Daniel Gautheret

The genome sequence of the model ascomycete fungus Podospora anserina

BackgroundThe dung-inhabiting ascomycete fungus Podospora anserina is a model used to study various aspects of eukaryotic and fungal biology, such as ageing, prions and sexual development.ResultsWe present a 10X draft sequence of P. anserina genome, linked to the sequences of a large expressed sequence tag collection. Similar to higher eukaryotes, the P. anserina transcription/splicing machinery generates numerous non-conventional transcripts. Comparison of the P. anserina genome and orthologous gene set with the one of its close relatives, Neurospora crassa, shows that synteny is poorly conserved, the main result of evolution being gene shuffling in the same chromosome. The P. anserina genome contains fewer repeated sequences and has evolved new genes by duplication since its separation from N. crassa, despite the presence of the repeat induced point mutation mechanism that mutates duplicated sequences. We also provide evidence that frequent gene loss took place in the lineages leading to P. anserina and N. crassa. P. anserina contains a large and highly specialized set of genes involved in utilization of natural carbon sources commonly found in its natural biotope. It includes genes potentially involved in lignin degradation and efficient cellulose breakdown.ConclusionThe features of the P. anserina genome indicate a highly dynamic evolution since the divergence of P. anserina and N. crassa, leading to the ability of the former to use specific complex carbon sources that match its needs in its natural biotope.

Novel selenoproteins identified in silico and in vivo by using a conserved RNA structural motif.

Selenocysteine is incorporated into selenoproteins by an in-frame UGA codon whose readthrough requires the selenocysteine insertion sequence (SECIS), a conserved hairpin in the 3′-untranslated region of eukaryotic selenoprotein mRNAs. To identify new selenoproteins, we developed a strategy that obviates the need for prior amino acid sequence information. A computational screen was used to scan nucleotide sequence data bases for sequences presenting a potential SECIS secondary structure. The computer-selected hairpins were then assayed in vivo for their functional capacities, and the cDNAs corresponding to the SECIS winners were identified. Four of them encoded novel selenoproteins as confirmed byin vivo experiments. Among these, SelZf1 and SelZf2 share a common domain with mitochondrial thioredoxin reductase-2. The three proteins, however, possess distinct N-terminal domains. We found that another protein, SelX, displays sequence similarity to a protein involved in bacterial pilus formation. For the first time, four novel selenoproteins were discovered based on a computational screen for the RNA hairpin directing selenocysteine incorporation.

ARNold: A web tool for the prediction of Rho-independent transcription terminators

Rho-independent termination is a major mechanism of transcriptional arrest in bacteria that controls both normal 3’ termination and a wide array of regulatory attenuation events. Detecting Rho-independent terminators is an obliged step in the annotation of bacterial operons. Yet, while several efficient algorithms are available for this purpose, there is no freely available web site enabling a rapid scanning of raw genomic sequence for the presence of terminators. Here we implemented such a web server, which combines two published prediction algorithms, Erpin and RNAmotif, and performs nearly as well as more complex procedures while being accessible to the non specialist. The ARNold Web server is available at : http://rna.igmors.u-psud.fr/toolbox/arnold/

Acquired initiating mutations in early hematopoietic cells of CLL patients

UNLABELLED Appropriate cancer care requires a thorough understanding of the natural history of the disease, including the cell of origin, the pattern of clonal evolution, and the functional consequences of the mutations. Using deep sequencing of flow-sorted cell populations from patients with chronic lymphocytic leukemia (CLL), we established the presence of acquired mutations in multipotent hematopoietic progenitors. Mutations affected known lymphoid oncogenes, including BRAF, NOTCH1, and SF3B1. NFKBIE and EGR2 mutations were observed at unexpectedly high frequencies, 10.7% and 8.3% of 168 advanced-stage patients, respectively. EGR2 mutations were associated with a shorter time to treatment and poor overall survival. Analyses of BRAF and EGR2 mutations suggest that they result in deregulation of B-cell receptor (BCR) intracellular signaling. Our data propose disruption of hematopoietic and early B-cell differentiation through the deregulation of pre-BCR signaling as a phenotypic outcome of CLL mutations and show that CLL develops from a pre-leukemic phase. SIGNIFICANCE The origin and pathogenic mechanisms of CLL are not fully understood. The current work indicates that CLL develops from pre-leukemic multipotent hematopoietic progenitors carrying somatic mutations. It advocates for abnormalities in early B-cell differentiation as a phenotypic convergence of the diverse acquired mutations observed in CLL.

Profile-based detection of microRNA precursors in animal genomes

MOTIVATION MicroRNAs (miRNA) are essential 21-22 nt regulatory RNAs produced from larger hairpin-like precursors. Local sequence alignment tools such as BLAST are able to identify new members of known miRNA families, but not all of them. We set out to estimate how many new miRNAs could be recovered using a profile-based strategy such as that implemented in the ERPIN program. RESULTS We constructed alignments for 18 miRNA families and performed ERPIN searches on animal genomes. Results were compared to those of a WU-BLAST search at the same E-value cutoff. The two combined approaches produced 265 new miRNA candidates that were not found in miRNA databases. About 17% of hits were ERPIN specific. They showed better structural characteristics than BLAST-specific hits and included interesting candidates such as members of the miR-17 cluster in Tetraodon. Profile-based RNA detection will be an important complement of similarity search programs in the completion of miRNA collections.

