A Comparative Study of the Structural Dynamics of Four Terminal Uridylyl Transferases.

African trypanosomiasis occurs in 36 countries in sub-Saharan Africa with 10,000 reported cases annually. No definitive remedy is currently available and if left untreated, the disease becomes fatal. Structural and biochemical studies of trypanosomal terminal uridylyl transferases (TUTases) demonstrated their functional role in extensive uridylate insertion/deletion of RNA. Trypanosoma brucei RNA Editing TUTase 1 (TbRET1) is involved in guide RNA 3' end uridylation and maturation, while TbRET2 is responsible for U-insertion at RNA editing sites. Two additional TUTases called TbMEAT1 and TbTUT4 have also been reported to share similar function. TbRET1 and TbRET2 are essential enzymes for the parasite viability making them potential drug targets. For this study, we clustered molecular dynamics (MD) trajectories of four TUTases based on active site shape measured by Pocket Volume Measurer (POVME) program. Among the four TUTases, TbRET1 exhibited the largest average pocket volume, while TbMEAT1's and TbTUT4's active sites displayed the most flexibility. A side pocket was also identified within the active site in all TUTases with TbRET1 having the most pronounced. Our results indicate that TbRET1's larger side pocket can be exploited to achieve selective inhibitor design as FTMap identifies it as a druggable pocket

The Function of a Central RNA Helicase RNP in Editosomes of Trypanosomes

Trypanosoma brucei requires extensive remodeling of its mitochondrial gene expression during different stages of its life cycle. RNA editing is one of the vital processes for these mitochondrial changes. This process involves insertions and deletions of uridylates in mitochondrial mRNAs that is governed by a multi-protein RNA editing core complex (RECC). Editing proceeds in small blocks from 3’ to 5’ direction leaded by small trans-acting guide RNAs (gRNAs). RECC appears to lack both, the mRNA substrates and gRNAs, indicating a role of accessory proteins in RNA editing. Many other non-RECC proteins have been discovered that directly impact the editing process, including mitochondrial RNA-binding complex 1(MRB1) that was shown to contain gRNAs. Despite of all the progress made, central long-standing question still remains unanswered including the mechanism for substrate delivery and regulation of RNA editing process. This dissertation presents the discovery of two variants of native MRB1 (mitochondrial RNA-binding complex 1) that we termed REH2-MRB and 3010-MRB. These MRB1 variants contain both mRNAs and gRNAs and show specialized functions, 3010-MRB and REH2-MRB seem to serve as scaffolds for RNA editing. REH2-MRB is defined by the critical RNA helicase termed REH2 (RNA editing helicase 2) that acts in trans, affecting multiple steps of the editing function in 3010-MRB. In addition, we discovered two cofactors of REH2. This novel RNA editing helicase 2-associated subcomplex (REH2C) binds mRNA substrates and products and therefore represents a stable mRNA-bound protein subcomplex (mRNP). Our working model is that MRB1 variant complexes are formed by the coupling of this mRNP with gRNA-bound subcomplexes (gRNPs). The mRNP/gRNP complexes form a platform for the assembly of functional mRNA-gRNA hybrids and catalytic RECC enzyme. Thus, in our proposed model editosomes are assembled in a stepwise process that involves the docking of mRNP and gRNP modules through specific base-pairing of respective mRNA and gRNAs. These subunits of the REH2C may control specific checkpoints in the editing pathway

RNA Editing in Trypanosomes: Substrate Recognition and its Integration to RNA Metabolism

RNA editing in trypanosomes is the post-transcriptional insertion or deletion of uridylates at specific sites in mitochondrial mRNAs. This process is catalyzed by a multienzyme, multisubunit complex through a series of enzymatic cycles directed by small, trans-acting RNA molecules. Despite impressive progress in our understanding of the mechanism of RNA editing and the composition of the editing complex, fundamental questions regarding RNP assembly and the regulation of catalysis remain. This dissertation presents studies of RNA-protein interactions between RNA editing complexes and substrate RNAs and the determination of substrate secondary structural determinants that govern them. Our results suggest that substrate association, cleavage and full-round editing by RNA editing complexes in vitro obey hierarchical determinants that increase in complexity as editing progresses and we propose a model for substrate recognition by RNA editing complexes. In addition, this dissertation also presents the characterization of a novel mitochondrial RNA helicase, named REH2 and its macromolecular interactions. Our data suggest that REH2 is intimately involved in interactions with macromolecular complexes that integrate diverse processes mediating mitochondrial gene expression. These results have implications for the mechanism of substrate RNA recognition by RNA editing complexes as well as for the integration of RNA editing to other facets of mitochondrial RNA metabolism

