Identification of a PRPF4 Loss-of-Function Variant That Abrogates U4/U6.U5 Tri-snRNP Integration and Is Associated with Retinitis Pigmentosa

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.     

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.     

A human homolog of yeast pre-mRNA splicing gene, PRP31, underlies autosomal dominant retinitis pigmentosa on chromosome 19q13.4 (RP11).

We report mutations in a gene (PRPF31) homologous to Saccharomyces cerevisiae pre-mRNA splicing gene PRP31 in families with autosomal dominant retinitis pigmentosa linked to chromosome 19q13.4 (RP11; MIM 600138). A positional cloning approach supported by bioinformatics identified PRPF31 comprising 14 exons and encoding a protein of 499 amino acids. The level of sequence identity to the yeast PRP31 gene indicates that PRPF31 is also likely to be involved in pre-mRNA splicing. Mutations that include missense substitutions, deletions, and insertions have been identified in four RP11-linked families and three sporadic RP cases. The identification of mutations in a pre-mRNA splicing gene implicates defects in the splicing process as a novel mechanism of photoreceptor degeneration.     

SANS (USH1G) regulates pre-mRNA splicing by mediating the intra-nuclear transfer of tri-snRNP complexes

Splicing is catalyzed by the spliceosome, a compositionally dynamic complex assembled stepwise on pre-mRNA. We reveal links between splicing machinery components and the intrinsically disordered ciliopathy protein SANS. Pathogenic mutations in SANS/USH1G lead to Usher syndrome—the most common cause of deaf-blindness. Previously, SANS was shown to function only in the cytosol and primary cilia. Here, we have uncovered molecular links between SANS and pre-mRNA splicing catalyzed by the spliceosome in the nucleus. We show that SANS is found in Cajal bodies and nuclear speckles, where it interacts with components of spliceosomal sub-complexes such as SF3B1 and the large splicing cofactor SON but also with PRPFs and snRNAs related to the tri-snRNP complex. SANS is required for the transfer of tri-snRNPs between Cajal bodies and nuclear speckles for spliceosome assembly and may also participate in snRNP recycling back to Cajal bodies. SANS depletion alters the kinetics of spliceosome assembly, leading to accumulation of complex A. SANS deficiency and USH1G pathogenic mutations affects splicing of genes related to cell proliferation and human Usher syndrome. Thus, we provide the first evidence that splicing dysregulation may participate in the pathophysiology of Usher syndrome.     

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.     

Six novel genes necessary for pre-mRNA splicing in Saccharomyces cerevisiae.

We have identified six new genes whose products are necessary for the splicing of nuclear pre-mRNA in the yeast Saccharomyces cerevisiae. A collection of 426 temperature-sensitive yeast strains was generated by EMS mutagenesis. These mutants were screened for pre-mRNA splicing defects by an RNA gel blot assay, using the intron- containing CRY1 and ACT1 genes as hybridization probes. We identified 20 temperature-sensitive mutants defective in pre-mRNA splicing. Twelve appear to be allelic to the previously identified prp2, prp3, prp6, prp16/prp23, prp18, prp19 or prp26 mutations that cause defects in spliceosome assembly or the first or second step of splicing. One is allelic to SNR14 encoding U4 snRNA. Six new complementation groups, prp29-prp34, were identified. Each of these mutants accumulates unspliced pre-mRNA at 37 degrees C and thus is blocked in spliceosome assembly or early steps of pre-mRNA splicing before the first cleavage and ligation reaction. The prp29 mutation is suppressed by multicopy PRP2 and displays incomplete patterns of complementation with prp2 alleles, suggesting that the PRP29 gene product may interact with that of PRP2. There are now at least 42 different gene products, including the five spliceosomal snRNAs and 37 different proteins that are necessary for pre-mRNA splicing in Saccharomyces cerevisiae. However, the number of yeast genes identifiable by this approach has not yet been exhausted.     

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.     

