Protein 61K, encoded by a gene (PRPF31) linked to autosomal dominant retinitis pigmentosa, is required for U4/U6*U5 tri-snRNP formation and pre-mRNA splicing

In each round of nuclear pre-mRNA splicing, the U4/U6*U5 tri-snRNP must be assembled from U4/U6 and U5 snRNPs, a reaction that is at present poorly understood. We have characterized a 61 kDa protein (61K) found in human U4/U6*U5 tri-snRNPs, which is homologous to yeast Prp31p, and show that it is required for this step. Immunodepletion of protein 61K from HeLa nuclear extracts inhibits tri-snRNP formation and subsequent spliceosome assembly and pre-mRNA splicing. Significantly, complementation with recombinant 61K protein restores each of these steps. Protein 61K is operationally defined as U4/U6 snRNP-specific as it remains bound to this particle at salt concentrations where the tri-snRNP dissociates. However, as shown by two-hybrid analysis and biochemical assays, protein 61K also interacts specifically with the U5 snRNP-associated 102K protein, indicating that it physically tethers U4/U6 to the U5 snRNP to yield the tri-snRNP. Interestingly, protein 61K is encoded by a gene (PRPF31) that has been shown to be linked to autosomal dominant retinitis pigmentosa. Thus, our studies suggest that disruptions in tri-snRNP formation and function resulting from mutations in the 61K protein may contribute to the manifestation of this disease.     

Structural basis for the bifunctionality of the U5 snRNP 52K protein (CD2BP2)

The bifunctional protein U5-52K is associated with the spliceosomal 20 S U5 snRNP, and it also plays a role in immune response as CD2 receptor binding protein 2 (CD2BP2). U5-52K binds to the CD2 receptor via its GYF-domain specifically recognizing a proline-rich motif on the cytoplasmic surface of the receptor. The GYF-domain is also mediating the interaction of the proteins U5-52K and U5-15K within the spliceosomal U5 snRNP. Here we report the crystal structure of the complex of GYF-domain and U5-15K protein revealing the structural basis for the bifunctionality of the U5-52K protein. The complex structure unveils novel interaction sites on both proteins, as neither the polyproline-binding site of the GYF-domain nor the common ligand-binding cleft of thioredoxin-like proteins, to which U5-15K belongs, are involved in the interaction of U5-15K and U5-52K.     

Human U4/U6.U5 and U4atac/U6atac.U5 tri-snRNPs exhibit similar protein compositions

In the U12-dependent spliceosome, the U4atac/U6atac snRNP represents the functional analogue of the major U4/U6 snRNP. Little information is available presently regarding the protein composition of the former snRNP and its association with other snRNPs. In this report we show that human U4atac/U6atac di-snRNPs associate with U5 snRNPs to form a 25S U4atac/U6atac.U5 trimeric particle. Comparative analysis of minor and major tri-snRNPs by using immunoprecipitation experiments revealed that their protein compositions are very similar, if not identical. Not only U5-specific proteins but, surprisingly, all tested U4/U6- and major tri-snRNP-specific proteins were detected in the minor tri-snRNP complex. Significantly, the major tri-snRNP-specific proteins 65K and 110K, which are required for integration of the major tri-snRNP into the U2-dependent spliceosome, were among those proteins detected in the minor tri-snRNP, raising an interesting question as to how the specificity of addition of tri-snRNP to the corresponding spliceosome is maintained. Moreover, immunodepletion studies demonstrated that the U4/U6-specific 61K protein, which is involved in the formation of major tri-snRNPs, is essential for the association of the U4atac/U6atac di-snRNP with U5 to form the U4atac/U6atac.U5 tri-snRNP. Subsequent immunoprecipitation studies demonstrated that those proteins detected in the minor tri-snRNP complex are also incorporated into U12-dependent spliceosomes. This remarkable conservation of polypeptides between minor and major spliceosomes, coupled with the absence of significant sequence similarity between the functionally analogous snRNAs, supports an evolutionary model in which most major and minor spliceosomal proteins, but not snRNAs, are derived from a common ancestor.     

