Pre-mRNA splicing by the spliceosome is an essential step in the maturation of nearly all human mRNAs. Mutations in six spliceosomal proteins, PRPF3, PRPF4, PRPF6, PRPF8, PRPF31 and SNRNP200, cause retinitis pigmentosa (RP), a disease characterized by progressive photoreceptor degeneration. All splicing factors linked to RP are constituents of the U4/U6.U5 tri-snRNP subunit of the spliceosome, suggesting that the compromised function of this particle may lead to RP. Here, we report the identification of the p.R192H variant of the tri-snRNP factor PRPF4 in a patient with RP. The mutation affects a highly conserved arginine residue that is crucial for PRPF4 function. Introduction of a corresponding mutation into the zebrafish homolog of PRPF4 resulted in a complete loss of function in vivo. A series of biochemical experiments suggested that p.R192H disrupts the binding interface between PRPF4 and its interactor PRPF3. This interferes with the ability of PRPF4 to integrate into the tri-snRNP, as shown in a human cell line and in zebrafish embryos. These data suggest that the p.R192H variant of PRPF4 represents a functional null allele. The resulting haploinsufficiency of PRPF4 compromises the function of the tri-snRNP, reinforcing the notion that this spliceosomal particle is of crucial importance in the physiology of the retina.

Retinitis pigmentosa (RP) is a common hereditary eye disease that causes blindness due to a progressive loss of photoreceptors in the retina. RP can be elicited by mutations that affect the tri-snRNP subunit of the pre-mRNA splicing machinery, but how defects in this essential macromolecular complex transform into a photoreceptor-specific phenotype is unknown. We have modeled the disease in zebrafish by silencing the RP-associated splicing factor Prpf31 and observed detrimental effects on visual function and photoreceptor morphology. Despite reducing the level of a constitutive splicing factor, no general defects in gene expression were found. Instead, retinal genes were selectively affected, providing the first in vivo link between mutations in splicing factors and the RP phenotype. Silencing of Prpf4, a splicing factor hitherto unrelated to RP, evoked the same defects in vision, photoreceptor morphology and retinal gene expression. Hence, various routes affecting the tri-snRNP can elicit tissue-specific gene expression defects and lead to the RP phenotype.

Distal spinal muscular atrophy type 1 (DSMA1) is an autosomal recessive disease that is clinically characterized by distal limb weakness and respiratory distress. In this disease, the degeneration of alpha-motoneurons is caused by mutations in the immunoglobulin mu-binding protein 2 (IGHMBP2). This protein has been implicated in DNA replication, pre-mRNA splicing and transcription, but its precise function in all these processes has remained elusive. We have purified catalytically active recombinant IGHMBP2, which has enabled us to assess its enzymatic properties and to identify its cellular targets. Our data reveal that IGHMBP2 is an ATP-dependent 5' --> 3' helicase, which unwinds RNA and DNA duplices in vitro. Importantly, this helicase localizes predominantly to the cytoplasm of neuronal and non-neuronal cells and associates with ribosomes. DSMA1-causing amino acid substitutions in IGHMBP2 do not affect ribosome binding yet severely impair ATPase and helicase activity. We propose that IGHMBP2 is functionally linked to translation, and that mutations in its helicase domain interfere with this function in DSMA1 patients.

Tudor domains are widespread among proteins involved in RNA metabolism, but only in a few cases their cellular function has been analyzed in detail. Here, we report on the characterization of the ubiquitously expressed Tudor domain containing protein Tdrd3. Apart from its Tudor domain, we show that Tdrd3 possesses an oligosaccharide/nucleotide binding fold (OB-fold) and an ubiquitin associated domain capable of binding tetra-ubiquitin. A set of biochemical experiments revealed an interaction of Tdrd3 with FMRP, the product of the gene affected in Fragile X syndrome, and its autosomal homologs FXR1 and FXR2. FMRP has been implicated in the translational regulation of target mRNAs and shown to be a component of stress granules (SG). We demonstrate that overexpression of Tdrd3 in cells induces the formation of SGs and as a result leads to its co-localization with endogenous FMRP in these structures. Interestingly, the disease-associated FMRP missense mutation I304N identified in a Fragile X patient severely impairs the interaction with Tdrd3 in biochemical experiments. We propose a contribution of Tdrd3 to FMRP-mediated translational repression and suggest that the loss of the FMRP-Tdrd3 interaction caused by the I304N mutation might contribute to the pathogenesis of Fragile X syndrome.