Arrested yeast splicing complexes indicate stepwise snRNP recruitment during in vivo spliceosome assembly

Pre-mRNA splicing is catalyzed by the spliceosome, a macromolecular machine dedicated to intron removal and exon ligation. Despite an abundance of in vitro information and a small number of in vivo studies, the pathway of yeast (Saccharomyces cerevisiae) in vivo spliceosome assembly remains uncertain. To address this situation, we combined in vivo depletions of U1, U2, or U5 snRNAs with chromatin immunoprecipitation (ChIP) analysis of other splicing snRNPs along an intron-containing gene. The data indicate that snRNP recruitment to nascent pre-mRNA predominantly proceeds via the canonical three-step assembly pathway: first U1, then U2, and finally the U4/U6*U5 tri-snRNP. Tandem affinity purification (TAP) using a U2 snRNP-tagged protein allowed the characterization of in vivo assembled higher-order splicing complexes. Consistent with an independent snRNP assembly pathway, we observed high levels of U1-U2 prespliceosomes under U5-depletion conditions, and we observed significant levels of a U2/U5/U6/Prp19-complex mature splicing complex under wild-type conditions. These complexes have implications for the steady-state distribution of snRNPs within nuclei and also reinforce the stepwise recruitment of U1, U2, and the tri-snRNP during in vivo spliceosome assembly.     

PRPF19 regulates p53-dependent cellular senescence by modulating alternative splicing of MDM4 mRNA

Alteration of RNA splicing is a hallmark of cellular senescence, which is associated with age-related disease and cancer development. However, the roles of splicing factors in cellular senescence are not fully understood. In this study, we identified the splicing factor PRPF19 as a critical regulator of cellular senescence in normal human diploid fibroblasts. PRPF19 was downregulated during replicative senescence, and PRPF19 knockdown prematurely induced senescence-like cell cycle arrest through the p53–p21 pathway. RNA-sequencing analysis revealed that PRPF19 knockdown caused a switch of the MDM4 splicing isoform from stable full-length MDM4-FL to unstable MDM4-S lacking exon 6. We also found that PRPF19 regulates MDM4 splicing by promoting the physical interaction of other splicing factors, PRPF3 and PRPF8, which are key components of the core spliceosome, U4/U6.U5 tri-snRNP. Given that MDM4 is a major negative regulator of p53, our findings imply that PRPF19 downregulation inhibits MDM4-mediated p53 inactivation, resulting in induction of cellular senescence. Thus, PRPF19 plays an important role in the induction of p53-dependent cellular senescence.     

SMNrp is an essential pre-mRNA splicing factor required for the formation of the mature spliceosome

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.     

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.     

Functional spliceosomal A complexes can be assembled in vitro in the absence of a penta-snRNP

Two different models currently exist for the assembly pathway of the spliceosome, namely, the traditional model, in which spliceosomal snRNPs associate in a stepwise, ordered manner with the pre-mRNA, and the holospliceosome model, in which all spliceosomal snRNPs preassemble into a penta-snRNP complex. Here we have tested whether the spliceosomal A complex, which contains solely U1 and U2 snRNPs bound to pre-mRNA, is a functional, bona fide assembly intermediate. Significantly, A complexes affinity-purified from nuclear extract depleted of U4/U6 snRNPs (and thus unable to form a penta-snRNP) supported pre-mRNA splicing in nuclear extract depleted of U2 snRNPs, whereas naked pre-mRNA did not. Mixing experiments with purified A complexes and naked pre-mRNA additionally confirmed that under these conditions, A complexes do not form de novo. Thus, our studies demonstrate that holospliceosome formation is not a prerequisite for generating catalytically active spliceosomes and that, at least in vitro, the U1 and U2 snRNPs can functionally associate with the pre-mRNA, prior to and independent of the tri-snRNP. The ability to isolate functional spliceosomal A complexes paves the way to study in detail subsequent spliceosome assembly steps using purified components.     

