A short motif in Drosophila SECIS Binding Protein 2 provides differential binding affinity to SECIS RNA hairpins

Selenoproteins contain the amino acid selenocysteine which is encoded by a UGA Sec codon. Recoding UGA Sec requires a complex mechanism, comprising the cis-acting SECIS RNA hairpin in the 3′UTR of selenoprotein mRNAs, and trans-acting factors. Among these, the SECIS Binding Protein 2 (SBP2) is central to the mechanism. SBP2 has been so far functionally characterized only in rats and humans. In this work, we report the characterization of the Drosophila melanogaster SBP2 (dSBP2). Despite its shorter length, it retained the same selenoprotein synthesis-promoting capabilities as the mammalian counterpart. However, a major difference resides in the SECIS recognition pattern: while human SBP2 (hSBP2) binds the distinct form 1 and 2 SECIS RNAs with similar affinities, dSBP2 exhibits high affinity toward form 2 only. In addition, we report the identification of a K (lysine)-rich domain in all SBP2s, essential for SECIS and 60S ribosomal subunit binding, differing from the well-characterized L7Ae RNA-binding domain. Swapping only five amino acids between dSBP2 and hSBP2 in the K-rich domain conferred reversed SECIS-binding properties to the proteins, thus unveiling an important sequence for form 1 binding

The Hsp90 chaperone controls the biogenesis of L7Ae RNPs through conserved machinery

RNA-binding proteins of the L7Ae family are at the heart of many essential ribonucleoproteins (RNPs), including box C/D and H/ACA small nucleolar RNPs, U4 small nuclear RNP, telomerase, and messenger RNPs coding for selenoproteins. In this study, we show that Nufip and its yeast homologue Rsa1 are key components of the machinery that assembles these RNPs. We observed that Rsa1 and Nufip bind several L7Ae proteins and tether them to other core proteins in the immature particles. Surprisingly, Rsa1 and Nufip also link assembling RNPs with the AAA + adenosine triphosphatases hRvb1 and hRvb2 and with the Hsp90 chaperone through two conserved adaptors, Tah1/hSpagh and Pih1. Inhibition of Hsp90 in human cells prevents the accumulation of U3, U4, and telomerase RNAs and decreases the levels of newly synthesized hNop58, hNHP2, 15.5K, and SBP2. Thus, Hsp90 may control the folding of these proteins during the formation of new RNPs. This suggests that Hsp90 functions as a master regulator of cell proliferation by allowing simultaneous control of cell signaling and cell growth