Degenerated motor neurons in the spinal cord are the pathological hallmark of spinal muscular atrophy (SMA). SMA is caused by mutations in the ubiquitously expressed survival motor neuron 1 (SMN1) gene, which lead to reduced levels of functional SMN protein. Many different functions have been assigned to SMN, including assembly of ribonucleoproteins (RNPs), splicing, transcription and axonal mRNA transport. Recently, tissue from SMA patients and animal models has been used to determine which function of SMN is affected in SMA patients. A surprising picture has emerged: the impaired assembly of RNP subunits of the spliceosome seems to be responsible for SMA pathogenesis. Here, we present a model of how this defect might cause motor-neuron degeneration and consider potential therapies.

The U5 snRNP plays an essential role in both U2- and U12-dependent splicing. Here, we have characterized a 52-kDa protein associated with the human U5 snRNP, designated U5-52K. Protein sequencing revealed that U5-52K is identical to the CD2BP2, which interacts with the cytoplasmic portion of the human T-cell surface protein CD2. Consistent with it associating with an snRNP, immunofluorescence studies demonstrated that the 52K protein is predominantly located in the nucleoplasm of HeLa cells, where it overlaps, at least in part, with splicing-factor compartments (or "speckles"). We further demonstrate that the 52K protein is a constituent of the 20S U5 snRNP, but is not found in U4/U6.U5 tri-snRNPs. Thus, it is the only 20S U5-specific protein that is not integrated into the tri-snRNP and resembles, in this respect, the U4/U6 di-snRNP assembly factor Prp24p/p110. Yeast two-hybrid screening and pulldown assays revealed that the 52K protein interacts with the U5-specific 102K and 15K proteins, suggesting that these interactions are responsible for its integration into the U5 particle. The N-terminal two-thirds of 52K interact with the 102K protein, whereas its C-terminal GYF-domain binds the 15K protein. As the latter lacks a proline-rich tract, our data indicate that a GYF-domain can also engage in specific protein-protein interactions in a polyproline-independent manner. Interestingly, the U5-102K protein has been shown previously to play an essential role in tri-snRNP formation, binding the U4/U6-61K protein. The interaction of 52K with a tri-snRNP bridging protein, coupled with its absence from the tri-snRNP, suggests it might function in tri-snRNP assembly.

Spinal muscular atrophy (SMA) is a motoneuron disease caused by reduced levels of survival motoneuron (SMN) protein. Previous studies have assigned SMN to uridine-rich small nuclear ribonucleoprotein particle (U snRNP) assembly, splicing, transcription, and RNA localization. Here, we have used gene silencing to assess the effect of SMN protein deficiency on U snRNP metabolism in living cells and organisms. In HeLa cells, we show that reduction of SMN to levels found in SMA patients impairs U snRNP assembly. In line with this, induced silencing of SMN expression in Xenopus laevis or zebrafish arrested embryonic development. Under less severe knock-down conditions, zebrafish embryos proceeded through development yet exhibited dramatic SMA-like motor axon degeneration. The same was observed after silencing two other essential factors in the U snRNP assembly pathway, Gemin2 and pICln. Importantly, the injection of purified U snRNPs into either SMN- or Gemin2-deficient embryos of Xenopus and zebrafish prevented developmental arrest and motoneuron degeneration, respectively. These findings suggest that motoneuron degeneration in SMA patients is a direct consequence of impaired production of U snRNPs.

SMNrp, also termed SPF30, has recently been identified in spliceosomes assembled in vitro. We have functionally characterized this protein and show that it is an essential splicing factor. We show that SMNrp is a 17S U2 snRNP‐associated protein that appears in the pre‐spliceosome (complex A) and the mature spliceosome (complex B) during splicing. Immunodepletion of SMNrp from nuclear extract inhibits the first step of pre‐mRNA splicing by preventing the formation of complex B. Re‐addition of recombinant SMNrp to immunodepleted extract reconstitutes both spliceosome formation and splicing. Mutations in two domains of SMNrp, although similarly deleterious for splicing, differed in their consequences on U2 snRNP binding, suggesting that SMNrp may also engage in interactions with splicing factors other than the U2 snRNP. In agreement with this, we present evidence for an additional interaction between SMNrp and the [U4/U6·U5] tri‐snRNP. A candidate that may mediate this interaction, namely the U4/U6‐90 kDa protein, has been identified. We suggest that SMNrp, as a U2 snRNP‐associated protein, facilitates the recruitment of the [U4/U6·U5] tri‐snRNP to the pre‐spliceosome.