ASTD: The Alternative Splicing and Transcript Diversity database

The Alternative Splicing and Transcript Diversity database (ASTD) gives access to a vast collection of alternative transcripts that integrate transcription initiation, polyadenylation and splicing variant data. Alternative transcripts are derived from the mapping of transcribed sequences to the complete human, mouse and rat genomes using an extension of the computational pipeline developed for the ASD (Alternative Splicing Database) and ATD (Alternative Transcript Diversity) databases, which are now superseded by ASTD. For the human genome, ASTD identifies splicing variants, transcription initiation variants and polyadenylation variants in 68%, 68% and 62% of the gene set, respectively, consistent with current estimates for transcription variation. Users can access ASTD through a variety of browsing and query tools, including expression state-based queries for the identification of tissue-specific isoforms. Participating laboratories have experimentally validated a subset of ASTD-predicted alternative splice forms and alternative polyadenylation forms that were not previously reported. The ASTD database can be accessed at http://www.ebi.ac.uk/astd.

RNAML: A Standard Syntax for Exchanging RNA Information

Analyzing a single data set using multiple RNA informatics programs often requires a file format conversion between each pair of programs, significantly hampering productivity. To facilitate the interoperation of these programs, we propose a syntax to exchange basic RNA molecular information. This RNAML syntax allows for the storage and the exchange of information about RNA sequence and secondary and tertiary structures. The syntax permits the description of higher level information about the data including, but not restricted to, base pairs, base triples, and pseudoknots. A class-oriented approach allows us to represent data common to a given set of RNA molecules, such as a sequence alignment and a consensus secondary structure. Documentation about experiments and computations, as well as references to journals and external databases, are included in the syntax. The chief challenge in creating such a syntax was to determine the appropriate scope of usage and to ensure extensibility as new needs will arise. The syntax complies with the eXtensible Markup Language (XML) recommendations, a widely accepted standard for syntax specifications. In addition to the various generic packages that exist to read and interpret XML formats, an XML processor was developed and put in the open-source MC-Core library for nucleic acid and protein structure computer manipulation.

Genome-wide discovery and analysis of microRNAs and other small RNAs from rice embryogenic callus

Small RNAs constitute a new and unanticipated layer of gene regulation present in the three domains of life. In plants, all organs are ultimately derived from a few pluripotent stem cells localized in specialized structures called apical meristems. The development of meristems involves a coordinated balance between undifferentiated growth and differentiation, a phenomenon requiring a tight regulation of gene expression. We used in vitro cultured embryogenic calli as a model to investigate the roles of meristem-associated small RNAs. Using high throughput sequencing, we sequenced 20 million short reads with size of 18-30nt from rice undifferentiated and differentiated calli. We confirmed 50 known microRNA families, representing one third of annotated rice microRNAs. Using a specific computational pipeline for plant microRNA identification, we identified 24 novel microRNA families. Among them, 53 microRNA or microRNA* sequences appear to vary in expression between differentiated and undifferentiated calli, suggesting a role in meristem development. Our analysis also revealed a new class of plant small RNAs derived from 5’ or 3’ ends of mature tRNA analogous to the tRFs in human cancer cell. We independently verified the expression of these small RNAs from 5’ end of mature tRNA using qRT-PCR.

The ERPIN server: an interface to profile-based RNA motif identification

ERPIN is an RNA motif identification program that takes an RNA sequence alignment as an input and identifies related sequences using a profile-based dynamic programming algorithm. ERPIN differs from other RNA motif search programs in its ability to capture subtle biases in the training set and produce highly specific and sensitive searches, while keeping CPU requirements at a practical level. In its latest version, ERPIN also computes E-values, which tell biologists how likely they are to encounter a specific sequence match by chance-a useful indication of biological significance. We present here the ERPIN online search interface (http://tagc.univ-mrs.fr/erpin/). This web server automatically performs ERPIN searches for different RNA genes or motifs, using predefined training sets and search parameters. With a couple of clicks, users can analyze an entire bacterial genome or a genomic segment of up to 5Mb for the presence of tRNAs, 5S rRNAs, SRP RNA, C/D box snoRNAs, hammerhead motifs, miRNAs and other motifs. Search results are displayed with sequence, score, position, E-value and secondary structure graphics. An example of a complete genome scan is provided, as well as an evaluation of run times and specificity/sensitivity information for all available motifs.

An RNA pattern matching program with enhanced performance and portability.

The identification of RNA genes in DNA sequences generally involves searching the sequence or database for consensus nucleotides. However, specific base pairing patterns rather than sequences provide a better characterization of an increasing number of functional RNA molecules. Computer programs that automatically recognize higher-order structural motifs not only facilitate the identification of RNA genes, but also find an important application in the refinement of RNA structure descriptors, an enlightening task which involves the inference of essential structural elements associated with a molecular function. We present here a signicant improvement of the program RNAMOT which allows searches of primary and secondary structural patterns in sequence databases (Gautheret et al., 1990). An important performance enhancement was achieved using a faster string-matching algorithm and more efficient sequence scans. RNAMOT can now perform complete GenBank searches for RNA motifs in a few hours. Other enhancements include an automatic determination of the optimal search order for structural motifs, the handling of sequences of virtually unlimited length and a full implementation of the IUPAC/IUB codes in either target sequences or RNA descriptors. RNAMOT is written in the ANSI C language and compiles on any workstation or personal computer. The memory requirement depends on the largest sequence used in the search (~ 3 Mbytes for a GenBank search); CPU time depends on the frequency of pattern elements in the target sequence.