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Biomedical subjects

B Michot

Publications and source records attributed to B Michot.

At least 19 recordsLinked to original sources
PubMed1999

Ribosomal internal transcribed spacer 2 (ITS2) exhibits a common core of secondary structure in vertebrates and yeast.

Molecular mechanisms of ITS2 processing, a eukaryotic insertion between the 5.8S and LSU rRNA, remain largely elusive even in yeast. To delineate ITS2 structural and functional features which could be common to eukaryotes, we first produced phylo-genetically supported folding models in the vertebrate lineage, then tested them in deeper branchings and, more particularly, among yeasts. ITS2 comparisons between four Teleostei, a Chondrichthyes specimen and two jawless organisms have revealed a common folding architecture in four to five domains of secondary structure emerging from a preserved structural core. This folding, largely reminiscent of ITS2 architecture in mammals, is also preserved in amphibia and in chicken, despite dramatic sequence variations. Preferential conservation is located around a central loop and at the apex of a long stem in the ITS2 3'-half. Interestingly, these two independent structural features contain, respectively, the 3'-ends of the two transient rRNA precursors 8S and 12S RNA identified in mammals, suggesting a preservation of these intermediates of processing over the entire vertebrate group. Surprising similarities between the vertebrate ITS2 folding shape and that of invertebrates as well as protista have made intriguing the significant differences from the yeast model. A detailed comparative analysis including four relatively close species and Schizosaccharomyces pombe, a deep yeast branching, has revealed an alternative phylogenetically supported four-domain folding presenting strong similarities to the vertebrate model. Remarkably, the two best conserved regions of vertebrates have unambiguously preserved counterparts which are also sites for internal processing in yeast. Therefore, molecular mechanisms involved in ITS2 excision in vertebrates and yeast might be more closely related than currently believed and might require a very similar trans -acting machinery.

Animals↗
PubMed1999

Evolutionarily conserved structural features in the ITS2 of mammalian pre-rRNAs and potential interactions with the snoRNA U8 detected by comparative analysis of new mouse sequences.

Mechanisms of ITS2 excision from pre-rRNA remain largely elusive. In mammals, at least two endonucleolytic cleavages are involved, which result in the transient accumulation of precursors to 5.8S rRNA termed 8S and 12S RNAs. We have sequenced ITS2 in four new species of the Mus genus and investigated its secondary structure using thermodynamic prediction and comparative approach. Phylogenetic evidence supports an ITS2 folding organized in four domains of secondary structure extending from a preserved structural core. This folding is also largely conserved for the previously available mammalian ITS2 sequences, rat and human, despite their extensive sequence divergence relative to the Mus species. Conserved structural features include the structural core, containing the 3' end of 8S pre-rRNA within a single-stranded sequence, and a stem containing the 3' end of the 12S pre-rRNA species. A putative, phylogenetically preserved pseudoknot has been detected 1 nt downstream from the 12S 3' end. Two long complementarities have also been identified, in sequences conserved among vertebrates, between the pre-rRNA 32S and the snoRNA (small nucleolar RNA) U8 which is required for the excision of Xenopus ITS2. The first complementarity involves the 5.8S-ITS2 junction and 13 nt at the 5' end of U8, whereas the other one occurs between a mature 28S rRNA segment known to be required for ITS2 excision and positions 15-25 of snoRNA U8. These two potential interactions, in combination with ITS2 folding, could organize a functional pocket containing three cleavage sites and key elements for pre-rRNA processing, suggesting a chaperone role for the snoRNA U8.

Animals↗
PubMed1998

Microsporidian Encephalitozoon cuniculi, a unicellular eukaryote with an unusual chromosomal dispersion of ribosomal genes and a LSU rRNA reduced to the universal core.

Microsporidia are eukaryotic parasites lacking mitochondria, the ribosomes of which present prokaryote-like features. In order to better understand the structural evolution of rRNA molecules in microsporidia, the 5S and rDNA genes were investigated in Encephalitozoon cuniculi . The genes are not in close proximity. Non-tandemly arranged rDNA units are on every one of the 11 chromosomes. Such a dispersion is also shown in two other Encephalitozoon species. Sequencing of the 5S rRNA coding region reveals a 120 nt long RNA which folds according to the eukaryotic consensus structural shape. In contrast, the LSU rRNA molecule is greatly reduced in length (2487 nt). This dramatic shortening is essentially due to truncation of divergent domains, most of them being removed. Most variable stems of the conserved core are also deleted, reducing the LSU rRNA to only those structural features preserved in all living cells. This suggests that the E.cuniculi LSU rRNA performs only the basic mechanisms of translation. LSU rRNA phylogenetic analysis with the BASEML program favours a relatively recent origin of the fast evolving microsporidian lineage. Therefore, the prokaryote-like ribosomal features, such as the absence of ITS2, may be derived rather than primitive characters.