Fragile X syndrome is a common form of inherited mental retardation. Most fragile X patients exhibit mutations in the fragile X mental retardation gene 1 (FMR1) that lead to transcriptional silencing and hence to the absence of the fragile X mental retardation protein (FMRP). Since FMRP is an RNA-binding protein which associates with polyribosomes, it had been proposed to function as a regulator of gene expression at the post-transcriptional level. In the present study, we show that FMRP strongly inhibits translation of various mRNAs at nanomolar concentrations in both rabbit reticulocyte lysate and microinjected Xenopus laevis oocytes. This effect is specific for FMRP, since other proteins with similar RNA-binding domains, including the autosomal homologues of FMRP, FXR1 and FXR2, failed to suppress translation in the same concentration range. Strikingly, a disease-causing Ile-->Asn substitution at amino acid position 304 (I304N) renders FMRP incapable of interfering with translation in both test systems. Initial studies addressing the underlying mechanism of inhibition suggest that FMRP inhibits the assembly of 80S ribosomes on the target mRNAs. The failure of FMRP I304N to suppress translation is not due to its reduced affinity for mRNA or its interacting proteins FXR1 and FXR2. Instead, the I304N point mutation severely impairs homo-oligomerization of FMRP. Our data support the notion that inhibition of translation may be a function of FMRP in vivo. We further suggest that the failure of FMRP to oligomerize, caused by the I304N mutation, may contribute to the pathophysiological events leading to fragile X syndrome.

Spinal muscular atrophy (SMA) is a neurodegenerative disease of motor neurons caused by reduced levels of functional survival of motor neurons (SMN) protein. Cytoplasmic SMN directly interacts with spliceosomal Sm proteins and facilitates their assembly onto U snRNAs. Nuclear SMN, in contrast, mediates recycling of pre-mRNA splicing factors. In this study, we have addressed the function of SMN in the nucleus. We show that a monoclonal antibody directed against SMN inhibits pre-mRNA splicing. Interestingly, the mode of inhibition suggests a novel role for SMN in splicing that occurs prior to, or in addition to, its role in recycling. Using biochemical fractionation and anti-SMN immunoaffinity chromatography, we identified two distinct nuclear SMN complexes termed NSC1 and NSC2. The biochemical properties and protein composition of NSC1 were determined in detail. NSC1 migrates in sucrose gradients as a U snRNA-free 20S complex containing at least 10 proteins. In addition to SMN, these include the SMN-interacting protein 1 (SIP-1), the putative helicase dp103/Gemin3, the novel dp103/Gemin3-interacting protein GIP1/Gemin4 and three additional proteins with apparent masses of 43, 33 and 18 kDa, respectively. Most surprisingly, NSC1 also contains a specific subset of spliceosomal Sm proteins. This shows that the SMN-Sm protein interaction is not restricted to the cytoplasm. Our data imply that nuclear SMN affects splicing by modulating the Sm protein composition of U snRNPs.

Splicing of nuclear precursors of mRNA (pre-mRNA) involves dynamic interactions between the RNA constituents of the spliceosome. The rearrangement of RNA-RNA interactions, such as the unwinding of the U4/U6 duplex, is believed to be driven by ATP-dependent RNA helicases. We recently have shown that spliceosomal U5 small nuclear ribonucleoproteins (snRNPs) from HeLa cells contain two proteins, U5-200kD and U5-100kD, which share homology with the DEAD/DEXH-box families of RNA helicases. Here we demonstrate that purified U5 snRNPs exhibit ATP-dependent unwinding of U4/U6 RNA duplices in vitro. To identify the protein responsible for this activity, U5 snRNPs were depleted of a subset of proteins under high salt concentrations and assayed for RNA unwinding. The activity was retained in U5 snRNPs that contain the U5-200kD protein but lack U5-100kD, suggesting that the U5-200kD protein could mediate U4/U6 duplex unwinding. Finally, U5-200kD was purified to homogeneity by glycerol gradient centrifugation of U5 snRNP proteins in the presence of sodium thiocyanate, followed by ion exchange chromatography. The RNA unwinding activity was found to reside exclusively with the U5-200kD DEXH-box protein. Our data raise the interesting possibility that this RNA helicase catalyzes unwinding of the U4/U6 RNA duplex in the spliceosome.