RNA editing in trypanosomes: a tale of two ligases

Uridylyl insertion/deletion mRNA editing is essential for mitochondrial gene expression in Trypanosoma brucei and governed by multi-protein complexes called editosomes. The final step in each cycle of this post-transcriptional process is that of re-ligating the edited mRNA fragments. The ~20S RNA editing core complex contains two RNA editing ligases, REL1 and REL2, located, respectively, in a deletion and an insertion subcomplex. While REL1 is clearly essential for RNA editing, REL2 knockdown by RNAi has not resulted in a detectable phenotype. To explain these findings, alternative scenarios have been suggested: (a) REL2 is not functional in vivo; (b) REL1 can function in both insertion and deletion editing, whereas REL2 can only function in insertion editing; (c) REL1 has an additional role in repairing erroneously cleaved mRNAs. To further investigate respective functions of the two RELs this study used three complimentary approaches: (i) genetic complementation with chimeric ligase enzymes, (ii) deep sequencing of RNA editing intermediates after ligase inactivation, and (iii) evolutionary analysis. In vivo expression of two chimeric ligases, providing a REL2 catalytic domain at REL1’s position in the deletion subcomplex and a REL1 catalytic domain at REL2’s position in the insertion subcomplex, did not rescue the growth defect caused by REL1 ablation. Although the results were not fully conclusive they suggest that it is the specific catalytic properties of REL1 rather than its position within the deletion subcomplex that makes it essential. In order to identify in vivo substrates of REL1, specific editing intermediates that accumulated after genetic knockdown of REL1 expression were captured by 5’ linker and deep sequenced using Ion Torrent and Illumina technology. Analyses of such unligated editing intermediates with bespoke bioinformatics tools suggest that REL1 functions in deletion editing as expected, but also in the repair of miscleaved mRNAs, implying a novel role for this ligase. Neither role can be fulfilled by REL2, at least not with sufficient efficiency. Sequencing data also suggest that either REL1 is not involved in ligation of addition editing substrates, or that REL2 in this case can fully compensate for loss of REL1. REL1, REL2 and KREPA3 sequences were subjected to analysis using MEGA5 and the HyPhy package available on the Datamonkey adaptive evolution server. Results indicated that all three editosome genes are under much stronger purifying than diversifying selective forces. In general this selection pressure to conserve protein sequence increased from KREPA3 to REL2 to REL1, suggesting a requirement to maintain catalytic function for both ligases. Taken together, these experiments reveal a novel function for REL1 during RNA editing, providing a rationale for its essentiality. Deductively, the results also suggest REL2, which was previously thought to be non-essential, may still be required by the cell at its position in the addition subcomplex. Evolutionary analysis suggests that the RELs and KREPA3 are under the same evolutionary forces to maintain their respective functions in RNA editing

Trypanosoma brucei editosomes have a single, bifunctional reaction center - Evidence for a non-collisional reaction mechanism

