DEAD-box proteins, like Leishmania eIF4A, modulate interleukin (IL)-12, IL-10 and tumour necrosis factor-alpha production by human monocytes.

Previously we showed that His-tagged, recombinant, Leishmania infantum eukaryotic initiation factor (LeIF) was both an RNA-dependent ATPase and an ATP-dependent RNA helicase in vitro, as described for other members of the DEAD-box helicase family. In addition, we showed that LeIF induces the production of IL-12, IL-10, and TNF-α by human monocytes. This study aims to characterize the cytokine-inducing activity in human monocytes of several proteins belonging to the DEAD-box family from mammals and yeast. All tested proteins contained the 11 conserved motifs (Q, I, Ia, GG Ib, II, III, IV, QxxR, V and VI) characteristic of DEAD-box proteins, but they have different biological functions and different percentages of identities with LeIF. We show that these mammalian or yeast recombinant proteins also are able to induce IL-12, IL-10 and TNF-α secretion by monocytes of healthy human subjects. This cytokine-inducing activity is proteinase K sensitive and polymyxin B resistant. Our results show that the induction of cytokines in human monocytes is not unique to the protein LeIF of Leishmania, and it suggests that the activity of certain DEAD-box proteins can be exploited as adjuvant and/or to direct immune responses towards a Th1 profile in vaccination or immunotherapy protocols.

From RNA helicases to RNPases.

In eukaryotic cells, all aspects of cellular RNA metabolism require putative RNA helicases of the DEAD and DExH protein families (collectively known as DExD/H families). Based on data from biochemical studies of a few of these RNA helicases, they are generally considered to be involved in the unwinding of duplex RNA molecules. However, recent reports provide evidence indicating that these proteins might also be involved in the active disruption of RNA-protein interactions.

Identification by mass spectrometry and functional analysis of novel proteins of the yeast [U4/U6.U5] tri-snRNP.

The 25S [U4/U6.U5] tri-snRNP (small nuclear ribonucleoprotein) is a central unit of the nuclear pre-mRNA splicing machinery. The U4, U5 and U6 snRNAs undergo numerous rearrangements in the spliceosome, and knowledge of all of the tri-snRNP proteins is crucial to the detailed investigation of the RNA dynamics during the spliceosomal cycle. Here we characterize by mass spectrometric methods the proteins of the purified [U4/U6.U5] tri-snRNP from the yeast Saccharomyces cerevisiae. In addition to the known tri-snRNP proteins (only one, Lsm3p, eluded detection), we identified eight previously uncharacterized proteins. These include four Sm-like proteins (Lsm2p, Lsm5p, Lsm6p and Lsm7p) and four specific proteins named Snu13p, Dib1p, Snu23p and Snu66p. Snu13p comprises a putative RNA-binding domain. Interestingly, the Schizosaccharomyces pombe orthologue of Dib1p, Dim1p, was previously assigned a role in cell cycle progression. The role of Snu23p, Snu66p and, additionally, Spp381p in pre-mRNA splicing was investigated in vitro and/or in vivo. Finally, we show that both tri-snRNPs and the U2 snRNP are co-precipitated with protein A-tagged versions of Snu23p, Snu66p and Spp381p from extracts fractionated by glycerol gradient centrifugation. This suggests that these proteins, at least in part, are also present in a [U2.U4/U6.U5] tetra-snRNP complex.

Functional and structural characterization of the prp3 binding domain of the yeast prp4 splicing factor.