The human homologue of the yeast splicing factor prp6p contains multiple TPR elements and is stably associated with the U5 snRNP via protein-protein interactions

An essential step of pre-mRNA spliceosome assembly is the interaction between the snRNPs U4/U6 and U5, to form the [U4/U6.U5] tri-snRNP. While the tri-snRNP protein Prp6p appears to play an important role for tri-snRNP formation in yeast, little is known about the interactions that connect the two snRNP particles in human tri-snRNPs. Here, we describe the molecular characterisation of a 102kD protein form HeLa tri-snRNPs. The 102kD protein exhibits a significant degree of overall homology with the yeast Prp6p, including the conservation of multiple tetratrico peptide repeats (TPR), making this the likely functional homologue of Prp6p. However, while the yeast Prp6p is considered to be a U4/U6-specific protein, the human 102kD protein was found to be tightly associated with purified 20 S U5 snRNPs. This association appears to be primarily due to protein-protein interactions. Interestingly, antibodies directed against the C-terminal TPR elements of the 102kD protein specifically and exclusively immunoprecipitate free U5 snRNPs, but not [U4/U6.U5] tri-snRNPs, from HeLa nuclear extract, suggesting that the C-terminal region of the 102kD protein is covered by U4/U6 or tri-snRNP-specific proteins. Since proteins containing TPR elements are typically involved in multiple protein-protein interactions, we suggest that the 102kD protein interacts within the tri-snRNP with both the U5 and U4/U6 snRNPs, thus bridging the two particles. Consistent with this idea, we show that in vitro translated U5-102kD protein binds to purified 13S U4/U6 snRNPs, which contain, in addition to the Sm proteins, all known U4/U6-specific proteins.     

The network of protein-protein interactions within the human U4/U6.U5 tri-snRNP

The human 25S U4/U6.U5 tri-snRNP is a major building block of the U2-type spliceosome and contains, in addition to the U4, U6, and U5 snRNAs, at least 30 distinct proteins. To learn more about the molecular architecture of the tri-snRNP, we have investigated interactions between tri-snRNP proteins using the yeast two-hybrid assay and in vitro binding assays, and, in addition, have identified distinct protein domains that are critical for the connectivity of this protein network in the human tri-snRNP. These studies revealed multiple interactions between distinct domains of the U5 proteins hPrp8, hBrr2 (a DExH/D-box helicase), and hSnu114 (a putative GTPase), which are key players in the catalytic activation of the spliceosome, during which the U4/U6 base-pairing interaction is disrupted and U4 is released from the spliceosome. Both the U5-specific, TPR/HAT-repeat-containing hPrp6 protein and the tri-snRNP-specific hSnu66 protein interact with several U5- and U4/U6-associated proteins, including hBrr2 and hPrp3, which contacts the U6 snRNA. Thus, both proteins are located at the interface between U5 and U4/U6 in the tri-snRNP complex, and likely play an important role in transmitting the activity of hBrr2 and hSnu114 in the U5 snRNP to the U4/U6 duplex during spliceosome activation. A more detailed analysis of these protein interactions revealed that different HAT repeats mediate interactions with specific hPrp6 partners. Taken together, data presented here provide a detailed picture of the network of protein interactions within the human tri-snRNP.     

RNAi knockdown of hPrp31 leads to an accumulation of U4/U6 di-snRNPs in Cajal bodies

Cajal bodies (CBs) are subnuclear organelles of animal and plant cells. A role of CBs in the assembly and maturation of small nuclear ribonucleoproteins (snRNP) has been proposed but is poorly understood. Here we have addressed the question where U4/U6.U5 tri-snRNP assembly occurs in the nucleus. The U4/U6.U5 tri-snRNP is a central unit of the spliceosome and must be re-formed from its components after each round of splicing. By combining RNAi and biochemical methods, we demonstrate that, after knockdown of the U4/U6-specific hPrp31 (61 K) or the U5-specific hPrp6 (102 K) protein in HeLa cells, tri-snRNP formation is inhibited and stable U5 mono-snRNPs and U4/U6 di-snRNPs containing U4/U6 proteins and the U4/U6 recycling factor p110 accumulate. Thus, hPrp31 and hPrp6 form an essential connection between the U4/U6 and U5 snRNPs in vivo. Using fluorescence microscopy, we show that, in the absence of either hPrp31 or hPrp6, U4/U6 di-snRNPs as well as p110 accumulate in Cajal bodies. In contrast, U5 snRNPs largely remain in nucleoplasmic speckles. Our data support the idea that CBs may play a role in tri-snRNP recycling.     