Assemblage et fonction de complexes ARN-protéines

Ribonucleoprotein particles (or RNPs) play essential roles in many fundamental cellular processes. Assembly of RNP particles is a complex process that requires several processing steps and multiple assembly factors. Proper formation of RNP particles is essential for their function. Thus, it is of fundamental importance to understand how RNPs are assembled within the cell. In my career I analyzed several aspects of these mechanisms. During my thesis directed by Chantal Ehresmann (1990-1994) in the team of Bernard Ehresmann (UPR 9002 du CNRS), I studied the 16S rRNA binding site of E. coli ribosomal protein S8. In the team of David Tollervey (EMBL, Heidelberg (1994-1996) and University d'Edinburg (1996-2001) I analyzed the mechanisms of maturation, assembly and degradation of various RNPs. I participated in the characterization of the exosome, a complex of 3'-5' exonucleases involved in the processing and degradation of various RNAs in yeast. I also studied the role of chaperone proteins in the biogenesis of snoRNAs (ribosome biogenesis), rRNAs and tRNAs.In 2001 I was recruited by the CNRS in the team of Alain Krol to study the mechanisms of selenoprotein synthesis. Selenoprotein synthesis requires co-translational recoding of in-frame UGA codons. In eukaryotes, this process involves the assembly of RNA-protein complexes to specific stem-loops located in the 3'UTR of selenoprotein mRNAs, called Selenocysteine Insertion Sequences (SECIS). SECIS-binding protein 2 binds the SECIS element and recruits the factors of the biosynthesis machinery. SBP2 is part of supramolecular complexes and undergoes nucleocytoplasmic shuttling, indicating a possible nuclear assembly of the SECIS mRNP. The RNA-binding domain of SBP2 belongs to the L7Ae family of proteins. Members of this family are at the core of many RNPs and are thus involved in many cellular functions. L7Ae proteins are part of the large and small ribosomal sub-units (translation), box C/D and H/ACA snoRNPs (ribosomal RNA biogenesis), spliceosomal RNPs (splicing), telomerase (telomere replication), and selenoprotein mRNPs (translation of selenoproteins). Our aims are to elucidate the principles of SBP2/SECIS interaction, to identify the components of the complexes that bind to the SECIS and understand their assembly pathway.In collaboration with Edouard Bertrand (Montpellier), Bruno Charpentier and Christiane Branlant (Nancy) we have identified a conserved molecular machinery for the assembly of RNPs of the L7Ae family. This machinery is conserved from yeast to human and of fundamental importance for the cell. It is composed of an adaptor protein and a complex of protein chaperones linked to HSP90. Our role is to understand it's specific role in selenoprotein mRNP formation.Les particules ribonucléoprotéiques (ou RNP) sont à la base de nombreuses fonctions cellulaires fondamentales. La formation de ces particules RNP est un processus très complexe qui nécessite de nombreuses étapes de maturation et de multiples facteurs d'assemblage. Par ailleurs, une structure correcte des particules RNP est essentielle à leur fonction. Il est donc critique de comprendre comment ces particules sont formées dans la cellule. Au cours de ma carrière, je me suis intéressée à plusieurs aspects de ces mécanismes. Au cours de ma thèse dirigée par Chantal Ehresmann (1990-1994), dans l'équipe de Bernard Ehresmann (UPR 9002 du CNRS) j'ai étudié le mode d'interaction de la protéine ribosomique S8 sur l'ARNr 16S d'E. coli. Mon travail post-doctoral dans l'équipe de David Tollervey (EMBL, Heidelberg (1994-1996) et Université d'Edimbourg (1996-2001) a porté sur l'étude des mécanismes de maturation, d'assemblage et de dégradation de diverses RNP. J'ai notamment contribué à la caractérisation de l'exosome, un complexe d'exonucléases 3'->5' impliqué dans la maturation et la dégradation de divers ARN chez la levure. J'ai également étudié le rôle de protéines chaperons dans la biogenèse des snoARN (biogenèse des ribosomes), des ARN ribosomiques et des ARNt. En 2001, j'ai été recrutée au grade de chargée de recherche au CNRS dans l'équipe d'Alain Krol où nous étudions les mécanismes de synthèse des sélénoprotéines. L'incorporation de sélénocystéine dans les sélénoprotéines fait appel au recodage co-traductionnel d'un codon UGASec en phase. Chez les eucaryotes, ce mécanisme implique l'assemblage d'un complexe ARN-protéine au niveau d'une structure en tige-boucle ou ARN SECIS (Selenocysteine Insertion Sequence) située dans la région 3' non codante de l'ARNm des sélénoprotéines. La protéine SBP2 se fixe spécifiquement à l'ARN SECIS et recrute les facteurs de la machinerie de biosynthèse. Elle fait également partie de complexes supramoléculaires dans le cytoplasme et le noyau, suggérant un possible assemblage nucléaire de la mRNP SECIS. Nous avons montré que la protéine SBP2 présentait une origine évolutive commune avec des protéines de la famille L7Ae. Ces protéines partagent un domaine de liaison à l'ARN similaire et participent à la construction de plusieurs RNP essentielles telles les sous-unités ribosomiques, les snoRNP (biogenèse des ribosomes), les snRNP (épissage), et les mRNP codant pour les sélénoprotéines. Nos objectifs sont d'élucider les principes d'interaction SBP2/SECIS, d'identifier les composants moléculaires des complexes qui se forment autour du SECIS et de comprendre leur assemblage.En collaboration avec Edouard Bertrand (Montpellier) et Bruno Charpentier et Christiane Branlant (Nancy) nous avons identifié une machinerie d'assemblage des RNP L7Ae conservée de la levure à l'homme et d'importance fondamentale pour la cellule. Elle est constituée d'une protéine adaptatrice et d'un complexe de protéines chaperons. Notre objectif est de comprendre son rôle dans l'assemblage des mRNP de sélénoprotéines