Nuclear pre-mRNA splicing occurs in a large RNA-protein complex containing four small nuclear ribonucleoprotein particles (snRNPs) and additional protein factors. The yeast Prp4 (yPrp4) protein is a specific component of the U4/U6 and U4/U6-U5 snRNPs, which associates transiently with the spliceosome before the first step of splicing. In this work, we used the in vivo yeast two-hybrid system and in vitro immunoprecipitation assays to show that yPrp4 interacts with yPrp3, another U4/U6 snRNP protein. To investigate the domain of yPrp4 that directly contacts yPrp3, we introduced deletions in the N-terminal half of yPrp4 and point mutations in the C-terminal half of the molecule, and we tested the resulting prp4 mutants for cell viability and for their ability to interact with yPrp3. We could not define any particular sequence in the first 161 amino acid residues that are specifically required for protein-protein interactions. However, deletion of a small basic-rich region of 30 amino acid residues is lethal to the cells. Analysis of the C terminus prp4 mutants obtained clearly shows that this region of yPrp4 represents the primary domain of interaction with yPrp3. Interestingly, yPrp4 shows significant similarity in its C-terminal half to the beta-subunits of G proteins. We have generated a three-dimensional computer model of this domain, consisting of a seven-bladed beta-propeller based on the crystalline structure of beta-transducin. Several lines of evidence suggested that yPrp4 is contacting yPrp3 through a large flat surface formed by the long variable loops linking the beta-strands of the propeller. This surface could be used as a scaffold for generating an RNA-protein complex.

Snt309p, a component of the Prp19p-associated complex that interacts with Prp19p and associates with the spliceosome simultaneously with or immediately after dissociation of U4 in the same manner as Prp19p.

The yeast protein Prp19p is essential for pre-mRNA splicing and is associated with the spliceosome concurrently with or just after dissociation of U4 small nuclear RNA. In splicing extracts, Prp19p is associated with several other proteins in a large protein complex of unknown function, but at least one of these proteins is also essential for splicing (W.-Y. Tarn, C.-H. Hsu, K.-T. Huang, H.-R. Chen, H.-Y. Kao, K.-R. Lee, and S.-C. Cheng, EMBO J. 13:2421-2431, 1994). To identify proteins in the Prp19p-associated complex, we have isolated trans-acting mutations that exacerbate the phenotypes of conditional alleles of prp19, using the ade2-ade3 sectoring system. A novel splicing factor, Snt309p, was identified through such a screen. Although the SNT309 gene was not essential for growth of Saccharomyces cerevisiae under normal conditions, yeast cells containing a null allele of the SNT309 gene were temperature sensitive and accumulated pre-mRNA at the nonpermissive temperature. Far-Western blot analysis revealed direct interaction between Prp19p and Snt309p. Snt309p was shown to be a component of the Prp19p-associated complex by Western blot analysis. Immunoprecipitation studies demonstrated that Snt309p was also a spliceosomal component and associated with the spliceosome in the same manner as Prp19p during spliceosome assembly. These results suggest that the functions of Prp19p and Snt309p in splicing may require coordinate action of these two proteins.

Mutations within the yeast U4/U6 snRNP protein Prp4 affect a late stage of spliceosome assembly.

We showed previously that the yeast Prp4 protein is a spliceosomal factor that is tightly associated with the U4, U5, and U6 small nuclear RNAs. Moreover, Prp4 appears to associate very transiently with the spliceosome before the U4 snRNA dissociates from the spliceosome. Prp4 belongs to the Gbeta-like protein family, which suggests that the Prp4 Gbeta motifs could mediate interactions with other components of the spliceosome. To investigate the function of the Gbeta motifs, we introduced mutations within the second WD-repeat of Prp4. Among the 35 new alleles found, 24 were pseudo wild-type mutants, 8 failed to grow at any temperature, and 3 were conditional sensitive mutants. The biochemical defects of the three thermosensitive prp4 mutants have been examined by immunoprecipitation, native gel electrophoresis, and glycerol gradient centrifugation. First, we show that snRNP formation is not impaired in these mutants and that Prp4 is present in the U4/U6 and U4/U6-U5 snRNP particles. We also demonstrate that spliceosome assembly is largely unaffected despite the fact that the first step of splicing does not occur. However, both Prp4 and U4 snRNA remain tightly associated with the spliceosome and this blocks the transition toward an active form of the spliceosome. Our results suggest a possible role of Prp4 in mediating important conformational rearrangements of proteins within the spliceosome that involve the region containing the Gbeta-repeats.

