”, I present evidence that the amino acid differences in S15 homologs contribute to differences in mRNA binding profiles, and likely lead to the development of the structurally diverse array of the regulatory RNAs we observe in diverse bacterial phyla. In Chapter IV, “Synthetic cis-regulatory RNAs for Ribosomal Protein S15”, I investigate the derivation of novel cis-regulatory RNAs for S15 and find novel structures are readily-derived, yet interact with the rRNA-binding face of S15. Together the work presented in this thesis advances our understanding of the co-evolution between ribosomal protein S15 and its regulatory RNAs in diverse bacterial phyla. / Thesis (PhD) — Boston College, 2016. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Translation initiation factor IF1 is a small, essential and ubiquitous protein factor encoded by a single infA gene in bacteria. Although several important functions have been attributed to IF1, the precise reason for its indispensability is yet to be defined. It is known that IF1 binds to the ribosomal A-site during initiation, where it primarily contacts ribosomal RNA (rRNA) and induces large scale conformational changes in the small ribosomal subunit. To shed more light on the function of IF1 and its interaction with the ribosome, we have employed a genetic approach to elucidate structure-function interactions between IF1 and rRNA. A selection has been used to isolate second site suppressor mutations in rRNA that restore the growth of a cold sensitive mutant IF1 with an arginine to leucine substitution in position 69 (R69L).  This yielded two classes of suppressors – one class that mapped to the processing stem of 23S rRNA – a transient structure important for proper maturation of 23S rRNA; and the other class to the functional sequence of 16S rRNA. Suppressor mutations in the processing stem of 23S rRNA were shown to disrupt efficient processing of 23S rRNA. In addition, we report that at least one of the manifestations of cold sensitivity associated with the mutant IF1 is at the level of ribosomal subunit association. These results led to a model whereby the cold sensitive R69L mutant IF1 results in aberrant ribosomal subunit association properties, while the 23S processing stem mutations indirectly suppress this effect by decreasing the pool of mature 50S subunits available for association.  Spontaneous suppressor mutations in 16S rRNA were diverse in position and phenotypic properties, but all mutations affected ribosomal subunit association, in most cases by directly decreasing the affinity of the 30S for 50S subunits. Site directed mutagenesis of select positions in 16S rRNA yielded additional suppressor mutations that were localized to the mRNA and streptomycin binding sites on the small ribosomal subunit. We suggest that the 16S rRNA suppressors occur in positions that affect the conformational dynamics brought about by IF1. Taken together, this work indicates that the major function of IF1 is the modulation of ribosomal subunit association brought about through conformational changes of the 30S subunit. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.</p>

In all life ribosomes are the ribonucloprotein machines in charge of decoding the genetic code and synthesizing proteins. In eukaryotes, ribosomes are pre-assembled in the nucleus and exported to the cytoplasm where the final maturation steps occur prior to their partaking in translation. My dissertation work focused on aspects of the last two known steps of the pre-60S subunit cytoplasmic maturation. In the penultimate step, the anti-association factor Tif6 is released from 60S by the concerted action of the translocase-like GTPase Efl1 and Sdo1. The release of Tif6 is necessary for the ultimate maturation step, which involves release of the export adaptor Nmd3 by the ribosomal protein Rpl10 and the putative GTPase Lsg1. Nmd3 is an essential export adaptor of the 60S subunit. Nmd3 binds to the ribosome in the nucleolus and is the last known trans-acting factor to be released from the subunit in the cytoplasm. In order to gain a better understanding of the molecular events leading to the release of Nmd3 from the 60S subunit I set out to identify the binding site of Nmd3 on 60S. In a collaboration with Dr Joachim Frank’s laboratory, we obtained a cryo-EM model of Nmd3 in a complex with 60S showing Nmd3 binding to the subunit joining face of the ribosome. This work provided the first visualization of an export factor on a ribosomal subunit. The release of the anti-association factor Tif6 requires the translocase-like GTPase Efl1. Mutations in a loop of Rpl10 which embraces the P site tRNA trapped Tif6 on the subunit. These Rpl10 mutants could be suppressed by Tif6 mutants which have weakened affinity for the subunit. Mutations in Efl1 which suppress these Rpl10 mutants were also obtained. These suppressing mutations in Efl1 mapped to regions on the translocases eEF2 and EF-G important for conformational changes during translation. These results highlight molecular signaling between the P site, involving a loop of Rpl10, and Tif6, 90Å away. I propose that Efl1 promotes a translocation-like event during biogenesis of the 60S subunit prior to its first round of bona fide translation. / text

