Florence Baudin

Bio: Florence Baudin is an academic researcher from European Bioinformatics Institute. The author has contributed to research in topics: RNA & Transcription (biology). The author has an hindex of 31, co-authored 56 publications receiving 5182 citations. Previous affiliations of Florence Baudin include Centre national de la recherche scientifique & Unit of Virus Host Cell Interactions.

The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit

Abstract: The influenza virus polymerase, a heterotrimer composed of three subunits, PA, PB1 and PB2, is responsible for replication and transcription of the eight separate segments of the viral RNA genome in the nuclei of infected cells. The polymerase synthesizes viral messenger RNAs using short capped primers derived from cellular transcripts by a unique 'cap-snatching' mechanism. The PB2 subunit binds the 5' cap of host pre-mRNAs, which are subsequently cleaved after 10-13 nucleotides by the viral endonuclease, hitherto thought to reside in the PB2 (ref. 5) or PB1 (ref. 2) subunits. Here we describe biochemical and structural studies showing that the amino-terminal 209 residues of the PA subunit contain the endonuclease active site. We show that this domain has intrinsic RNA and DNA endonuclease activity that is strongly activated by manganese ions, matching observations reported for the endonuclease activity of the intact trimeric polymerase. Furthermore, this activity is inhibited by 2,4-dioxo-4-phenylbutanoic acid, a known inhibitor of the influenza endonuclease. The crystal structure of the domain reveals a structural core closely resembling resolvases and type II restriction endonucleases. The active site comprises a histidine and a cluster of three acidic residues, conserved in all influenza viruses, which bind two manganese ions in a configuration similar to other two-metal-dependent endonucleases. Two active site residues have previously been shown to specifically eliminate the polymerase endonuclease activity when mutated. These results will facilitate the optimisation of endonuclease inhibitors as potential new anti-influenza drugs.

Probing the structure of RNAs in solution

Abstract: During these last years, a powerful methodology has been developed to study the secondary and tertiary structure of RNA molecules either free or engaged in complex with proteins. This method allows to test the reactivity of every nucleotide towards chemical or enzymatic probes. The detection of the modified nucleotides and RNase cleavages can be conducted by two different paths which are oriented both by the length of the studied RNA and by the nature of the probes used. The first one uses end-labeled RNA molecule and allows to detect only scissions in the RNA chain. The second approach is based on primer extension by reverse transcriptase and detects stops of transcription at modified or cleaved nucleotides. The synthesized cDNA fragments are then sized by electrophoresis on polyacrylamide:urea gels. In this paper, the various structure probes used so far are described, and their utilization is discussed.

Structure and Nuclear Import Function of the C- Terminal Domain of Influenza Virus Polymerase Pb2 Subunit

Abstract: The trimeric influenza virus polymerase, comprising subunits PA, PB1 and PB2, is responsible for transcription and replication of the segmented viral RNA genome. Using a novel library-based screening technique called expression of soluble proteins by random incremental truncation (ESPRIT), we identified an independently folded C-terminal domain from PB2 and determined its solution structure by NMR. Using green fluorescent protein fusions, we show that both the domain and the full-length PB2 subunit are efficiently imported into the nucleus dependent on a previously overlooked bipartite nuclear localization sequence (NLS). The crystal structure of the domain complexed with human importin alpha5 shows how the last 20 residues unfold to permit binding to the import factor. The domain contains three surface residues implicated in adaptation from avian to mammalian hosts. One of these tethers the NLS-containing peptide to the core of the domain in the unbound state.

Structure of influenza virus RNP. I. Influenza virus nucleoprotein melts secondary structure in panhandle RNA and exposes the bases to the solvent.

Abstract: The influenza virus genome consists of eight segments of negative-sense RNA, i.e. the viral (v) RNA forms the template for the mRNA. Each segment is encapsidated by the viral nucleoprotein to form a ribonucleoprotein (RNP) particle and each RNP carries its own polymerase complex. We studied the interaction of purified nucleoprotein with RNA in vitro, by using a variety of enzymatic and chemical probes for RNA conformation. Our results suggest that the nucleoprotein binds to the vRNA backbone without apparent sequence specificity, exposing the bases to the outside and melting all secondary structure. In this way, the viral polymerase may transcribe the RNA without the need for dissociating the nucleoprotein and without being stopped by RNA secondary structure, and the viral RNPs are ready to start transcription as soon as they enter the host cell.