Animals↗
PubMed1997

ESSA: an integrated and interactive computer tool for analysing RNA secondary structure.

With ESSA, we propose an approach of RNA secondary structure analysis based on extensive viewing within a friendly graphical interface. This computer program is organized around the display of folding models produced by two complementary methods suitable to draw long RNA molecules. Any feature of interest can be managed directly on the display and highlighted by a rich combination of colours and symbols with emphasis given to structural probe accessibilities. ESSA also includes a word searching procedure allowing easy visual identification of structural features even complex and degenerated. Analysis functions make it possible to calculate the thermodynamic stability of any part of a folding using several models and compare homologous aligned RNA both in primary and secondary structure. The predictive capacities of ESSA which brings together the experimental, thermodynamic and comparative methods, are increased by coupling it with a program dedicated to RNA folding prediction based on constraints management and propagation. The potentialities of ESSA are illustrated by the identification of a possible tertiary motif in the LSU rRNA and the visualization of a pseudoknot in S15 mRNA.

Base Composition↗
PubMed1996

Intron-encoded, antisense small nucleolar RNAs: the characterization of nine novel species points to their direct role as guides for the 2'-O-ribose methylation of rRNAs.

A growing number of small nucleolar RNAs (snoRNAs) are intron-encoded, contain the characteristic box C (UGAUGA) and box D (CUGA) motifs and exhibit long complementarities to conserved sequences in mature rRNAs. We have identified nine additional members of this family, U32 to U40. All but one are encoded in introns of ribosomal protein genes in vertebrates: U32 to U35 in rpL13a, U36 in rpL7a and U38 to U40 in rpS8. By contrast, U37 is encoded in elongation factor 2 gene. Interestingly, U32 and U36 each contain two complementarities (one to 18 S and the other to 28 S rRNA). U32 to U40 are fibrillarin-associated, devoid of a 5'-trimethyl-cap and display an exclusively nucleolar localization. They are all metabolically stable and roughly as abundant as previously reported members of this family. Characterization of their homologs in distant species shows that their 10 to 14 nt long rRNA complementarities are conserved. A clue on the function of this snoRNA family is provided by the comparative analysis of the largely expanded collection of their conserved duplexes with rRNA. Not only does each duplex span at least one site of 2'-O-ribose methylation in the rRNA but the modification site is always at the same position in the duplex, paired to the fifth nucleotide upstream from a box D motif in the snoRNA. Consistent with the notion that each snoRNA of this family guides one particular methylation along the rRNA sequence, we have detected several pairs of snoRNAs with overlapping complementarities to rRNA tracts with vicinal sites of ribose methylations. In each case, the two overlapping complementarities are shifted from each other by a distance equal to the spacing between the methylated sites which are thus found at the same position within each of the mutually exclusive duplexes. Finally, we have also identified, within three previously known snoRNAs, novel antisense elements able to form a canonical duplex around ribose-methylated sites in rRNA, which further supports the conclusion that the duplex structure provides the 2'-O-methyltransferase with the appropriate site-specificity on the substrate.

Animals↗
PubMed1996

Sequence and characterisation of a ribosomal RNA operon from Agrobacterium vitis.

One of the four ribosomal RNA operons (rrnA) from the Agrobacterium vitis vitopine strain S4 was sequenced, rrnA is most closely related to the rrn operons of Bradyrhizobium japonicum and Rhodobacter sphaeroides and carries an fMet-tRNA gene downstream of its 5S gene, as in the case of R. sphaeroides. The 16S rRNA sequence of S4 differs from the A. vitis K309 type strain sequence by only one nucleotide, in spite of the fact that S4 and K309 have very different Ti plasmids. The predicted secondary structure of the S4 23S rRNA shows several features that are specific for the alpha proteobacteria, and an unusual branched structure in the universal B8 stem. The 3' ends of the three other rrn copies of S4 were also cloned and sequenced. Sequence comparison delimits the 3' ends of the four repeats and defines two groups: rrnA/rrnB and rrnC/rrnD.