The [U4/U6.U5] tri-snRNP-specific 27K protein is a novel SR protein that can be phosphorylated by the snRNP-associated protein kinase.

SR proteins play important roles in the recognition and selection of the 3' and 5' splice site of a given intron and contribute to the phosphorylation/dephosphorylation-mediated regulation of pre-mRNA splicing. Recent studies have demonstrated that the U1 snRNP is recruited to the 5' splice site by protein/protein interactions involving the SR domains of the U1-70K protein and SF2/ASF. Recently, it was suggested that SR proteins might also contribute to the binding of the [U4/U6.U5] tri-snRNP to the pre-spliceosome (Roscigno RF, Garcia-Blanco MA, 1995, RNA 1:692-706), although it remains unclear whether these SR proteins interact with proteins of the tri-snRNP complex. As a first step toward the identification of proteins that could potentially mediate the integration of the [U4/U6.U5] tri-snRNP complex into the spliceosome, we investigated whether purified [U4/U6.U5] tri-snRNP complexes contain SR proteins. Three proteins in the tri-snRNP complex with approximate molecular weights of 27, 60, and 100 kDa were phosphorylated by purified snRNP-associated protein kinase, which has been shown previously to phosphorylate the serine/ arginine-rich domains of U1-70K and SF2/ASF (Woppmann A et al., 1993, Nucleic Acids Res 21:2815-2822). These proteins are thus prime candidates for novel tri-snRNP SR proteins. Here, we describe the biochemical and molecular characterization of the 27K protein. Analysis of a cDNA encoding the 27K protein revealed an N-terminal SR domain strongly homologous (54% identity) to the SR domain of the U1 snRNP-specific 70K protein. In contrast to many other SR proteins, the 27K protein does not contain an RNA-binding domain. The 27K protein can be phosphorylated in vitro by the snRNP-associated protein kinase and exhibits several isoelectric variants upon 2D gel electrophoresis. Thus, the tri-snRNP-specific 27K protein could potentially be involved in SR protein-mediated protein/protein interactions and, additionally, its phosphorylation state could modulate pre-mRNA splicing.     

The yeast Prp3 protein is a U4/U6 snRNP protein necessary for integrity of the U4/U6 snRNP and the U4/U6.U5 tri-snRNP.

Previously, yeast prp3 mutants were found to be blocked prior to the first catalytic step of pre-mRNA splicing. No splicing intermediates or products are formed from pre-mRNA in heat-inactivated prp3 mutants or prp3 mutant extracts. Here we show that Prp3p is a component of the U4/U6 snRNP and is also present in the U4/U6.U5 tri-snRNP. Heat inactivation of prp3 extracts results in depletion of free U6 snRNPs and U4/U6.U5 tri-snRNPs, but not U4/U6 snRNPs or U5 snRNPs. Free U4 snRNP, normally not present in wild-type extracts, accumulates under these conditions. Assays of in vivo levels of snRNAs in a prp3 mutant revealed that amounts of free U6 snRNA decreased, free U4 snRNA increased, and U4/U6 hybrids decreased slightly. These results suggest that Prp3p is required for formation of stable U4/U6 snRNPs and for assembly of the U4/U6.U5 tri-snRNP from its component snRNPs. Upon inactivation of Prp3p, spliceosomes cannot assemble from prespliceosomes due to the absence of intact U4/U6.U5 tri-snRNPs. Prp3p is homologous to a human protein that is a component of U4/U6 snRNPs, exemplifying the conservation of splicing factors between yeast and metazoans.     

Biochemical and genetic analyses of the U5, U6, and U4/U6 x U5 small nuclear ribonucleoproteins from Saccharomyces cerevisiae.

We have purified the yeast U5 and U6 pre-mRNA splicing small nuclear ribonucleoproteins (snRNPs) by affinity chromatography and analyzed the associated polypeptides by mass spectrometry. The yeast U5 snRNP is composed of the two variants of U5 snRNA, six U5-specific proteins and the 7 proteins of the canonical Sm core. The U6 snRNP is composed of the U6 snRNA, Prp24, and the 7 Sm-Like (LSM) proteins. Surprisingly, the yeast DEAD-box helicase-like protein Prp28 is stably associated with the U5 snRNP, yet is absent from the purified U4/U6 x U5 snRNP. A novel yeast U5 and four novel yeast U4/U6 x U5 snRNP polypeptides were characterized by genetic and biochemical means to demonstrate their involvement in the pre-mRNA splicing reaction. We also show that, unlike the human tri-snRNP, the yeast tri-snRNP dissociated upon addition of ATP or dATP.     