Most mitochondrial transcripts in African trypanosomes are edited to generate translatable transcripts. The reaction is catalyzed by a macromolecular protein complex, the 20S editosome. Editing is characterized by the site-specific insertion and/or deletion of exclusively U nucleotides and in order to catalyze the reaction, editosomes must bind a panel of different substrate pre-mRNAs. The experiments documented in chapter one verify that 20S editosomes bind different “in vivo-sized” transcripts with nanomolar affinities and association/dissociation rate constants typical for RNA/protein complexes. The editosome/RNA interaction is non-discriminative, thus enabling the interaction with different pre-edited mRNAs as well as with partially edited mRNAs and guide RNAs. Using immunogold-labeling in combination with transmission electron microscopy (TEM) I was able to demonstrate that editosomes have only one RNA substrate-binding site, which suggests that both subtypes of the RNA editing reaction (U-insertion and U-deletion) are catalyzed within a single, bifunctional reaction center. In chapter two I present the first atomicforce microscopy (AFM)-based pictures of 20S editosomes and 20S editosome/RNA complexes. The data confirm that editosomes have a single RNA binding domain and further demonstrate that editosomes contain a so far unknown “chaperone-type” RNA unwinding activity. Upon RNA binding, transcripts become progressively unwound, ultimately enabling multiple 20S editosomes to interact with one substrate RNA. RNA editing is a pre-requisite for the survival of Trypanosoma brucei. The life cycle of the parasite involves the cyclic transmission between a mammalian host and the Tsetse fly as the insect vector. Since RNA editing has been shown to be regulated between the two developmental stages, I analyzed in chapter three whether RNA editing is also regulated within the cell cycle of the parasite. Editosome isolates from the G1- and G2-phase of the trypanosome cell cycle were tested for their RNA editing activity. The experiments identified catalytic activity in both phases thus demonstrating that the processing reaction is not cell cycle-regulated. The basic steps of the editing reaction cycle have been unraveled with the help of an in vitro assay that is per-formed at dilute solvent conditions. However, in vivo the reaction takes place inside the highly “crowded” mitochondrial environment. In chapter four I analyzed the effects of macro-molecular crowding on RNA editing using defined conditions from dilute to semidilute to crowded solvent proper-ties. I was able to demonstrate that the thermodynamic stabilities of the pre-mRNA/gRNA hybrid RNAs differ at these conditions. Crowded solvent properties stabilize the RNA molecules and alter the rate constants for the association and dissociation of the substrate RNAs to editosomes. Ultimately, the processing reaction is inhibited. These results imply that the in vivo reaction cannot rely on a diffusionally-controlled, collision-based mechanism. The data advocate a scenario in which RNA editing is conducted by a “hand-over” or “channeling” of substrate RNAs from one processing machinery to the next

Duplication and Diversification of Arabidopsis thaliana Telomerase RNP Components

Telomerase is a highly regulated ribonucleoprotein complex that stabilizes eukaryotic genomes by replenishing telomeric repeats on chromosome ends. Defects in telomerase RNP components involving the catalytic subunit TERT or the RNA template TER lead to stem cell-related diseases such as dyskeratosis congenita and idiopathic pulmonary fibrosis, while inappropriate telomerase expression is a rate-limiting step in carcinogenesis. In this study we report the discovery of a novel negative regulatory mechanism for telomerase that stems from duplication and diversification of key components of the telomerase RNP in the flowering plant Arabidopsis thaliana. We show that Arabidopsis encodes three distinct TERs: TER1, TER2 and a processed form of TER2 termed TER2S. Although all three RNAs can serve as templates for telomerase in vitro, in vivo they have different expression patterns, assemble into distinct RNPs with different protein binding partners, and play opposing roles in telomere maintenance. The TER1 RNP is analogous to the telomerase enzyme previously described in other eukaryotes, but the TER2 RNP is a negative regulator of telomerase activity and telomere maintenance in vivo. Furthermore, we demonstrate that the Protection Of Telomeres (POT1) paralogs in Arabidopsis (POT1a, POT1b and POT1c) are novel TER binding proteins. This finding is striking because in yeast and vertebrates, POT1 is an essential component of the telomere capping complex and functions to distinguish the chromosome terminus from a double-strand break. Thus, our data argue that Arabidopsis POT1 proteins have migrated off of the chromosome terminus and onto the telomerase RNP, indicating that duplication and diversification of Arabidopsis telomerase may be the end result of the co-evolution of the TER and POT1 RNP components. Additionally, given the dire consequences of misregulating telomerase in human cells, our discovery of a novel negative regulatory mechanism for telomerase in plants strongly suggests that additional modes of telomerase control remain to be elucidated in vertebrates