eIF3 Interacts with Selenoprotein mRNAs

International audienceThe synthesis of selenoproteins requires the co-translational recoding of an in-frame UGASec codon. Interactions between the Selenocysteine Insertion Sequence (SECIS) and the SECIS binding protein 2 (SBP2) in the 3′untranslated region (3′UTR) of selenoprotein mRNAs enable the recruitment of the selenocysteine insertion machinery. Several selenoprotein mRNAs undergo unusual cap hypermethylation and are not recognized by the translation initiation factor 4E (eIF4E) but nevertheless translated. The human eukaryotic translation initiation factor 3 (eIF3), composed of 13 subunits (a-m), can selectively recruit several cellular mRNAs and plays roles in specialized translation initiation. Here, we analyzed the ability of eIF3 to interact with selenoprotein mRNAs. By combining ribonucleoprotein immunoprecipitation (RNP IP) in vivo and in vitro with cross-linking experiments, we found interactions between eIF3 and a subgroup of selenoprotein mRNAs. We showed that eIF3 preferentially interacts with hypermethylated capped selenoprotein mRNAs rather than m7G-capped mRNAs. We identified direct contacts between GPx1 mRNA and eIF3 c, d, and e subunits and showed the existence of common interaction patterns for all hypermethylated capped selenoprotein mRNAs. Differential interactions of eIF3 with selenoprotein mRNAs may trigger specific translation pathways independent of eIF4E. eIF3 could represent a new player in the translation regulation and hierarchy of selenoprotein expression

Mise en évidence d'une voie commune pour la maturité et l'assemblage en particules ribonucléoprotéiques des ARN massagers de sélénoprotéines et de petits ARN non-codants

La synthèse des sélénoprotéines fait appel à une machinerie de traduction spécialisée pour incorporer l acide aminé sélénocystéine en réponse à un codon UGASec, habituellement reconnu comme un codon stop. Ce mécanisme de recodage nécessite la présence d une structure en tige-boucle (SECIS) dans la région 3 UTR des ARNm de sélénoprotéines. La protéine SBP2 se lie au SECIS et recrute le complexe facteur d élongation spécialisé EFSec-ARNt Sec. Nous avons révélé que l assemblage des ARNm de sélénoprotéines présentait de surprenantes similarités avec celui d autres complexes ARN-protéines (RNP) essentiels de la cellule: snoRNP nucléolaires (maturation des ribosomes) et snRNP nucléaires (épissage des pré-ARNm) L assemblage requiert un complexe lié au chaperon protéique Hsp90, composé de co-chaperons et de la protéine adaptatrice Nufip. Ce complexe conservé de la levure à l homme est d importance fondamentale pour la cellule. Nous avons également mis en évidence que: (1) le méthylosome et le complexe SMN impliqués dans la biogenèse des snRNP sont certainement aussi requis pour l assemblage des mRNP de sélénoprotéines; (2) des protéines coeur des snoRNP, Nop56 et 58, font également partie des mRNP de sélénoprotéines dont l assemblage a vraisemblablement lieu dans le noyau; (3) le résultat le plus frappant de ma thèse indique que les ARNm de sélénoprotéines portent une coiffe hyperméthylée. Cette découverte montre pour la première fois qu il existe des ARNm de mammifères comportant une telle coiffe. Les spécificités de ces ARN messagers permettent vraisemblablement un mode de régulation plus fin de leur traduction qui reste à être élucidé.Selenocysteine is incorporated into selenoproteins in response to a reprogrammed UGA stop codon. This recoding process requires the presence of a secondary structure called SECIS in the 3 UTR of selenoprotein mRNAs. The protein SBP2 binds specifically the SECIS element and recruits the specialized elongation factor EFsec when complexed with tRNASec. In this thesis we showed that the assembly of selenoprotein mRNPs shows striking similarities with that of other essential RNPs of the cell such as snoRNPs (ribosome maturation) and snRNP (pre-mRNA splicing). We have characterized a conserved assembly machinery implicated in the biogenesis of selenoprotein mRNPs, sn/snoRNPs and telomerase. This conserved machinery is composed of the adaptor Nufip and a co-chaperone complex associated to the protein chaperone Hsp90. My results further indicate that: (1) two complexes implicated in snRNP biogenesis, the methylosome and the SMN complex, are also required for selenoprotein mRNP assembly; (2) core proteins of snoRNPs (Nop56 and Nop58) are also associated to selenoprotein mRNPs and that the assembly of these mRNPs seems to take place in the nucleus; (3) the most striking result of my thesis indicates that selenoprotein mRNAs have a hypermethylated cap. This represents the first demonstration of the existence of messenger RNAs with such a modified cap in mammals. The specificities of selenoprotein mRNAs may probably induce a fine regulation of their translation.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Degradation of ribosomal RNA precursors by the exosome