Sad1 Counteracts Brr2-Mediated Dissociation of U4/U6.U5 in Tri-snRNP Homeostasis

The yeast Sad1 protein was previously identified in a screen for factors involved in the assembly of the U4/U6 di-snRNP particle. Sad1 is required for pre-mRNA splicing both in vivo and in vitro, and its human orthologue has been shown to associate with U4/U6.U5 tri-snRNP. We show here that Sad1 plays a role in maintaining a functional form of the tri-snRNP by promoting the association of U5 snRNP with U4/U6 di-snRNP. In the absence of Sad1, the U4/U6.U5 tri-snRNP dissociates into U5 and U4/U6 upon ATP hydrolysis and cannot bind to the spliceosome. The separated U4/U6 and U5 can reassociate upon incubation more favorably in the absence of ATP and in the presence of Sad1. Brr2 is responsible for mediating ATP-dependent dissociation of the tri-snRNP. Our results demonstrate a role of Sad1 in maintaining the integrity of the tri-snRNP by counteracting Brr2-mediated dissociation of tri-snRNP and provide insights into homeostasis of the tri-snRNP.     

Direct probing of RNA structure and RNA-protein interactions in purified HeLa cell's and yeast spliceosomal U4/U6.U5 tri-snRNP particles

The U4/U6.U5 tri-snRNP is a key component of spliceosomes. By using chemical reagents and RNases, we performed the first extensive experimental analysis of the structure and accessibility of U4 and U6 snRNAs in tri-snRNPs. These were purified from HeLa cell nuclear extract and Saccharomyces cerevisiae cellular extract. U5 accessibility was also investigated. For both species, data demonstrate the formation of the U4/U6 Y-shaped structure. In the human tri-snRNP and U4/U6 snRNP, U6 forms the long range interaction, that was previously proposed to be responsible for dissociation of the deproteinized U4/U6 duplex. In both yeast and human tri-snRNPs, U5 is more protected than U4 and U6, suggesting that the U5 snRNP-specific protein complex and other components of the tri-snRNP wrapped the 5' stem-loop of U5. Loop I of U5 is partially accessible, and chemical modifications of loop I were identical in yeast and human tri-snRNPs. This reflects a strong conservation of the interactions of proteins with the functional loop I. Only some parts of the U4/U6 Y-shaped motif (the 5' stem-loop of U4 and helix II) are protected. Due to difference of protein composition of yeast and human tri-snRNP, the U6 segment linking the 5' stem-loop to the Y-shaped structure and the U4 central single-stranded segment are more accessible in the yeast than in the human tri-snRNP, especially, the phylogenetically conserved ACAGAG sequence of U6. Data are discussed taking into account knowledge on RNA and protein components of yeast and human snRNPs and their involvement in splicesome assembly.     

SPF30 is an essential human splicing factor required for assembly of the U4/U5/U6 tri-small nuclear ribonucleoprotein into the spliceosome

Spliceosome assembly involves the sequential recruitment of small nuclear ribonucleoproteins (snRNPs) onto a pre-mRNA substrate. Although several non-snRNP proteins function during the binding of U1 and U2 snRNPs, little is known about the subsequent binding of the U4/U5/U6 tri-snRNP. A recent proteomic analysis of the human spliceosome identified SPF30 (Neubauer, G., King, A., Rappsilber, J., Calvio, C., Watson, M., Ajuh, P., Sleeman, J., Lamond, A., and Mann, M. (1998) Nat. Genet. 20, 46-50), a homolog of the survival of motor neurons (SMN) protein, as a spliceosome factor. We show here that SPF30 is a nuclear protein that associates with both U4/U5/U6 and U2 snRNP components. In the absence of SPF30, the preformed tri-snRNP fails to assemble into the spliceosome. Mass spectrometric analysis shows that a recombinant glutathione S-transferase-SPF30 fusion protein associates with complexes containing core Sm and U4/U5/U6 tri-snRNP proteins when added to HeLa nuclear extract, most strongly to U4/U6-90. The data indicate that SPF30 is an essential human splicing factor that may act to dock the U4/U5/U6 tri-snRNP to the A complex during spliceosome assembly or, alternatively, may act as a late assembly factor in both the tri-snRNP and the A-complex.     