FgPrp4 Kinase Is Important for Spliceosome B-Complex Activation and Splicing Efficiency in Fusarium graminearum

PRP4 encodes the only kinase among the spliceosome components. Although it is an essential gene in the fission yeast and other eukaryotic organisms, the Fgprp4 mutant was viable in the wheat scab fungus Fusarium graminearum. Deletion of FgPRP4 did not block intron splicing but affected intron splicing efficiency in over 60% of the F. graminearum genes. The Fgprp4 mutant had severe growth defects and produced spontaneous suppressors that were recovered in growth rate. Suppressor mutations were identified in the PRP6, PRP31, BRR2, and PRP8 orthologs in nine suppressor strains by sequencing analysis with candidate tri-snRNP component genes. The Q86K mutation in FgMSL1 was identified by whole genome sequencing in suppressor mutant S3. Whereas two of the suppressor mutations in FgBrr2 and FgPrp8 were similar to those characterized in their orthologs in yeasts, suppressor mutations in Prp6 and Prp31 orthologs or FgMSL1 have not been reported. Interestingly, four and two suppressor mutations identified in FgPrp6 and FgPrp31, respectively, all are near the conserved Prp4-phosphorylation sites, suggesting that these mutations may have similar effects with phosphorylation by Prp4 kinase. In FgPrp31, the non-sense mutation at R464 resulted in the truncation of the C-terminal 130 aa region that contains all the conserved Prp4-phosphorylation sites. Deletion analysis showed that the N-terminal 310-aa rich in SR residues plays a critical role in the localization and functions of FgPrp4. We also conducted phosphoproteomics analysis with FgPrp4 and identified S289 as the phosphorylation site that is essential for its functions. These results indicated that FgPrp4 is critical for splicing efficiency but not essential for intron splicing, and FgPrp4 may regulate pre-mRNA splicing by phosphorylation of other components of the tri-snRNP although itself may be activated by phosphorylation at S289.     

Regulation of mammalian spliceosome assembly by a protein phosphorylation mechanism.

Splicing of mRNA precursors (pre-mRNA) is preceded by assembly of the pre-mRNA with small nuclear ribonucleoprotein particles (snRNPs) and protein factors to form a splicesome. Here we show that stimulating Ser/Thr-specific protein dephosphorylation selectively inhibits an early step during mammalian spliceosome assembly. Treatment of HeLa nuclear splicing extracts with human protein phosphatase 1 (PP1) expressed in Escherichia coli, or PP1 purified from rabbit skeletal muscle, prevents pre-spliceosome E complex (early complex) formation and stable binding of U2 and U4/U6.U5 snRNPs to the pre-mRNA. PP1 does not inhibit splicing catalysis if added after spliceosome assembly has taken place. Addition of purified SR protein splicing factors restores spliceosome formation and splicing to PP1-inhibited extracts, consistent with SR proteins being targets regulated by phosphorylation. These data extend earlier observations showing that splicing catalysis, but not spliceosome assembly, is blocked by inhibiting protein phosphatases. It therefore appears that pre-mRNA splicing, in common with other biological processes, can be regulated both positively and negatively by reversible protein phosphorylation.     

Identification and functional characterization of a novel splicing mutation in RP gene PRPF31

A six-generation Chinese family with autosomal dominant retinitis pigmentosa (adRP) was identified and characterized. Genome-wide linkage analysis linked the family to markers D19S601 to D19S605, which span the PRPF31 gene on chromosome 19q13.33-13.43 (RP11) (LOD = 5.03). Direct DNA sequence analysis identified a novel splicing mutation (IVS1+1G>T) in affected family members and carriers, but not in unaffected family members and 200 normal controls. The splicing mutation occurs at the splicing donor of intron 1. Real time PCR with lymphoblastoid RNA samples from family members showed that in comparison to normal family members, the splicing mutation reduced the expression level of the PRPF31 mRNA by 57% in symptomatic patients and by 28% in clinically asymptomatic carriers. Our studies identify a novel splicing mutation in PRPF31 associated with adRP and suggest that the penetrance of RP11 mutations may be correlated with the expression level of the PRPF31 mRNA.     