Base Sequence↗
PubMed1995

U24, a novel intron-encoded small nucleolar RNA with two 12 nt long, phylogenetically conserved complementarities to 28S rRNA.

Following computer searches of sequence banks, we have positively identified a novel intronic snoRNA, U24, encoded in the ribosomal protein L7a gene in humans and chicken. Like previously reported intronic snoRNAs, U24 is devoid of a 5'-trimethyl-cap. U24 is immunoprecipitated by an antifibrillarin antibody and displays an exclusively nucleolar localization by fluorescence microscopy after in situ hybridization with antisense oligonucleotides. In vertebrates, U24 is a 76 nt long conserved RNA which is metabolically stable, present at approximately 14,000 molecules per human HeLa cell. U24 exhibits a 5'-3' terminal stem-box C-box D structure, typical for several snoRNAs, and contains two 12 nt long conserved sequences complementary to 28S rRNA. It is, therefore, strikingly related to U14, U20 and U21 snoRNAs which also possess long sequences complementary to conserved sequences of mature 18S or 28S rRNAs. In 28S rRNA the two tracts complementary to U24 are adjacent to each other, they involve several methylated nucleotides and are surprisingly close, within the rRNA secondary structure, to complementarities to snoRNAs U18 and U21. Identification of the yeast Saccharomyces cerevisiae U24 gene directly confirms the outstanding conservation of the complementarity to 28S rRNA during evolution, suggesting a key role of U24 pairing to pre-rRNA during ribosome biogenesis, possible in the control of pre-rRNA folding. Yeast S.cerevisiae U24 is also intron-encoded but not in the same host-gene as in humans or chicken.

Animals↗
PubMed1995

Antisense snoRNAs: a family of nucleolar RNAs with long complementarities to rRNA.

A growing subset of small nucleolar RNAs (snoRNAs) contains long stretches of sequence complementarity to conserved sequences in mature ribosomal RNA (rRNA). This article reviews current knowledge about these complementarities and proposes that these antisense snoRNAs might function in pre-rRNA folding, base modification and ribosomal ribonucleoprotein assembly, in some cases acting as RNA chaperones.

Animals↗
PubMed1995

RNA-d2: a computer program for editing and display of RNA secondary structures.

RNA-d2 is a user-friendly program developed for interactively generating aesthetic and non-overlapping drawings of RNA secondary structures. It designed so that the drawings can be edited in a very natural and intuitive way, in order to emphasize structural homologies between several molecules, as well as the foldings themselves to update the base-pair sets according to new data. The program automatically produces a polygonal display in which the unpaired nucleotides are regularly positioned on circles and the stems harmoniously distributed on their periphery. RNA secondary structures can be encoded via the keyboard, but the program also automatically draws output files from thermodynamic prediction programs. The user interacts directly on different screen displays according to the editing functions. Rotation/translation of any subdomain and deletion of stems are performed on a coloured backbone view to make easier the identification of structural features, whereas addition of new base-pairings and numbering manipulation are realized on a complete polygonal view. Each modification is displayed in real time on the screen. When the display is obscured by numerous overlaps despite the colour code of the backbone view, an automatic function progressively straightens the subdomains which are highly compacted by very dissymmetric internal loops. RNA-d2 allows easy untangling and editing of RNA molecules > 1000 nucleotides long.

Algorithms↗
PubMed1994

U21, a novel small nucleolar RNA with a 13 nt. complementarity to 28S rRNA, is encoded in an intron of ribosomal protein L5 gene in chicken and mammals.