Prp2p, Prp16p, Prp22p, and Prp43p are members of the DEAH-box family of ATP-dependent putative RNA helicases required for pre-mRNA splicing in Saccharomyces cerevisiae. Recently, mammalian homologues of Prp43p and Prp22p have been described, supporting the idea that splicing in yeast and man is phylogenetically conserved. In this study, we show that a murine cell line resistant to the novel immunoregulatory drug Leflunomide (Arava) overexpresses a 135-kDa protein that is a putative DEAH-box RNA helicase. We have cloned the human counterpart of this protein and show that it shares pronounced sequence homology with Prp16p. Apart from its N-terminal domain, which is rich in RS, RD, and RE dipeptides, this human homologue of Prp16p (designated hPrp16p) is 41% identical to Prp16p. Significantly, homology is not only observed within the phylogenetically conserved helicase domain, but also in Prp16p-specific sequences. Immunofluorescence microscopy studies demonstrated that hPrp16p co-localizes with snRNPs in subnuclear structures referred to as speckles. Antibodies specific for hPrp16p inhibited pre-mRNA splicing in vitro prior to the second step. Thus, like its yeast counterpart, hPrp16p also appears to be required for the second catalytic step of splicing. Taken together, our data indicate that the human 135-kDa protein identified here is the structural and functional homologue of the yeast putative RNA helicase, Prp16p.

Splicing of nuclear precursors of mRNA (pre-mRNA) involves dynamic interactions between the RNA constituents of the spliceosome. The rearrangement of RNA- RNA interactions, such as the unwinding of the U4yU6 duplex, is believed to be driven by ATP-dependent RNA helicases. We recently have shown that spliceosomal U5 small nuclear ribonucleoproteins (snRNPs) from HeLa cells contain two proteins, U5-200kD and U5-100kD, which share homology with the DEADyDEXH-box families of RNA helicases. Here we demonstrate that purified U5 snRNPs exhibit ATP-dependent unwinding of U4yU6 RNA duplices in vitro. To identify the protein responsible for this activity, U5 snRNPs were depleted of a subset of proteins under high salt concentrations and assayed for RNA unwinding. The activity was retained in U5 snRNPs that contain the U5-200kD protein but lack U5- 100kD, suggesting that the U5-200kD protein could mediate U4yU6 duplex unwinding. Finally, U5-200kD was purified to homogeneity by glycerol gradient centrifugation of U5 snRNP proteins in the presence of sodium thiocyanate, followed by ion exchange chromatography. The RNA unwinding activity was found to reside exclusively with the U5-200kD DEXH-box protein. Our data raise the interesting possibility that this RNA helicase catalyzes unwinding of the U4yU6 RNA duplex in the spliceosome.

The driving forces behind the many RNA conformational changes occurring in the spliceosome are not well understood. Here we characterize an evolutionarily conserved human U5 small nuclear ribonucleoprotein (snRNP) protein (U5‐116kD) that is strikingly homologous to the ribosomal elongation factor EF‐2 (ribosomal translocase). A 114 kDa protein (Snu114p) homologous to U5‐116kD was identified in Saccharomyces cerevisiae and was shown to be essential for yeast cell viability. Genetic depletion of Snu114p results in accumulation of unspliced pre‐mRNA, indicating that Snu114p is essential for splicing in vivo. Antibodies specific for U5‐116kD inhibit pre‐mRNA splicing in a HeLa nuclear extract in vitro. In HeLa cells, U5‐116kD is located in the nucleus and co‐localizes with snRNP‐containing subnuclear structures referred to as speckles. The G domain of U5‐116kD/Snu114p contains the consensus sequence elements G1–G5 important for binding and hydrolyzing GTP. Consistent with this, U5‐116kD can be cross‐linked specifically to GTP by UV irradiation of U5 snRNPs. Moreover, a single amino acid substitution in the G1 sequence motif of Snu114p, expected to abolish GTP‐binding activity, is lethal, suggesting that GTP binding and probably GTP hydrolysis is important for the function of U5‐116kD/Snu114p. This is to date the first evidence that a G domain‐containing protein plays an essential role in the pre‐mRNA splicing process.

