PRP4: a protein of the yeast U4/U6 small nuclear ribonucleoprotein particle

The Saccharomyces cerevisiae prp mutants (prp2 through prp11) are known to be defective in pre-mRNA splicing at nonpermissive temperatures. We have sequenced the PRP4 gene and shown that it encodes a 52-kilodalton protein. We obtained PRP4 protein-specific antibodies and found that they inhibited in vitro pre-mRNA splicing, which confirms the essential role of PRP4 in splicing. Moreover, we found that PRP4 is required early in the spliceosome assembly pathway. Immunoprecipitation experiments with anti-PRP4 antibodies were used to demonstrate that PRP4 is a protein of the U4/U6 small nuclear ribonucleoprotein particle (snRNP). Furthermore, the U5 snRNP could be immunoprecipitated through snRNP-snRNP interactions in the large U4/U5/U6 complex

Mechanistic characterization of the DEAD-box RNA helicase Ded1 from yeast as revealed by a novel technique using single-molecule magnetic tweezers

International audienceDEAD-box helicases are involved in all steps of RNA metabolism. They are ATP-dependent RNA binding proteins and RNA-dependent ATPases. They can displace short duplexes, but they lack processivity. Their mechanism and functioning are not clearly understood; classical or bulk biochemical assays are not sufficient to answer these questions. Single-molecule techniques provide useful tools, but they are limited in cases where the proteins are nonprocessive and give weak signals. We present here a new, magnetic-tweezers-based, single-molecule assay that is simple and that can sensitively measure the displacement time of a small, hybridized, RNA oligonucleotide. Tens of molecules can be analyzed at the same time. Comparing the displacement times with and without a helicase gives insights into the enzymatic activity of the protein. We used this assay to study yeast Ded1, which is orthologous to human DDX3. Although Ded1 acts on a variety of substrates, we find that Ded1 requires an RNA substrate for its ATP-dependent unwinding activity and that ATP hydrolysis is needed to see this activity. Further, we find that only intramolecular single-stranded RNA extensions enhance this activity. We propose a model where ATP-bound Ded1 stabilizes partially unwound duplexes and where multiple binding events may be needed to see displacement

Recherche des partenaires de l'ARN hélicase à boîte DEAD de levure Ded1

L ARN hélicase à boite DEAD de la levure S.cerevisiae Ded1 est une protéine essentielle dont la fonction a été conservée au cours de l évolution. Ses homologues fonctionnels sont impliqués dans le développement et le cycle cellulaire. Ded1 a longtemps été associée à l étape de scanning de la région 5 UTR des ARNm au niveau de l initiation de la traduction. Nous avons utilisé différentes approches comme les co-immunoprécipitations, des analyses de spectrométrie de masse, des tests de complémentation génétique, de séparation des complexes sur gradients de saccharose, des expériences de localisation in situ et d enzymologie pour montrer que Ded1 interagissait physiquement avec des complexes cytoplasmique et nucléaire de liaison à la coiffe des ARNm. Nous avons également montré que Ded1 peut passer du noyau vers le cytoplasme par différentes voies d export nucléaire. De façon intéressante, ses partenaires protéines sont capables de stimuler son activité ATPase. De plus, nous avons montré qu il existait un lien génétique entre Ded1 et ses partenaires. Nous avons également montré que Ded1 colocalise partiellement avec ses partenaires dans des gradients de saccharose, suggérant que Ded1 pourrait être associée à certains mRNPs. Nos résultats encore préliminaires indiquent que Ded1 pourrait s associer à d autre ARNs coiffés. Ainsi, Ded1 pourrait remodeler les complexes associés à différentes étapes de la vie des ARN coiffés.The budding yeast DEAD-box RNA helicase Ded1 is an essential yeast protein that is closely related to a subfamily of DEAD-box proteins that are involved in developmental and cell-cycle regulation. Ded1 is generally considered to be a translation-initiation factor that helps the 40S ribosome scan the mRNA from the 5' 7-methylguanosine cap to the AUG start codon. We have used IgG pulldown experiments, mass spectroscopy analyses, genetic experiments, saccharose gradients, in situ localizations, and enzymatic assays to show that Ded1 is a cap-associated factor that actively shuttles between the cytoplasm and the nucleus. We show that Ded1 physically interacts with various cap-associated factors and that its enzymatic activity is stimulated by these factors. By using various mutated proteins, we show that Ded1 is genetically linked to these factors. Ded1 comigrates with these factors on saccharose gradients, but the peak of Ded1 sediments slightly heavier than for the other factors, which suggests that Ded1 is predominately associated with a subset of the mRNPs. Finally, purification of the protein complexes associated with Ded1 and subsequent analysis by nanoLC-MS/MS indicates that Ded1 is associated with both nuclear and cytoplasmic mRNPs. Preliminary experiements showed that Ded1 can associate with other capped RNA. We conclude that Ded1 may function as a remodeling factor that is needed to form the different complexes associated with the different processing steps of the capped RNA.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