Spontaneous mutations in FgSAD1 suppress the growth defect of the Fgprp4 mutant by affecting tri-snRNP stability and its docking in Fusarium graminearum

FgPrp4, the only kinase in the spliceosome, is not essential for viability, but is important for splicing efficiency in Fusarium graminearum. The Fgprp4 deletion mutant had severe growth defects but often produced spontaneous suppressors with faster growth rate. To better understand the suppression mechanism, we identified and characterized spontaneous mutations in the tri-snRNP-specific protein, FgSad1, which suppressed the growth defects of Fgprp4. The L512P mutation was verified for its suppressive effects on Fgprp4, suggesting that mutations in FgSad1 may have effects involving FgPrp4 phosphorylation on FgSad1. Phosphoproteomics analysis showed that FgSad1 may not be the direct substrate of FgPrp4 kinase. Furthermore, truncation analysis showed that the N-terminal, extra RS-rich region of FgSad1 is critical for its function and is post-translationally modified. The P258S or S269P mutations in FgSad1 increased its interactions with the U5 protein FgPrp8 and the U4/U6 protein FgPrp31, which may result in tri-snRNP stabilization. Additionally, the D76N mutation increased the association of FgSad1 with the U2 snRNP. These data indicate that suppressor mutations in FgSad1 increase the stability of the tri-snRNP and/or the affinity of FgSad1 with U2 snRNP and therefore potentially facilitate the docking of tri-snRNP into the spliceosome.     

The differential interaction of snRNPs with pre-mRNA reveals splicing kinetics in living cells

Precursor messenger RNA (pre-mRNA) splicing is catalyzed by the spliceosome, a large ribonucleoprotein (RNP) complex composed of five small nuclear RNP particles (snRNPs) and additional proteins. Using live cell imaging of GFP-tagged snRNP components expressed at endogenous levels, we examined how the spliceosome assembles in vivo. A comprehensive analysis of snRNP dynamics in the cell nucleus enabled us to determine snRNP diffusion throughout the nucleoplasm as well as the interaction rates of individual snRNPs with pre-mRNA. Core components of the spliceosome, U2 and U5 snRNPs, associated with pre-mRNA for 15-30 s, indicating that splicing is accomplished within this time period. Additionally, binding of U1 and U4/U6 snRNPs with pre-mRNA occurred within seconds, indicating that the interaction of individual snRNPs with pre-mRNA is distinct. These results are consistent with the predictions of the step-wise model of spliceosome assembly and provide an estimate on the rate of splicing in human cells.     

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.     

Remodeling of U2-U6 snRNA helix I during pre-mRNA splicing by Prp16 and the NineTeen Complex protein Cwc2

Removal of intron regions from pre-messenger RNA (pre-mRNA) requires spliceosome assembly with pre-mRNA, then subsequent spliceosome remodeling to allow activation for the two steps of intron removal. Spliceosome remodeling is carried out through the action of DExD/H-box ATPases that modulate RNA–RNA and protein–RNA interactions. The ATPase Prp16 remodels the spliceosome between the first and second steps of splicing by catalyzing release of first step factors Yju2 and Cwc25 as well as destabilizing U2-U6 snRNA helix I. How Prp16 destabilizes U2-U6 helix I is not clear. We show that the NineTeen Complex (NTC) protein Cwc2 displays genetic interactions with the U6 ACAGAGA, the U6 internal stem loop (ISL) and the U2-U6 helix I, all RNA elements that form the spliceosome active site. We find that one function of Cwc2 is to stabilize U2-U6 snRNA helix I during splicing. Cwc2 also functionally cooperates with the NTC protein Isy1/NTC30. Mutation in Cwc2 can suppress the cold sensitive phenotype of the prp16-302 mutation indicating a functional link between Cwc2 and Prp16. Specifically the prp16-302 mutation in Prp16 stabilizes Cwc2 interactions with U6 snRNA and destabilizes Cwc2 interactions with pre-mRNA, indicating antagonistic functions of Cwc2 and Prp16. We propose that Cwc2 is a target for Prp16-mediated spliceosome remodeling during pre-mRNA splicing.