Secondary structure of the yeast Saccharomyces cerevisiae pre-U3A snoRNA and its implication for splicing efficiency.

The Saccharomyces cerevisiae U3 snoRNA genes contain long spliceosomal introns with noncanonical branch site sequences. By using chemical and enzymatic methods to probe the RNA secondary structure and site-directed mutagenesis, we established the complete secondary structure of the U3A snoRNA precursor. This is the first determination of the complete secondary structure of an RNA spliced in a spliceosome. The peculiar cruciform structure of the U3A snoRNA 3'-terminal region is formed in the precursor RNA and the conserved Boxes B and C are accessible for binding the U3 snoRNP proteins. The intron forms a highly folded structure with a long central stem-loop structure that brings the 5' box and the branch site together. This is in agreement with the idea that secondary structure interactions are necessary for efficient splicing of long introns in yeast. The 3' splice site is in a bulged loop and the branch site sequence is single-stranded. Surprisingly, the 5' splice site is involved in a 6-base pair interaction. We used in vitro splicing experiments to show that, despite a noncanonical branch site sequence and a base paired 5' splice site, transcripts that mimic the authentic pre-U3A snoRNA are spliced very efficiently in vitro. Sequestering the 5' splice site in a more stable structure had a negative effect on splicing, which was partially compensated by converting the branch site sequence into a canonical sequence. Analysis of spliceosomal complex formation revealed a cumulative negative effect of a base pair interaction at the 5' splice site and of a deviation to the consensus sequence at the branch site on the efficiency of spliceosome formation in vitro.

An experimental study of Saccharomyces cerevisiae U3 snRNA conformation in solution.

The conformation of Saccharomyces cerevisiae U3 snRNA (snR17A RNA) in solution was studied using enzymatic and chemical probes. In vitro synthesized and authentic snR17A RNAs have a similar conformation in solution. The S. cerevisiae U3 snRNA is folded in two distinct domains. The 5'-domain has a low degree of compactness; it is constituted of two stem-loop structures separated by a single-stranded segment, which has recently been proposed to basepair with the 5'-ETS of pre-ribosomal RNA. We demonstrate that, as previously proposed, the 5'-terminal region of U3 snRNA has a different structure in higher and lower eukaryotes and that this may be related to pre-rRNA 5'-ETS evolution. The S. cerevisiae U3 snRNA 3'-domain has a cruciform secondary structure and a compact conformation resulting from an higher order structure involving the single-stranded segments at the center of the cross and the bottom parts of helices. Compared to tRNA, where long range interactions take place between terminal loops, this represents another kind of tertiary folding of RNA molecules that will deserve further investigation, especially since the implicated single-strands have highly evolutionarily conserved primary structures that are involved in snRNP protein binding.

Domains of yeast U4 spliceosomal RNA required for PRP4 protein binding, snRNP-snRNP interactions, and pre-mRNA splicing in vivo.

U4 small nuclear RNA (snRNA) contains two intramolecular stem-loop structures, located near each end of the molecule. The 5' stem-loop is highly conserved in structure and separates two regions of U4 snRNA that base-pair with U6 snRNA in the U4/U6 small nuclear ribonucleoprotein particle (snRNP). The 3' stem-loop is highly divergent in structure among species and lies immediately upstream of the binding site for Sm proteins. To investigate the function of these two domains, mutants were constructed that delete the yeast U4 snRNA 5' stem-loop and that replace the yeast 3' stem-loop with that from trypanosome U4 snRNA. Both mutants fail to complement a null allele of the yeast U4 gene. The defects of the mutants have been examined in heterozygous strains by native gel electrophoresis, glycerol gradient centrifugation, and immunoprecipitation. The chimeric yeast-trypanosome RNA does not associate efficiently with U6 snRNA, suggesting that the 3' stem-loop of yeast U4 snRNA might be a binding site for a putative protein that facilitates assembly of the U4/U6 complex. In contrast, the 5' hairpin deletion mutant associates efficiently with U6 snRNA. However, it does not bind the U4/U6-specific protein PRP4 and does not assemble into a U4/U5/U6 snRNA. Thus, we propose that the role of the PRP4 protein is to promote interactions between the U4/U6 snRNP and the U5 snRNP.