Incorporating chemical modification constraints into a dynamic programming algorithm for prediction of RNA secondary structure

Abstract: A dynamic programming algorithm for prediction of RNA secondary structure has been revised to accommodate folding constraints determined by chemical modification and to include free energy increments for coaxial stacking of helices when they are either adjacent or separated by a single mismatch. Furthermore, free energy parameters are revised to account for recent experimental results for terminal mismatches and hairpin, bulge, internal, and multibranch loops. To demonstrate the applicability of this method, in vivo modification was performed on 5S rRNA in both Escherichia coli and Candida albicans with 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate, dimethyl sulfate, and kethoxal. The percentage of known base pairs in the predicted structure increased from 26.3% to 86.8% for the E. coli sequence by using modification constraints. For C. albicans, the accuracy remained 87.5% both with and without modification data. On average, for these sequences and a set of 14 sequences with known secondary structure and chemical modification data taken from the literature, accuracy improves from 67% to 76%. This enhancement primarily reflects improvement for three sequences that are predicted with <40% accuracy on the basis of energetics alone. For these sequences, inclusion of chemical modification constraints improves the average accuracy from 28% to 78%. For the 11 sequences with <6% pseudoknotted base pairs, structures predicted with constraints from chemical modification contain on average 84% of known canonical base pairs.

N 6 -methyladenosine-dependent RNA structural switches regulate RNA–protein interactions

Abstract: RNA-binding proteins control many aspects of cellular biology through binding single-stranded RNA binding motifs (RBMs). However, RBMs can be buried within their local RNA structures, thus inhibiting RNA-protein interactions. N(6)-methyladenosine (m(6)A), the most abundant and dynamic internal modification in eukaryotic messenger RNA, can be selectively recognized by the YTHDF2 protein to affect the stability of cytoplasmic mRNAs, but how m(6)A achieves its wide-ranging physiological role needs further exploration. Here we show in human cells that m(6)A controls the RNA-structure-dependent accessibility of RBMs to affect RNA-protein interactions for biological regulation; we term this mechanism 'the m(6)A-switch'. We found that m(6)A alters the local structure in mRNA and long non-coding RNA (lncRNA) to facilitate binding of heterogeneous nuclear ribonucleoprotein C (HNRNPC), an abundant nuclear RNA-binding protein responsible for pre-mRNA processing. Combining photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) and anti-m(6)A immunoprecipitation (MeRIP) approaches enabled us to identify 39,060 m(6)A-switches among HNRNPC-binding sites; and global m(6)A reduction decreased HNRNPC binding at 2,798 high-confidence m(6)A-switches. We determined that these m(6)A-switch-regulated HNRNPC-binding activities affect the abundance as well as alternative splicing of target mRNAs, demonstrating the regulatory role of m(6)A-switches on gene expression and RNA maturation. Our results illustrate how RNA-binding proteins gain regulated access to their RBMs through m(6)A-dependent RNA structural remodelling, and provide a new direction for investigating RNA-modification-coded cellular biology.

How TCRs Bind MHCs, Peptides, and Coreceptors

Abstract: Since the first crystal structure determinations of alphabeta T cell receptors (TCRs) bound to class I MHC-peptide (pMHC) antigens in 1996, a sizable database of 24 class I and class II TCR/pMHC complexes has been accumulated that now defines a substantial degree of structural variability in TCR/pMHC recognition. Recent determination of free and bound gammadelta TCR structures has enabled comparisons of the modes of antigen recognition by alphabeta and gammadelta T cells and antibodies. Crystal structures of TCR accessory (CD4, CD8) and coreceptor molecules (CD3epsilondelta, CD3epsilongamma) have further advanced our structural understanding of most of the components that constitute the TCR signaling complex. Despite all these efforts, the structural basis for MHC restriction and signaling remains elusive as no structural features that define a common binding mode or signaling mechanism have yet been gleaned from the current set of TCR/pMHC complexes. Notwithstanding, the impressive array of self, foreign (microbial), and autoimmune TCR complexes have uncovered the diverse ways in which antigens can be specifically recognized by TCRs.