Step 2 of pre-mRNA splicing has characteristics that are suggestive of a 5' to 3' scanning process from the branch point to locate the 3' splice site. Specifically, the 3' splice site is almost always at the first AG downstream of the branch point even when the two elements are separated by hundreds of nucleotides. Insertion of new AGs between the branch and 3' splice site, or mutation of the wild-type 3' splice site, usually results in use of the new first AG as the 3' splice site. Finally, insertion of stable secondary structure between the branch point and 3' splice site, but distant from both elements, results in a block to step 2. We have sought to complement this circumstantial evidence by detecting physical contacts between the spliceosome and the RNA substrate in regions that are not themselves important for splicing, other than that they lie between the branch point/polypyrimidine tract and the 3' splice site. We have blocked step 2 of splicing by insertion of hairpin structures between the branch point and 3' splice site and applied methylene blue-mediated crosslinking, which is specific for protein-dsRNA interactions. Using this approach, we have detected a 116-kDa crosslinked protein that appears after step 1 of splicing with all transcripts containing a hairpin downstream of the branch point. The protein was identified as the 116-kDa U5 snRNP protein, which is a GTP-binding protein involved in step 2 of splicing. The crosslinking characteristics of U5 p116 are consistent with it having a role in locating the 3' splice site AG prior to step 2 of splicing.

Nuclear pre-mRNA splicing requires ATP at several steps from spliceosome assembly to product release. Here, we demonstrate that an integral component of the 20S U5 snRNP is an RNA-dependent ATPase. The ATPase activity of 20S US and 25S [U4/U6,U5] snRNPs purified by glycerol gradient centrifugation is strongly stimulated by homopolymeric RNA but not ssDNA, Purified 12S U1 and U2 snRNPs do not exhibit ATPase activity. Moreover the US-associated NTPase specifically hydrolyzes ATP and dATP The additional purification of 20S U5 snRNPs by Mono Q chromatography does not affect the efficiency of ATP hydrolysis. Both US and tri-snRNPs bind ATP stoichiometrically in an RNA-independent manner. A candidate ATPase was identified by UV-irradiation of purified snRNPs with radiolabeled ATP. In the presence of homopolymeric RNA, the 200 kDa US-specific protein is the major crosslinked protein, even in Mono Q-purified U5 snRNPs. The correlation between RNA-dependent ATPase activity in the U5 snRNP and the RNA-dependent onset of this crosslink strongly suggests that the 200 kDa protein is an RNA-dependent ATPase. Furthermore, both the formation of the crosslink and ATPase activity appear with a similar substrate specificity for ATP.

The L‐21 Tetrahymena ribozyme, an RNA molecule with sequence‐specific endoribonuclease activity derived from a self‐splicing group I intron, provides a model system for studying the RNA folding problem. A 160 nucleotide, independently folding domain of tertiary structure (the P4‐P6 domain) comprises about half of the ribozyme. We now apply Fe(II)‐EDTA cleavage to mutants of the ribozyme to explore the role of individual structural elements in tertiary folding of the RNA at equilibrium. Deletion of peripheral elements near the 3′ end of the ribozyme destabilizes a region of the catalytic core (P3‐P7) without altering the folding of the P4‐P6 domain. Three different mutations within the P4‐P6 domain that destabilize its folding also shift the folding of the P3‐P7 region of the catalytic core to higher MgCl2 concentrations. We conclude that the role of the extended P4‐P6 domain and of the 3′‐terminal peripheral elements is at least in part to stabilize the catalytic core. The organization of RNA into independently folding domains of tertiary structure may be common in large RNAs, including ribosomal RNAs. Furthermore, the observation of domain‐domain interactions in a catalytic RNA supports the feasibility of a primitive spliceosome without any proteins.