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.

Site-specific DNA endonuclease and RNA maturase activities of two homologous intron-encoded proteins from yeast mitochondria.

Two introns of the mitochondrial genome 777-3A of S. cerevisiae, bl4 in cob and al4 in coxl genes, contain ORFs that can be translated into two homologous proteins. We changed the UGA, AUA, and CUN codons of these ORFs to the universal genetic code, in order to study the functions of their translated products in E. coli and in yeast, by retargeting the nuclear encoded protein into mitochondria. The p27bl4 protein has been shown to be required for the splicing of both introns bl4 and al4. The homologous p28al4 protein is highly toxic to E. coli. It can specifically cleave double-stranded DNA at a sequence representing the junction of the two fused flanking exons. We present evidence that this system is a good model for studying the role of mitochondrial intron-encoded proteins in the rearrangement of genetic information at both the RNA (RNA splicing-bl4 maturase) and DNA levels (intron transposition-al4 transposase).

Efficient splicing of two yeast mitochondrial introns controlled by a nuclear-encoded maturase.

bI4 maturase encoded by the fourth intron of the yeast mitochondrial cytochrome b gene, controls the splicing of both the fourth intron of the cytochrome b gene and the fourth intron of the gene encoding subunit I of cytochrome oxidase. It has been shown previously that a cytoplasmically translated hybrid protein composed of the pre-sequence of subunit 9 of Neurospora ATPase fused to a part of the bI4 maturase can be guided to mitochondria where it could compensate maturase deficiencies. This in vivo complementation of maturase mutants can be easily estimated by restoration of respiration. This work examines the efficiency of different bI4 maturase constructions to restore respiration in different yeast maturase-deficient strains. It is shown that the N-terminal end of the bI4 maturase plays a crucial role in the maturase activity. Moreover, the 12 N-terminal amino acids of the mitochondrial outer membrane protein constitute the most efficient mitochondrial targeting sequence in this system. Surprisingly enough, it was found that the cytoplasmically translated bI4 maturase containing the 254 C-terminal amino acid coded by the intron open reading frame can complement maturase mutations without any added mitochondrial-targeting sequence.

A mitochondrial RNA maturase gene transferred to the yeast nucleus can control mitochondrial mRNA splicing.

bI4 maturase, encoded by the fourth intron of the yeast mitochondrial cytochrome b gene, controls the splicing of both the fourth intron of the cytochrome b gene and the fourth intron of the gene encoding subunit I of cytochrome oxidase. By fusing the encoding presequence of subunit 9 of the Neurospora ATPase to a restriction fragment containing the bI4 maturase coding sequence, we have constructed a hybrid gene that can be translated on yeast cytosolic ribosomes. The resulting protein is imported into mitochondria, which was revealed by its ability to restore to respiratory competence a yeast mutant defective in the bI4 maturase. Moreover, a protein reacting with antimaturase antibodies was detected in the mitochondria of the transformed cells; this imported maturase functioned similarly to the endogenous maturase.

Antibodies against a fused 'lacZ-yeast mitochondrial intron' gene product allow identification of the mRNA maturase encoded by the fourth intron of the yeast cob-box gene.

