Les ARNnc sont impliqués dans la régulation de l’expression des gènes via divers mécanismes. Ils adoptent des structures 3D composées à 70% de pb WC formant des hélices de types A liées entre elles par des jonctions modulaires ayant des caractéristiques géométriques spécifiques. Nous avons identifié un nouveau motif 3D d’ARN apparenté au kturn, le pk-turn. Le pk-turn, situé dans la RNase P bactérienne permet, comme le k-turn, la formation d’un angle de 60° entre les hélices P16 & P17 avec cependant des exigences de séquences et de structure différentes. Le 2nd ribozyme qui a focalisé mon attention est le LCrz observé dans l’intron siamois (GIR2/LCrz) identifié dans le pré-ARNr 18S de la petite sous unité du ribosome eucaryote du myxomycète D. iridis. LCrz catalyse une réaction de branchement, équivalente à la première étape de l’épissage par les introns de groupe II, dans un contexte structural proche des introns du groupe I. Nous avons résolu la structure cristallographique du LCrz à une résolution de 2.5Å révélant un repliement inattendu. Cette structure a été confirmée par des expériences de SAXS. Ce travail nous permet de souligner la relation entre structure et fonction dans l'évolution des ribozymes. / NcRNA represent most of primary transcripts RNA in higher eukaryotes and tune gene expression via diverse mechanisms. They adopt 3D structures composed at 70% by WC bp forming A-form helices linked by RNA motifs. We identified the pk-turn, a new RNA motif related to k-turns that allow for the formation of a bend of 60° between stems P16 and P17 from the bacterial RNaseP. Yet it features different sequence and structural requirements than k-turns. The 2nd ribozyme which got my attention is the LCrz inserted in GIR2, a group I intron. This twintron is observed in the pre-rRNA 18S of the small subunit of the eukaryoteD. iris. LCrz catalyzes a reaction equivalent to the first step of splicing by group II introns, but in a structural context related to group I introns. We solved the 2.5 Å crystal structure of the LCrz and confirmed the unexpected shape by means of SAXS experiments. This work emphasizes the relationship between structure and function in the evolution of ribozymes.

The mitochondrial small-subunit ribosomal RNA (mt. SSU rRNA = rns) gene appears to be a reservoir for a number of group I and II introns along with the intron- encoded proteins (IEPs) such as homing endonucleases (HEases) and reverse transcriptases. The key objective for this thesis was to examine the rns gene among different groups of ophiostomatoid fungi for the presence of introns and IEPs. Overall the distribution of the introns does not appear to follow evolutionary lineages suggesting the possibility of rare horizontal gains and frequent loses. Some of the novel findings of this work were the discovery of a twintron complex inserted at position S1247 within the rns gene, here a group IIA1 intron invaded the ORF embedded within a group IC2 intron. Another new element was discovered within strains of Ophiostoma minus where a group II introns has inserted at the rns position S379; the mS379 intron represents the first mitochondrial group II intron that has an RT-ORF encoded outside Domain IV and it is the first intron reported to at position S379. The rns gene of O. minus WIN(M)371 was found to be interrupted with a group IC2 intron at position mS569 and a group IIB1 intron at position mS952 and they both encode double motif LAGLIDADG HEases referred as I-OmiI and I-OmiII respectively. These IEPs were examined in more detail to evaluate if these proteins represent functional HEases. To express I-OmiI and I-OmiII in Escherichia. coli, a codon-optimized versions of I-OmiI and I-OmiII sequences were synthesized based on differences between the fungal mitochondrial and bacterial genetic code. The optimized I-OmiI and I-OmiII sequences were cloned in the pET200/D TOPO expression vector system and transformed into E. coli BL21 (DE3). These two proteins were biochemically characterized and the results showed that: both I-OmiI and I-OmiII are functional HEases. Detailed data for I-OmiII showed that this endonuclease cleaves the target site two nucleotides upstream of the intron insertion site generating 4 nucleotide 3’overhangs.