Décodage de l'expression de gènes cryptiques

Pour certaines espèces, les nouvelles technologies de séquençage à haut débit et les pipelines automatiques d'annotation permettent actuellement de passer du tube Eppendorf au fichier genbank en un clic de souris, ou presque. D'autres organismes, en revanche, résistent farouchement au bio-informaticien le plus acharné en leur opposant une complexité génomique confondante. Les diplonémides en font partie. Ma thèse est centrée sur la découverte de nouvelles stratégies d'encryptage de l'information génétique chez ces eucaryotes, et l'identification des processus moléculaires de décodage. Les diplonémides sont des protistes marins qui prospèrent à travers tous les océans de la planète. Ils se distinguent par une diversité d'espèces riche et inattendue. Mais la caractéristique la plus fascinante de ce groupe est leur génome mitochondrial en morceaux dont les gènes sont encryptés. Ils sont décodés au niveau ARN par trois processus: (i) l'épissage en trans, (ii) l'édition par polyuridylation à la jonction des fragments de gènes, et (iii) l'édition par substitution de A-vers-I et C-vers-T; une diversité de processus posttranscriptionnels exceptionnelle dans les mitochondries. Par des méthodes bio-informatiques, j'ai reconstitué complètement le transcriptome mitochondrial à partir de données de séquences ARN à haut débit. Nous avons ainsi découvert six nouveaux gènes dont l'un présente des isoformes par épissage alternatif en trans, 216 positions éditées par polyuridylation sur 14 gènes (jusqu'à 29 uridines par position) et 114 positions éditées par déamination de A-vers-I et C-vers-T sur sept gènes (nad4, nad7, rns, y1, y2, y3, y5). Afin d'identifier les composants de la machinerie réalisant la maturation des ARNs mitochondriaux, le génome nucléaire a été séquencé, puis je l'ai assemblé et annoté. Cette machinerie est probablement singulière et complexe car aucun signal en cis ni acteur en trans caractéristiques des machineries d'épissage connues n'a été trouvé. J'ai identifié plusieurs candidats prometteurs qui devront être validés expérimentalement: des ARN ligases, un nombre important de protéines de la famille des PPR impliquées dans l'édition des ARNs dans les organites de plantes, ainsi que plusieurs déaminases. Durant ma thèse, nous avons mis en évidence de nouveaux types de maturation posttranscriptionnelle des ARNs dans la mitochondrie des diplonémides et identifié des candidats prometteurs de la machinerie. Ces composants, capables de lier précisément des fragments d'ARN et de les éditer pourraient trouver des applications biotechnologique. Au niveau évolutif, la caractérisation de nouvelles excentricités moléculaires de ce type nous donne une idée des processus de recrutement de gènes, de leur adaptation à de nouvelles fonctions, et de la mise en place de machineries moléculaires complexes.Thanks to new high throughput sequencing technologies and automatic annotation pipelines, proceeding from an eppendorf tube to a genbank file can be achieved in a single mouse click or so, for some species. Others, however, fiercely resist bioinformaticians with their confounding genomic complexity. Diplonemids are one of them. My thesis is centered on the discovery of new strategies for encrypting genetic information in eukaryotes, and the identification of molecular decoding processes. Diplonemids are a group of poorly studied marine protists. Unexpectedly, metagenomic studies have recently ranked this group as one of the most diverse in the oceans. Yet, their most distinctive feature is their multipartite mitochondrial genome with genes in pieces, and encryption by nucleotide deletions and substitutions. Genes are decrypted at the RNA level through three processes: (i) trans-splicing, (ii) polyuridylation at the junction of gene pieces and (iii) substitutions of A-to-I and C-to-T. Such a diverse arsenal of mitochondrial post-transcriptional processes is highly exceptional. Using a bioinformatics approach, I have reconstructed the mitochondrial transcriptome from RNA-seq libraries. We have identified six new genes including one that presents alternative trans-splicing isoforms. In total, there are 216 uridines added in 14 genes with up to 29 U insertions, and 114 positions edited by deamination (A-to-I or C-to-T) among seven genes (nad4, nad7, rns, y1, y2, y3, y5). In order to identify the machinery that processes mitochondrial RNAs, the nuclear genome has been sequenced. I have then assembled and annotated the genome. This machinery is probably unique and complex because no cis signal or trans actor typical for known splicing machineries have been found. I have identified promising protein candidates that are worth to be tested experimentally, notably RNA ligases, numerous members of the PPR family involved in plants RNA editing and deaminases. During my thesis, we have identified new types of post-transcriptional RNA processing in diplonemid mitochondria and identified new promising candidates for the machinery. A system capable of joining precisely or editing RNAs could find biotechnological applications. From an evolutionary perspective, the discovery of new molecular systems gives insight into the process of gene recruitment, adaptation to new functions and establishment of complex molecular machineries