Several missense or nonsense mutations have been localized in the fourth intron open reading frame (ORF) of the yeast mitochondrial cytochrome b gene. These results and the phenotypes of mutants strongly suggested that a mRNA maturase, controlling the expression of both cytochrome b and cytochrome oxidase subunit I (COXI) genes, is encoded in this ORF. To investigate more directly the biosynthesis of mRNA maturase we raised antibodies against a part of the putative ORF translation product. For that purpose we inserted a fragment of the ORF sequence, in phase, into the C-terminal EcoRI site of lacZ gene. The hybrid gene was then expressed in Escherichia coli under the control of either the wild-type lac promoter or the thermoregulated lambda system PR/cI857. The hybrid protein was partially purified and antibodies were raised against it. These antibodies recognized a mitochondrially coded protein, p27, in intron mutants, whereas no such protein was detected in the wild-type cell. These results demonstrate that the p27 protein, previously shown to be associated with the mRNA maturase activity, is actually translated from the intron ORF. The autoregulated mRNA maturase synthesis model is discussed in relation to these results.

Molecular studies on galactose 1 phosphate uridylyl transferase from normal and mutant subjects. An immunological approach.

The mutant forms of uridylyl transferase of eight galactosemic patients and two 'Rennes' variants were characterized with regard to the presence and level of immunoreactive protein, the apparent subunit molecular weight and the isoelectric point. Semi-purified haemolysates were studied by various electrophoretic techniques, then proteins were electrophoretically transferred on to nitrocellulose filters. They were treated with specific anti-transferase antibodies, and then with radioiodinated protein A, followed by autoradiography. We have found that: in all cases, a cross-reacting material was detectable, with a molecular subunit size of 46 000, indistinguishable from that of controls. a biochemical heterogeneity of the mutant enzyme was found: the amount of apparent immunologically reactive protein varied from 20 to 100% of that of controls; electrophoretic experiments performed on two 'Rennes' variants showed an increased negative charge.

Purification of human liver uridylyl transferase and comparison with the erythrocyte enzyme.

Uridylyl transferase (UDP glucose: alpha-D-galactose 1 phosphate uridylyl transferase, EC 2.7.7.12) has been purified 1350-fold from human liver to complete homogeneity. The purification procedure involved ammonium sulfate fractionation, batch treatment, chromatography on DEAE-cellulose, hexylagarose and hydroxylapatite. The specific activity of the homogeneous enzyme was 27 units/mg protein. The liver enzyme was compared to the red cell enzyme previously purified by us. The liver enzyme was similar to the red cell enzyme with respect to subunit molecular weight, kinetic studies and immunological properties. Differences in electrophoretic behaviour were found: the liver transferase has a more basic pI (between 5.5 and 5.8) than that of the erythrocyte enzyme (pI between 5.0 and 5.45). It is very likely that the liver uridylyl transferase and the red blood cell transferase are the same enzymes with post-translational modifications.

Purification and characterization of human erythrocyte uridylyl transferase.

A new method for the purification of human erythrocyte uridylyl transferase (UDPglucose: alpha-D-galactose-1-phosphate uridylyltransferase EC 2.7.7.12) is described. It consists of a hydrophobic purification step associated with hydroxyapatite chromatography and provided for the first time a purification of more than 45 000-fold with a high activity (15 I.U/mg) and a yield of 32%. We show that the enzyme is a dimer and has a molecular weight of 88 000. It can be resolved into three bands by isoelectric focusing with an apparent pI between 5.0 and 5.4. It could be shown by steady-state initial rate measurements that the interconversion of the two substrates of human transferase (Gal-1-P and UDP-glucose) follows ping-pong bi-bi kinetics, with Km values of 0.2 and 0.065 mM, respectively.

Isoelectrofocusing of erythrocyte galactose 1 phospho uridyl transferase in a family with both galactosemia and Duarte variants.

A family with the presence of the genes for both galactosemia and the Duarte variant is described. Galactose 1 phospho uridyl transferase has been studied not only by electrophoresis on starch gel, but also by isoelectro-focusing on thin-layer acrylamide. Normal and variant transferases were resolved into three bands, the isoelectric point of which was between 5.40 and 5.10 for the normal subjects, and between 5.25 and 4.95 for subjects with the Duarte variant.