Following a search of sequence data bases for intronic sequences exhibiting structural features typical of snoRNAs, we have positively identified by Northern assays and sequence analysis another intron-encoded snoRNA, termed U21. U21 RNA is a 93 nt. long, metabolically stable RNA, present at about 10(4) molecules per HeLa cell. It is encoded in intron 5 of the ribosomal protein L5 gene, both in chicken and in the two mammals studied so far, human and mouse. U21 RNA is devoid of a 5'-trimethyl-cap and is likely to result from processing of intronic RNA. The nucleolar localization of U21 has been established by fluorescence microscopy after in situ hybridization with digoxigenin-labeled oligonucleotide probes. Like most other snoRNAs U21 contains the box C and box D motifs and is precipitated by anti-fibrillarin antibodies. By the presence of a typical 5'-3' terminal stem, U21 appears more particularly related to U14, U15, U16 and U20 intron-encoded snoRNAs. Remarkably, U21 contains a long stretch (13 nt.) of complementarity to a highly conserved sequence in 28S rRNA. Sequence comparisons between chicken and mammals, together with Northern hybridizations with antisense oligonucleotides on cellular RNAs from more distant vertebrates, point to the preferential preservation of this segment of U21 sequence during evolution. Accordingly, this complementarity, which overlaps the complementarity of 28S rRNA to another snoRNA, U18, could reflect an important role of U21 snoRNA in the biogenesis of large ribosomal subunit.

Animals↗
PubMed1994

RNAlign program: alignment of RNA sequences using both primary and secondary structures.

We have developed an algorithm and a computer program for aligning new RNA sequences with a bank of aligned homologous RNA sequences. Given a common folding structure for the bank, the program performs an alignment between the bank and a new sequence, optimal both in terms of primary and secondary structure. This method is useful to align sequences that present a common folding structure despite extensive divergence of their primary structures. It allows these preserved regions to be precisely distinguished from domains with more variable secondary structure. An optimal alignment of a sequence of length N with a bank of homologous sequences of length M is produced in O (M2N3) time and O(M2N2) space. For sequences that are too long for an algorithm of this complexity, a proposed strategy is to use a classical alignment (using only primary structure data) then improve it with the new algorithm in the regions where the bank stems are not aligned with possible stems in the new sequence. The algorithm has been implemented in Turbo Pascal on a PC, and has been used to align RNA sequences of eubacterial large ribosomal subunit.

Algorithms↗
PubMed1994

U20, a novel small nucleolar RNA, is encoded in an intron of the nucleolin gene in mammals.

We have found that intron 11 of the nucleolin gene in humans and rodents encodes a previously unidentified small nucleolar RNA, termed U20. The single-copy U20 sequence is located on the same DNA strand as the nucleolin mRNA. U20 RNA, which does not possess a trimethyl cap, appears to result from intronic RNA processing and not from transcription of an independent gene. In mammals, U20 RNA is an 80-nucleotide-long, metabolically stable species, present at about 7 x 10(3) molecules per exponentially growing HeLa cell. It has a nucleolar localization, as indicated by fluorescence microscopy following in situ hybridization with digoxigenin-labeled oligonucleotides. U20 RNA contains the box C and box D sequence motifs, hallmarks of most small nucleolar RNAs reported to date, and is immunoprecipitated by antifibrillarin antibodies. It also exhibits a 5'-3' terminal stem bracketing the box C-box D motifs like U14, U15, U16, or Y RNA. A U20 homolog of similar size has been detected in all vertebrate classes by Northern (RNA) hybridization with mammalian oligonucleotide probes. U20 RNA contains an extended region (21 nucleotides) of perfect complementarity with a phylogenetically conserved sequence in 18S rRNA. This complementarity is strongly preserved among distant vertebrates, suggesting that U20 RNA may be involved in the formation of the small ribosomal subunit like nucleolin, the product of its host gene.

Base Sequence↗
PubMed1993

Conserved secondary structures in the ITS2 of trematode pre-rRNA.

Biological functions of transcribed spacer regions in eucaryotic pre-rRNAs remain elusive so far. Utilization of the comparative approach to analyse their secondary structure has been impeded by the extensive sequence divergence observed among most of the specimens available to date. However, we have taken advantage of the recent derivation of a set of largely similar sequences for different schistosome species to look for the presence of constrained secondary structures within the internally transcribed spacer 2 (ITS2). We show that several common features of secondary structure are shared by these species despite sequence variation, with some of them supported by compensatory changes, suggesting a significant role of ITS2 as an RNA domain during ribosome biogenesis.

Animals↗
PubMed1992

An algorithm for comparing RNA secondary structures and searching for similar substructures.

