Bruno Paquin

THE FUNGAL MITOCHONDRIAL GENOME PROJECT : EVOLUTION OF FUNGAL MITOCHONDRIAL GENOMES AND THEIR GENE EXPRESSION

Abstract The goal of the fungal mitochondrial genome project (FMGP) is to sequence complete mitochondrial genomes for a representative sample of the major fungal lineages; to analyze the genome structure, gene content, and conserved sequence elements of these sequences; and to study the evolution of gene expression in fungal mitochondria. By using our new sequence data for evolutionary studies, we were able to construct phylogenetic trees that provide further solid evidence that animals and fungi share a common ancestor to the exclusion of chlorophytes and protists. With a database comprising multiple mitochondrial gene sequences, the level of support for our mitochondrial phylogenies is unprecedented, in comparison to trees inferred with nuclear ribosomal RNA sequences. We also found several new molecular features in the mitochondrial genomes of lower fungi, including: (1) tRNA editing, which is the same type as that found in the mitochondria of the amoeboid protozoan Acanthamoeba castellanii; (2) two novel types of putative mobile DNA elements, one encoding a site-specific endonuclease that confers mobility on the element, and the other constituting a class of highly compact, structured elements; and (3) a large number of introns, which provide insights into intron origins and evolution. Here, we present an overview of these results, and discuss examples of the diversity of structures found in the fungal mitochondrial genome.

Molecular phylogeny of Allomyces macrogynus: Congruency between nuclear ribosomal RNA- and mitochondrial protein-based trees

We have sequenced the nuclear and mitochondrial small subunit rRNA genes (rns) and the mitochondrial genes coding for subunits 1 and 3 of the cytochrome oxidase (cox1 and cox3, respectively) of the chytridiomycete Allomyces macrogynus. Phylogenetic trees inferred from the derived COX1 and COX3 proteins and the nuclear rns sequences show with good bootstrap support that A. macrogynus is an early diverging fungus. The trees inferred from mitochondrial rns sequences do not yield a topology that is supported by bootstrap analysis. The similarity and the relative robustness of the nuclear rns and the mitochondrial protein-derived phylogenetic trees suggest that protein sequences are of higher value than rRNA sequences for reconstructing mitochondrial evolution. In addition, our trees support a monophyletic origin of mitochondria for the range of analyzed eukaryotes.

The recognition of a noncanonical RNA base pair by a zinc finger protein

BACKGROUNDnThe zinc finger (ZF) is the most abundant nucleic-acid-interacting protein motif. Although the interaction of ZFs with DNA is reasonably well understood, little is known about the RNA-binding mechanism. We investigated RNA binding to ZFs using the Zif268-DNA complex as a model system. Zif268 contains three DNA-binding ZFs; each independently binds a 3 base pair (bp) subsite within a 9 bp recognition sequence.nnnRESULTSnWe constructed a library of phage-displayed ZFs by randomizing the alpha helix of the Zif268 central finger. Successful selection of an RNA binder required a noncanonical base pair in the middle of the RNA triplet. Binding of the Zif268 variant to an RNA duplex containing a G.A mismatch (rG.A) is specific for RNA and is dependent on the conformation of the mismatched middle base pair. Modeling and NMR analyses revealed that the rG.A pair adopts a head-to-head configuration that counterbalances the effect of S-puckered riboses in the backbone. We propose that the structure of the rG.A duplex is similar to the DNA in the original Zif268-DNA complex.nnnCONCLUSIONSnIt is possible to change the specificity of a ZF from DNA to RNA. The ZF motif can use similar mechanisms in binding both types of nucleic acids. Our strategy allowed us to rationalize the interactions that are possible between a ZF and its RNA substrate. This same strategy can be used to assess the binding specificity of ZFs or other protein motifs for noncanconical RNA base pairs, and should permit the design of proteins that bind specific RNA structures.

Intron-encoded open reading frame of the GIY-YIG subclass in a plastid gene

Group-I introns, containing open reading frames (ORFs) that code for homing endonucleases, are widely distributed amongst eukaryotic organellar genomes. However, endonucleases of the GIY-YIG subclass have a restricted distribution in mitochondria and bacteriophages, and have never been observed in plastids. We have found the GIY-YIG motif in an intronic ORF within the previously published psbA gene sequence from Chlamydomonas reinhardtii chloroplasts. Based on phylogenetic analysis and an evaluation of amino-acid substitutions, this ORF is not closely related to any of the other GIY-YIG ORFs. These results suggest that GIY-YIG ORFs have a longer evolutionary history than previously assumed.

Cleavage of mitochondria-like transfer RNAs expressed in Escherichia coli

Mitochondrial (mt) transfer RNAs (tRNAs) often harbor unusual structural features causing their secondary structure to differ from the conventional cloverleaf. tRNAs designed with such irregularities, termed mt‐like tRNAs, are active in Escherichia coli as suppressors of reporter genes, although they display low steady‐state levels. Characterization of fragments produced during mt‐like tRNA processing in vitro and in vivo suggests that these RNAs are not fully processed at their 5′ ends and are cleaved internally. These abnormal processing events may account for the low levels of mature mt‐like RNAs in vivo and are most likely related to defective processing by RNase P.