Roles for Terminal Uridyl Transferases in the Post-Transcriptional Regulation of Developmental miRNAs

MicroRNAs (miRNAs) are a diverse and evolutionarily conserved class of non-coding RNAs that play a multitude of roles in many branches of eukaryotic biology. The regulation of miRNAs is dynamically controlled both spatially and temporally, and the expression of miRNAs can be modulated at the level of transcription or at points downstream of the miRNA maturation process. A relevant example of post-transcriptional miRNA regulation is the blockade of let-7 precursor miRNAs by Lin28 in embryonic stem cells. This pathway, which is initiated by the small RNA-binding protein Lin28, recruits the terminal uridyl transferase (TUTase) Zcchc11 to add a non-templated oligouridine tail to the miRNAs 3' end, and signals it for degradation by the cytoplasmic exonuclease Dis3l2. The Lin28/let-7 axis is essential for development and metabolic homeostasis, and is reactivated in a subset of human cancers. This thesis describes the biochemical mechanism underlying Lin28-mediated degradation of let-7, as well as a novel role for Zcchc11 and the related TUTase Zcchc6 in targeting mature developmental miRNAs in a Lin28-independent manner

Elements of Nucleotide Specificity in the <i>Trypanosoma brucei</i> Mitochondrial RNA Editing Enzyme RET2

The causative agent of African sleeping sickness, Trypanosoma brucei, undergoes an unusual mitochondrial RNA editing process that is essential for its survival. RNA editing terminal uridylyl transferase 2 of T. brucei (TbRET2) is an indispensable component of the editosome machinery that performs this editing. TbRET2 is required to maintain the vitality of both the insect and bloodstream forms of the parasite, and with its high-resolution crystal structure, it poses as a promising pharmaceutical target. Neither the exclusive requirement of uridine 5'-triphosphate (UTP) for catalysis, nor the RNA primer preference of TbRET2 is well-understood. Using all-atom explicitly solvated molecular dynamics (MD) simulations, we investigated the effect of UTP binding on TbRET2 structure and dynamics, as well as the determinants governing TbRET2’s exclusive UTP preference. Through our investigations of various nucleoside triphosphate substrates (NTPs), we show that UTP preorganizes the binding site through an extensive water-mediated H-bonding network, bringing Glu424 and Arg144 side chains to an optimum position for RNA primer binding. In contrast, cytosine 5'-triphosphate (CTP) and adenosine 5'-triphosphate (ATP) cannot achieve this preorganization and thus preclude productive RNA primer binding. Additionally, we have located ligand-binding “hot spots” of TbRET2 based on the MD conformational ensembles and computational fragment mapping. TbRET2 reveals different binding pockets in the apo and UTP-bound MD simulations, which could be targeted for inhibitor design

Elements of Nucleotide Specificity in the <i>Trypanosoma brucei</i> Mitochondrial RNA Editing Enzyme RET2

The causative agent of African sleeping sickness, Trypanosoma brucei, undergoes an unusual mitochondrial RNA editing process that is essential for its survival. RNA editing terminal uridylyl transferase 2 of T. brucei (TbRET2) is an indispensable component of the editosome machinery that performs this editing. TbRET2 is required to maintain the vitality of both the insect and bloodstream forms of the parasite, and with its high-resolution crystal structure, it poses as a promising pharmaceutical target. Neither the exclusive requirement of uridine 5'-triphosphate (UTP) for catalysis, nor the RNA primer preference of TbRET2 is well-understood. Using all-atom explicitly solvated molecular dynamics (MD) simulations, we investigated the effect of UTP binding on TbRET2 structure and dynamics, as well as the determinants governing TbRET2’s exclusive UTP preference. Through our investigations of various nucleoside triphosphate substrates (NTPs), we show that UTP preorganizes the binding site through an extensive water-mediated H-bonding network, bringing Glu424 and Arg144 side chains to an optimum position for RNA primer binding. In contrast, cytosine 5'-triphosphate (CTP) and adenosine 5'-triphosphate (ATP) cannot achieve this preorganization and thus preclude productive RNA primer binding. Additionally, we have located ligand-binding “hot spots” of TbRET2 based on the MD conformational ensembles and computational fragment mapping. TbRET2 reveals different binding pockets in the apo and UTP-bound MD simulations, which could be targeted for inhibitor design