The mitochondrial small-subunit ribosomal RNA (mt. SSU rRNA = rns) gene appears to be a reservoir for a number of group I and II introns along with the intron- encoded proteins (IEPs) such as homing endonucleases (HEases) and reverse transcriptases. The key objective for this thesis was to examine the rns gene among different groups of ophiostomatoid fungi for the presence of introns and IEPs. Overall the distribution of the introns does not appear to follow evolutionary lineages suggesting the possibility of rare horizontal gains and frequent loses. Some of the novel findings of this work were the discovery of a twintron complex inserted at position S1247 within the rns gene, here a group IIA1 intron invaded the ORF embedded within a group IC2 intron. Another new element was discovered within strains of Ophiostoma minus where a group II introns has inserted at the rns position S379; the mS379 intron represents the first mitochondrial group II intron that has an RT-ORF encoded outside Domain IV and it is the first intron reported to at position S379. The rns gene of O. minus WIN(M)371 was found to be interrupted with a group IC2 intron at position mS569 and a group IIB1 intron at position mS952 and they both encode double motif LAGLIDADG HEases referred as I-OmiI and I-OmiII respectively. These IEPs were examined in more detail to evaluate if these proteins represent functional HEases. To express I-OmiI and I-OmiII in Escherichia. coli, a codon-optimized versions of I-OmiI and I-OmiII sequences were synthesized based on differences between the fungal mitochondrial and bacterial genetic code. The optimized I-OmiI and I-OmiII sequences were cloned in the pET200/D TOPO expression vector system and transformed into E. coli BL21 (DE3). These two proteins were biochemically characterized and the results showed that: both I-OmiI and I-OmiII are functional HEases. Detailed data for I-OmiII showed that this endonuclease cleaves the target site two nucleotides upstream of the intron insertion site generating 4 nucleotide 3’overhangs.

NcRNA represent most of primary transcripts RNA in higher eukaryotes and tune gene expression via diverse mechanisms. They adopt 3D structures composed at 70% by WC bp forming A-form helices linked by RNA motifs. We identified the pk-turn, a new RNA motif related to k-turns that allow for the formation of a bend of 60° between stems P16 and P17 from the bacterial RNaseP. Yet it features different sequence and structural requirements than k-turns. The 2nd ribozyme which got my attention is the LCrz inserted in GIR2, a group I intron. This twintron is observed in the pre-rRNA 18S of the small subunit of the eukaryoteD. iris. LCrz catalyzes a reaction equivalent to the first step of splicing by group II introns, but in a structural context related to group I introns. We solved the 2.5 Å crystal structure of the LCrz and confirmed the unexpected shape by means of SAXS experiments. This work emphasizes the relationship between structure and function in the evolution of ribozymes.

The small ribosomal subunit gene of the Chaetomium thermophilum DSM 1495 is invaded by a nested intron at position mS1247, which is composed of a group I intron encoding a LAGLIDADG open reading frame interrupted by an internal group II intron. The first objective was to examine if splicing of the internal intron could reconstitute the coding regions and facilitate the expression of an active homing endonuclease. Using in vitro transcription assays, the group II intron was shown to self-splice only under high salt concentration. Both in vitro endonuclease and cleavage mapping assays suggested that the nested intron encodes an active homing endonuclease which cleaves near the intron insertion site. This composite arrangement hinted that the group II intron could be regulatory with regards to the expression of the homing endonuclease. Constructs were generated where the codon-optimized open reading frame was interrupted with group IIA1 or IIB introns. The concentration of the magnesium in the media sufficient for splicing was determined by the Reverse Transcriptase-Polymerase Chain Reaction analyses from the bacterial cells grown under various magnesium concentrations. Further, the in vivo endonuclease assay showed that magnesium chloride stimulated the expression of a functional protein but the addition of cobalt chloride to the growth media antagonized the expression. This study showed that the homing endonuclease expression in Escherichia coli can be regulated by manipulating the splicing efficiency of the group II introns which may have implications in genome engineering as potential ‘on/off switch’ for temporal regulation of homing endonuclease expression . Another objective was to characterize native homing endonucleases, cytb.i3ORF and I-OmiI encoded within fungal mitochondrial DNAs, which were difficult to express and purify. For these, an alternative approach was used where two compatible plasmids, HEase.pET28b (+)-kanamycin and substrate.pUC57-chloramphenicol, based on the antibiotic markers were maintained in Escherichia coli BL21 (DE3). The in vivo endonuclease assays demonstrated that these homing endonucleases were able to cleave the substrate plasmids when expressed, leading to the loss of the antibiotic markers and thereby providing an indirect approach to screen for potential active homing endonucleases before one invests effort into optimizing protein overexpression and purification strategies. / October 2016