The biochemical defects of prp4-1 and prp6-1 yeast splicing mutants reveal that the PRP6 protein is required for the accumulation of the [U4/U6.U5] tri-snRNP.

We have raised specific antibodies against the PRP6 protein and shown that the U4, U5 and U6 snRNAs are co-precipitated with this protein. Using splicing extracts prepared from in vivo heat-inactivated cells, we have characterized the prp4-1 and prp6-1 biochemical defects. In inactivated prp4-1 cell extracts, the U6 snRNA content as well as the U6, U4/U6 snRNPs and the [U4/U6.U5] tri-snRNP particles amounts are severely reduced. In inactivated prp6-1 cell extracts, the PRP6 mutant protein is barely detectable. Glycerol gradient analyses indicate that, in these extracts, the [U4/U6.U5] tri-snRNPs are present in very low amounts, but U4/U6 snRNP particles are normally represented. These results establish that the PRP6 protein is required for the accumulation of the [U4/U6.U5] tri-snRNP. We found no evidence for the presence of the PRP6 protein in the U4/U6 particle.     

Functional interaction of a novel 15.5kD [U4/U6.U5] tri-snRNP protein with the 5' stem-loop of U4 snRNA.

Activation of the spliceosome for splicing catalysis requires the dissociation of U4 snRNA from the U4/U6 snRNA duplex prior to the first step of splicing. We characterize an evolutionarily conserved 15.5 kDa protein of the HeLa [U4/U6.U5] tri-snRNP that binds directly to the 5' stem-loop of U4 snRNA. This protein shares a novel RNA recognition motif with several RNP-associated proteins, which is essential, but not sufficient for RNA binding. The 15.5kD protein binding site on the U4 snRNA consists of an internal purine-rich loop flanked by the stem of the 5' stem-loop and a stem comprising two base pairs. Addition of an RNA oligonucleotide comprising the 5' stem-loop of U4 snRNA (U4SL) to an in vitro splicing reaction blocked the first step of pre-mRNA splicing. Interestingly, spliceosomal C complex formation was inhibited while B complexes accumulated. This indicates that the 15.5kD protein, and/or additional U4 snRNP proteins associated with it, play an important role in the late stage of spliceosome assembly, prior to step I of splicing catalysis. Our finding that the 15.5kD protein also efficiently binds to the 5' stem-loop of U4atac snRNA indicates that it may be shared by the [U4atac/U6atac.U5] tri-snRNP of the minor U12-type spliceosome.     

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 recognition of 5' splice site by U4/U6.U5 tri-snRNP defines a novel ATP-dependent step in early spliceosome assembly

A sensitive assay based on competition between cis-and trans-splicing suggested that factors in addition to U1 snRNP were important for early 5' splice site recognition. Cross-linking and physical protection experiments revealed a functionally important interaction between U4/U6.U5 tri-snRNP and the 5' splice site, which unexpectedly was not dependent upon prior binding of U2 snRNP to the branch point. The early 5' splice site/tri-snRNP interaction requires ATP, occurs in both nematode and HeLa cell extracts, and involves sequence-specific interactions between the highly conserved splicing factor Prp8 and the 5' splice site. We propose that U1 and U5 snRNPs functionally collaborate to recognize and define the 5' splice site prior to establishment of communication with the 3' splice site.     

Prp31p promotes the association of the U4/U6 x U5 tri-snRNP with prespliceosomes to form spliceosomes in Saccharomyces cerevisiae.