Le nucléole de la levure contient près de 100 petites ribonucléoprotéines nucléolaires (snoRNP), qui sont constituées d'une molécule d'ARN (snoRNA) et de quelques protéines. Les snoRNA forment deux grandes familles : la famille à boîtes C/D et à boîtes H/ACA. Ces snoRNA servent de guides pour des modifications post-transcriptionnelles des ARN ribosomiques (ARNr), c'est-à-dire la méthylation en 2'-OH de riboses (C/D) et la pseudouridylation (H/ACA). Certains de ces snoRNA sont requis pour des réactions de clivage sur le précurseur des ARNr; ces clivages servent à éliminer des régions espaceur dans le pré-ARNr. Contrairement aux autres snoRNA H/ACA, snR30 n'a pas de cible pour la pseudouridylation. Par contre, la snoRNP snR30 est essentielle aux clivages précoces des pré-ARNr menant à la production de l'ARNr 18S. Le but de ces travaux de recherche est de purifier le complexe snR30 afin d'identifier et caractériser ses composantes protéiques pour déterminer leur rôle dans la fonction particulière de snR30. De plus, nous voulons mieux saisir le rôle de snR30 dans la maturation de l'ARNr. Afin d'identifier le contenu protéique de snR30, nous avons mis au point un système de purification permettant d'isoler de façon spécifique snR30; d'une part, nous avons enrichi les échantillons en snoRNP H/ACA en purifiant la protéine H/ACA Garl et, d'autre part, nous avons isolé spécifiquement snR30 grâce à un aptamère d'ARN introduit dans la séquence de snR30. Cette méthode de purification nous a permis de montrer une association entre snR30 et la protéine nucléolaire Nop6, les protéines ribosomiques S9 et S18, ainsi que les histones H2B et H4. Ces protéines interagissent aussi avec d'autres snoRNA. Comme plusieurs éléments suggéraient que Nop6 était impliquée dans la biogenèse des ribosomes, nous avons voulu définir son rôle dans la maturation des ARNr. Nos analyses ont montré que Nop6 localise au nucléole et que cette protéine cosédimente avec snR30 dans un gradient de sucrose. Grâce à des expériences d'extension d'amorces, nous avons démontré que snR30 est nécessaire pour les clivages aux sites A0, A1 et A2 et que l'absence de Nop6 diminue l'efficacité de clivage au site A2. De plus, une analyse par microscopie électronique de cellules déplétées de snR30 indique que ce snoRNA est requis pour la formation du SSU processome, un complexe ribonucléoprotéique nécessaire à la biogenèse de la petite sous-unité du ribosome. L'ensemble de ces résultats suggère que le SSU processome n'est pas seulement composé du snoRNA U3 et de ses protéines associées, mais pourrait contenir plusieurs autres snoRNP, dont snR30. On sait que certaines protéines ribosomiques ont des rôles « extraribosomiques », c'est-à-dire qu'en plus d'être des constituants du ribosome, elles ont d'autres fonctions cellulaires. Comme la protéine ribosomique Rps18 peut être associée à snR30, ceci laisse supposer qu'elle pourrait avoir un rôle dans la maturation des ARNr. Chez la levure, Rps18 est exprimée à partir de gènes dupliqués (RPS18A et RPS18B) qui encodent des pré-ARNm dont la séquence diffère (principalement au niveau de l'intron) mais qui génèrent des protéines de séquences identiques. Afin d'étudier la fonction de Rps18, nous avons généré des souches de levures où l'expression de RPS18A ou RPS18B est modifiée. Nos résultats indiquent que ces protéines sont régulées de manières différentes et qu'elles ont des fonctions cellulaires distinctes. Nous montrons aussi qu'en plus d'être un constituant du ribosome, la protéine S18 est essentielle à la maturation de l'ARNr 18S. Somme toute, nos résultats suggèrent que Rps18B a un rôle plus important dans la maturation des ARNr tandis que Rps18A serait plus impliquée dans l'assemblage des ribosomes. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Nucléole, Ribosome, Ribonucléoprotéine, Ribosomopathies, ARNr

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The mammalian mitochondria contain their own genome which encodes thirteen polypeptide components of the oxidative phosphorylation (OxPhos) system, and the mitochondrial (mt-) rRNAs and tRNAs required for their translation. The maturation of the mitochondrial ribosome requires both mt-rRNAs to undergo post-transcriptional chemical modifications, folding of the rRNA and assembly of the protein components assisted by numerous biogenesis factors. The post-transcriptional modifications of the mt-rRNAs include base methylations, 2’-O-ribose methylations and pseudouridylation. However, the exact function of these modifications is unknown. Many mitoribosome biogenesis factors still remain to be identified and characterised. This work aims to broaden our understanding of two proteins involved in mitoribosome biogenesis through the study of the function of an rRNA methyltransferase and a novel biogenesis factor. Firstly, we characterised MRM1 (mitochondrial rRNA methyltransferase 1), a highly conserved 2’-O-ribose methyltransferase. We confirmed that MRM1 modifies a guanine in the peptidyl (P) transferase region of the 16S mt-rRNA that specifically interacts with the 3’ end of the tRNA at the ribosomal P-site. In bacteria, the modification is dispensable for ribosomal biogenesis and cell viability under standard conditions. However, in yeast mitochondria, Mrm1p is vital for ribosomal assembly and function. We generated knockout cells lines using programmable nuclease technology, and characterised the possible effects of MRM1 depletion on mitochondrial translation and mitoribosome biogenesis. We demonstrated that neither the enzyme nor the modification is required for human mitoribosomal assembly and translation in our experimental setup. Secondly, we identified a novel mitochondrially-targeted putative RNA endonuclease, YbeY. Using YbeY knockout cell lines, we showed that depletion of YbeY leads to loss of cell viability and OxPhos function as a consequence of a severe decrease in mitochondrial translation. Northern blotting and transcriptomic analysis using next generation RNA-Seq revealed transcript-specific changes to steady state levels. This analysis identified mt-tRNASer as a potential target of YbeY. We investigated the effect of YbeY deficiency on mitoribosomal assembly by quantitative sucrose gradient fractionation and mass spectrometry. This analysis showed that the mt-SSU is depleted in YbeY knockout cells. Further, immunoaffinity purification identified MRPS11 as a key interactor of YbeY. We propose that YbeY is a multifunctional protein that performs endonucleolytic functions in the mitochondria and also acts as a mitochondrial ribosome biogenesis factor, assisting small subunit assembly through its interaction with MRPS11.

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