Caractérisation du motif Q des protéines à boîte DEAD

RNA helicases of the DEAD-box protein family are associated with nearly every processes that involve RNA in a cell. Members of the DEAD-box protein family possess eight conserved motifs that participate in the ATPase and helicase activities. In the course of my PhD work, we highlighted the presence of a new conserved motif, that we called the Q-motif, and we characterised this motif. It is specific to, and distinctive of the DEAD-box proteins, and it is essential for their in vivo activity. Using several in vitro assays, we showed that the Q-motif is necessary both for the ATPase activity and the helicase activity. It is required for binding adenine nucleotides as well as for regulating the binding of RNA. Our data indicate that this motif could act as a molecular switch of the binding and the hydrolysis of ATP in response to RNA binding, and as a regulator of the affinity for RNA through the binding of an adenine nucleotide. In the context of a dynamic RNP complex, the Q-motif could be required for the tight regulation of the enzymatic activities of DEAD-box proteins.Les hélicases à ARN à boîte DEAD participent à la plupart des mécanismes impliquant l'ARN chez les procaryotes, les eucaryotes et chez de nombreux virus. Ces protéines possèdent huit motifs conservés qui sont nécessaires aux activités ATPase et hélicase, ainsi qu'à leur(s) régulation(s). Dans le cadre des travaux de ma thèse, nous avons montré l'existence d'un nouveau motif hautement conservé et spécifique des protéines à boîte DEAD. Son étude in vivo a permit de montrer que ce motif, que nous avons appelé le motif Q, est nécessaire à l'activité des protéines à boîte DEAD. In vitro, le motif Q est indispensable à la liaison et à l'hydrolyse de l'ATP, ainsi qu'à l'activité hélicase et à la régulation de la liaison de l'ARN. Le motif Q pourrait agir à la façon d'un interrupteur moléculaire des activités ATPase et hélicase et ainsi participer à la régulation des activités enzymatiques des protéines à boîte DEAD in vivo.VERSAILLES-BU Sciences et IUT (786462101) / SudocSudocFranceF

A Conserved Phenylalanine of Motif IV in Superfamily 2 Helicases Is Required for Cooperative, ATP-Dependent Binding of RNA Substrates in DEAD-Box Proteins▿ †

We have identified a highly conserved phenylalanine in motif IV of the DEAD-box helicases that is important for their enzymatic activities. In vivo analyses of essential proteins in yeast showed that mutants of this residue had severe growth phenotypes. Most of the mutants also were temperature sensitive, which suggested that the mutations altered the conformational stability. Intragenic suppressors of the F405L mutation in yeast Ded1 mapped close to regions of the protein involved in ATP or RNA binding in DEAD-box crystal structures, which implicated a defect at this level. In vitro experiments showed that these mutations affected ATP binding and hydrolysis as well as strand displacement activity. However, the most pronounced effect was the loss of the ATP-dependent cooperative binding of the RNA substrates. Sequence analyses and an examination of the Protein Data Bank showed that the motif IV phenylalanine is conserved among superfamily 2 helicases. The phenylalanine appears to be an anchor that maintains the rigidity of the RecA-like domain. For DEAD-box proteins, the phenylalanine also aligns a highly conserved arginine of motif VI through van der Waals and cation-π interactions, thereby helping to maintain the network of interactions that exist between the different motifs involved in ATP and RNA binding

The DEAD-Box RNA Helicase Ded1 Is Associated with Translating Ribosomes

DEAD-box RNA helicases are ATP-dependent RNA binding proteins and RNA-dependent ATPases that possess weak, nonprocessive unwinding activity in vitro, but they can form long-lived complexes on RNAs when the ATPase activity is inhibited. Ded1 is a yeast DEAD-box protein, the functional ortholog of mammalian DDX3, that is considered important for the scanning efficiency of the 48S pre-initiation complex ribosomes to the AUG start codon. We used a modified PAR-CLIP technique, which we call quicktime PAR-CLIP (qtPAR-CLIP), to crosslink Ded1 to 4-thiouridine-incorporated RNAs in vivo using UV light centered at 365 nm. The irradiation conditions are largely benign to the yeast cells and to Ded1, and we are able to obtain a high efficiency of crosslinking under physiological conditions. We find that Ded1 forms crosslinks on the open reading frames of many different mRNAs, but it forms the most extensive interactions on relatively few mRNAs, and particularly on mRNAs encoding certain ribosomal proteins and translation factors. Under glucose-depletion conditions, the crosslinking pattern shifts to mRNAs encoding metabolic and stress-related proteins, which reflects the altered translation. These data are consistent with Ded1 functioning in the regulation of translation elongation, perhaps by pausing or stabilizing the ribosomes through its ATP-dependent binding