The PRP31 gene encodes a factor essential for the splicing of pre-mRNA in Saccharomyces cerevisiae. Cell extracts derived from a prp31-1 strain fail to form mature spliceosomes upon heat inactivation, although commitment complexes and prespliceosome complexes are detected under these conditions. Coimmunoprecipitation experiments indicate that Prp31p is associated both with the U4/U6 x U5 tri-snRNP and, independently, with the prespliceosome prior to assembly of the tri-snRNP into the splicing complex. Nondenaturing gel electrophoresis and glycerol gradient analyses demonstrate that while Prp31p may play a role in maintaining the assembly or stability of tri-snRNPs, functional protein is not essential for the formation of U4/U6 or U4/U6 x U5 snRNPs. These results suggest that Prp31p is involved in recruiting the U4/U6 x U5 tri-snRNP to prespliceosome complexes or in stabilizing these interactions.     

Hierarchical,clustered protein interactions with U4/U6 snRNA: a biochemical role for U4/U6 proteins

During activation of the spliceosome, the U4/U6 snRNA duplex is dissociated, releasing U6 for subsequent base pairing with U2 snRNA. Proteins that directly bind the U4/U6 interaction domain potentially could mediate these structural changes. We thus investigated binding of the human U4/U6-specific proteins, 15.5K, 61K and the 20/60/90K protein complex, to U4/U6 snRNA in vitro. We demonstrate that protein 15.5K is a nucleation factor for U4/U6 snRNP assembly, mediating the interaction of 61K and 20/60/90K with U4/U6 snRNA. A similar hierarchical assembly pathway is observed for the U4atac/U6atac snRNP. In addition, we show that protein 61K directly contacts the 5' portion of U4 snRNA via a novel RNA-binding domain. Furthermore, the 20/60/90K heteromer requires stem II but not stem I of the U4/U6 duplex for binding, and this interaction involves a direct contact between protein 90K and U6. This uneven clustering of the U4/U6 snRNP-specific proteins on U4/U6 snRNA is consistent with a sequential dissociation of the U4/U6 duplex prior to spliceosome catalysis.     

The 65 and 110 kDa SR-related proteins of the U4/U6.U5 tri-snRNP are essential for the assembly of mature spliceosomes

The association of the U4/U6.U5 tri-snRNP with pre-spliceosomes is a poorly understood step in the spliceosome assembly pathway. We have identified two human tri-snRNP proteins (of 65 and 110 kDa) that play an essential role in this process. Characterization by cDNA cloning of the 65 and 110 kDa proteins revealed that they are likely orthologues of the yeast spliceosomal proteins Sad1p and Snu66p, respectively. Immunodepletion of either protein from the HeLa cell nuclear extracts inhibited pre-mRNA splicing due to a block in the formation of mature spliceosomes, but had no effect on the integrity of the U4/U6.U5 tri-snRNP. Spliceosome assembly and splicing catalysis could be restored to the respective depleted extract by the addition of recombinant 65 or 110 kDa protein. Our data demonstrate that both proteins are essential for the recruitment of the tri-snRNP to the pre-spliceosome but not for the maintenance of the tri-snRNP stability. Moreover, since both proteins contain an N-terminal RS domain, they could mediate the association of the tri-snRNP with pre-spliceosomes by interaction with members of the SR protein family.     

New insights into trypanosomatid U5 small nuclear ribonucleoproteins

Several protozoan parasites exist in the Trypanosomatidae family, including various agents of human diseases. Multiple lines of evidence suggest that important differences are present between the translational and mRNA processing (trans splicing) systems of trypanosomatids and other eukaryotes. In this context, certain small complexes of RNA and protein, which are named small nuclear ribonucleoproteins (U snRNPs), have an essential role in pre-mRNA processing, mainly during splicing. Even though they are well defined in mammals, snRNPs are still not well characterized in trypanosomatids. This study shows that a U5-15K protein is highly conserved among various trypanosomatid species. Tandem affinity pull-down assays revealed that this protein interacts with a novel U5-102K protein, which suggests the presence of a sub-complex that is potentially involved in the assembly of U4/U6-U5 tri-snRNPs. Functional analyses showed that U5-15K is essential for cell viability and is somehow involved with the trans and cis splicing machinery. Similar tandem affinity experiments with a trypanonosomatid U5-Cwc21 protein led to the purification of four U5 snRNP specific proteins and a Sm core, suggesting U5-Cwc-21 participation in the 35S U5 snRNP particle. Of these proteins, U5-200K was molecularly characterized. U5-200K has conserved domains, such as the DEAD/DEAH box helicase and Sec63 domains and displays a strong interaction with U5 snRNA.