The yeast exosome is a complex of 3′→5′ exonucleases involved in RNA processing and degradation. All 11 known components of the exosome are required during 3′ end processing of the 5.8S rRNA. Here we report that depletion of each of the individual components inhibits the early pre-rRNA cleavages at sites A(0), A(1), A(2) and A(3), reducing the levels of the 32S, 20S, 27SA(2) and 27SA(3) pre-rRNAs. The levels of the 27SB pre-rRNAs were also reduced. Consequently, both the 18S and 25S rRNAs were depleted. Since none of these processing steps involves 3′→5′ exonuclease activities, the requirement for the exosome is probably indirect. Correct assembly of trans-acting factors with the pre-ribosomes may be monitored by a quality control system that inhibits pre-rRNA processing. The exosome itself degrades aberrant pre-rRNAs that arise from such inhibition. Exosome mutants stabilize truncated versions of the 23S, 21S and A(2)-C(2) RNAs, none of which are observed in wild-type cells. The putative helicase Dob1p, which functions as a cofactor for the exosome in pre-rRNA processing, also functions in these pre-rRNA degradation activities

Modification of Selenoprotein mRNAs by Cap Tri-methylation

International audienc

A Nuclear Surveillance Pathway for mRNAs with Defective Polyadenylation

The pap1-5 mutation in poly(A) polymerase causes rapid depletion of mRNAs at restrictive temperatures. Residual mRNAs are polyadenylated, indicating that Pap1-5p retains at least partial activity. In pap1-5 strains lacking Rrp6p, a nucleus-specific component of the exosome complex of 3′-5′ exonucleases, accumulation of poly(A)(+) mRNA was largely restored and growth was improved. The catalytically inactive mutant Rrp6-1p did not increase growth of the pap1-5 strain and conferred much less mRNA stabilization than rrp6Δ. This may indicate that the major function of Rrp6p is in RNA surveillance. Inactivation of core exosome components, Rrp41p and Mtr3p, or the nuclear RNA helicase Mtr4p gave different phenotypes, with accumulation of deadenylated and 3′-truncated mRNAs. We speculate that slowed mRNA polyadenylation in the pap1-5 strain is detected by a surveillance activity of Rrp6p, triggering rapid deadenylation and exosome-mediated degradation. In wild-type strains, assembly of the cleavage and polyadenylation complex might be suboptimal at cryptic polyadenylation sites, causing slowed polyadenylation