To access the functional informations carried by RNA molecules at the level of their secondary structure interactions, we propose a comparison method based on a tree edit algorithm which takes into account the tree structure of RNA foldings. Any secondary structure is translated into a tree involving all its elementary substructures; then a shorter condensed tree is built in which any unbranched helix interspersed with bulges and interior loops is taken as a single node. This method includes several parameters: a comparison matrix between structural units, gap penalties, and the scoring between nodes of the condensed trees. Their effects have been analysed using as a model a rapidly divergent domain of the large ribosomal RNA, for which structural variation during evolution is well known. This method allows one to recognize precisely, in large target molecules, definite substructures that present with the query molecules only a limited set of closely related secondary structure features; it is still efficient if intervening features, which can correspond to insertion/deletion of entire stem regions, separate such structural elements. When coupled with a hierarchical clustering algorithm, this method is suitable for classifying RNA molecules according to their secondary structure homologies.

Algorithms↗
PubMed1991

Interaction of cis-diamminedichloroplatinum (II) with single-stranded DNA in the presence or absence of Escherichia coli single-stranded binding protein.

We have compared the mode of fixation in vitro of the antitumor drug cis-diamminedichloroplatinum (II) (cis-DDP) to single-stranded M13mp10 DNA either in the presence or absence of the Escherichia coli single-stranded binding protein (SSB). Platinum binding sites have been identified by taking advantage of their capacity to inhibit DNA replication of primed M13 DNA catalysed by E. coli DNA polymerase I large fragment. We report here that the presence of SSB increases the number of platinum-DNA lesions and alters their distribution. We also present evidence that SSB allows cis-DDP to bind to DNA sequences otherwise less accessible.

Bacterial Proteins↗
PubMed1991

Secondary structure of the 5' external transcribed spacer of vertebrate pre-rRNA. Presence of phylogenetically conserved features.

Eucaryotic pre-rRNA spacers are evolutionarily highly variable in sequence and size, and are markedly expanded in vertebrates, particularly in mammals. The longest mammalian spacer by far is the 3.5-4-kb 5' external transcribed spacer (5'-ETS), which is excised in two steps. We present a folding model for the entire mammalian 5'-ETS, derived from comparative analyses and thermodynamic predictions for mouse, rat and human sequences, which should prove helpful in identifying cis-acting processing signals, particularly those involved in its early internal cleavage, for which U3 RNA is an essential factor. Although the rodent and primate sequences have extensively diverged, a series of relatively conserved sequence tracts can nevertheless be identified: they participate in base-pairing, preserved through the occurrence of compensatory base changes, which delineate four independent domains of secondary structure. The first domain is located entirely upstream from the site of internal cleavage. The second domain, immediately downstream from this cleavage site, encompasses most of the region required for faithful and efficient in vitro processing at this site. Phylogenetically supported conserved structures also define two other independent domains, encompassing most of the 5'-ETS length, with the presence of giant hairpins (extending from the conserved core elements) which exhibit both some analogous features and substantial differences between man and mouse. The comparative analysis was extended to the two other vertebrate sequences available so far, amphibians Xenopus laevis and Xenopus borealis. The amphibian folding model, supported by comparative evidence between these two species, displays some features in common with the mammalian model, with a similar organization into four separate domains of secondary structure, suggesting the functional relevance of these structures in the process of ribosome formation.

Amphibians↗
PubMed1990

Evolution of large-subunit rRNA structure. The diversification of divergent D3 domain among major phylogenetic groups.

During evolution, the potential for sequence (and length) variation of large-subunit rRNA has been mostly restricted over 12 divergent domains (termed D1-D12) interspersed along the molecule. Here, we have focused our attention onto the D3 divergent domain, through a detailed analysis of its pattern of variation in the phylogeny, both in terms of primary and secondary structures. We have systematically compared all the procaryotic and eucaryotic sequences published so far (i.e. 36 species), together with a series of 10 additional eucaryotic specimens, which were determined by direct RNA sequencing. Secondary structures supported by comparative evidence have been derived for archaebacteria, eubacteria and eucaryotes respectively, which shows that the D3 domain contains a subset of universally conserved structural features interspersed with four variable subdomains. Within the four portions where a structural diversification has taken place, elementary structures specific of large phylogenetic groups can be identified. Remarkably such diversified structures appear to be preserved despite sequence divergence, suggesting they correspond to functionally important structures. Accordingly, the mode of sequence variation of the D3 domain suggests this region of the molecule may encode elementary functions of rRNA which could have significantly diversified during the evolution of the major groups of organisms.

Animals↗