Spliceosomal Small Nuclear Ribonucleoprotein Particles Research Articles

Identification of Coilin Mutants in a Screen for Enhanced Expression of an Alternatively Spliced GFP Reporter Gene in Arabidopsis thaliana

Coilin is a marker protein for subnuclear organelles known as Cajal bodies, which are sites of various RNA metabolic processes including the biogenesis of spliceosomal small nuclear ribonucleoprotein particles. Through self-associations and interactions with other proteins and RNA, coilin provides a structural scaffold for Cajal body formation. However, despite a conspicuous presence in Cajal bodies, most coilin is dispersed in the nucleoplasm and expressed in cell types that lack these organelles. The molecular function of coilin, particularly of the substantial nucleoplasmic fraction, remains uncertain. We identified coilin loss-of-function mutations in a genetic screen for mutants showing either reduced or enhanced expression of an alternatively spliced GFP reporter gene in Arabidopsis thaliana. The coilin mutants feature enhanced GFP fluorescence and diminished Cajal bodies compared with wild-type plants. The amount of GFP protein is several-fold higher in the coilin mutants owing to elevated GFP transcript levels and more efficient splicing to produce a translatable GFP mRNA. Genome-wide RNA-sequencing data from two distinct coilin mutants revealed a small, shared subset of differentially expressed genes, many encoding stress-related proteins, and, unexpectedly, a trend toward increased splicing efficiency. These results suggest that coilin attenuates splicing and modulates transcription of a select group of genes. The transcriptional and splicing changes observed in coilin mutants are not accompanied by gross phenotypic abnormalities or dramatically altered stress responses, supporting a role for coilin in fine tuning gene expression. Our GFP reporter gene provides a sensitive monitor of coilin activity that will facilitate further investigations into the functions of this enigmatic protein.

SMN affects membrane remodelling and anchoring of the protein synthesis machinery.

Disconnection between membrane signalling and actin networks can have catastrophic effects depending on cell size and polarity. The survival motor neuron (SMN) protein is ubiquitously involved in assembly of spliceosomal small nuclear ribonucleoprotein particles. Other SMN functions could, however, affect cellular activities driving asymmetrical cell surface expansions. Genes able to mitigate SMN deficiency operate within pathways in which SMN can act, such as mRNA translation, actin network and endocytosis. Here, we found that SMN accumulates at membrane protrusions during the dynamic rearrangement of the actin filaments. In addition to localization data, we show that SMN interacts with caveolin-1, which mediates anchoring of translation machinery components. Importantly, SMN deficiency depletes the plasma membrane of ribosomes, and this correlates with the failure of fibroblasts to extend membrane protrusions. These findings strongly support a relationship between SMN and membrane dynamics. We propose that SMN could assembly translational platforms associated with and governed by the plasma membrane. This activity could be crucial in cells that have an exacerbated interdependence of membrane remodelling and local protein synthesis.

SART3-Dependent Accumulation of Incomplete Spliceosomal snRNPs in Cajal Bodies

Cajal bodies (CBs) are evolutionarily conserved nuclear structures involved in the metabolism of spliceosomal small nuclear ribonucleoprotein particles (snRNPs). CBs are not present in all cell types, and the trigger for their formation is not yet known. Here, we depleted cells of factors required for the final steps of snRNP assembly and assayed for the presence of stalled intermediates in CBs. We show that depletion induces formation of CBs in cells that normally lack these nuclear compartments, suggesting that CB nucleation is triggered by an imbalance in snRNP assembly. Accumulation of stalled intermediates in CBs depends on the di-snRNP assembly factor SART3. SART3 is required for both the induction of CB formation as well as the tethering of incomplete snRNPs to coilin, the CB scaffolding protein. We propose a model wherein SART3 monitors tri-snRNP assembly and sequesters incomplete particles in CBs, thereby allowing cells to maintain ahomeostatic balance of mature snRNPs in the nucleoplasm.

Presynaptic localization of Smn and hnRNP R in axon terminals of embryonic and postnatal mouse motoneurons.

Spinal muscular atrophy (SMA) is caused by deficiency of the ubiquitously expressed survival motoneuron (SMN) protein. SMN is crucial component of a complex for the assembly of spliceosomal small nuclear ribonucleoprotein (snRNP) particles. Other cellular functions of SMN are less characterized so far. SMA predominantly affects lower motoneurons, but the cellular basis for this relative specificity is still unknown. In contrast to nonneuronal cells where the protein is mainly localized in perinuclear regions and the nucleus, Smn is also present in dendrites, axons and axonal growth cones of isolated motoneurons in vitro. However, this distribution has not been shown in vivo and it is not clear whether Smn and hnRNP R are also present in presynaptic axon terminals of motoneurons in postnatal mice. Smn also associates with components not included in the classical SMN complex like RNA-binding proteins FUS, TDP43, HuD and hnRNP R which are involved in RNA processing, subcellular localization and translation. We show here that Smn and hnRNP R are present in presynaptic compartments at neuromuscular endplates of embryonic and postnatal mice. Smn and hnRNP R are localized in close proximity to each other in axons and axon terminals both in vitro and in vivo. We also provide new evidence for a direct interaction of Smn and hnRNP R in vitro and in vivo, particularly in the cytosol of motoneurons. These data point to functions of SMN beyond snRNP assembly which could be crucial for recruitment and transport of RNA particles into axons and axon terminals, a mechanism which may contribute to SMA pathogenesis.

Phosphoregulation of the human SMN complex

The survival motor neuron (SMN) complex is a macromolecular machine comprising 9 core proteins: SMN, Gemins2–8 and unrip in vertebrates. It performs tasks in RNA metabolism including the cytoplasmic assembly of spliceosomal small nuclear ribonucleoprotein particles (snRNPs). The SMN complex also localizes to the nucleus, where it accumulates in Cajal Bodies (CB) and may function in transcription and/or pre-mRNA splicing.The SMN complex is subject to extensive phosphorylation. Detailed understanding of SMN complex regulation necessitates a comprehensive analysis of these post-translational modifications. Here, we report on the first comprehensive phosphoproteome analysis of the intact human SMN complex, which identify 48 serine/threonine phosphosites in the complex. We find that 7 out of 9 SMN components of the intact complex are phosphoproteins and confidently place 29 phosphorylation sites, 12 of them in SMN itself.By the generation of multi non-phosphorylatable or phosphomimetic variants of SMN, respectively, we address to which extent phosphorylation regulates SMN complex function and localization. Both phosphomimetic and non-phosphorylatable variants assemble into intact SMN complexes and can compensate the loss of endogenous SMN in snRNP assembly at least to some extent. However, they partially or completely fail to target to nuclear Cajal bodies. Moreover, using a mutant of SMN, which cannot be phosphorylated on previously reported tyrosine residues, we provide first evidence that this PTM regulates SMN localization and nuclear accumulation. Our data suggest complex regulatory cues mediated by phosphorylation of serine/threonine and tyrosine residues, which control the subcellular localization of the SMN complex and its accumulation in nuclear CB.

Peripheral SMN restoration is essential for long-term rescue of a severe spinal muscular atrophy mouse model

Spinal muscular atrophy (SMA) is a motor neuron disease and the leading genetic cause of infant mortality; it results from loss-of-function mutations in the survival motor neuron 1 (SMN1) gene. Humans have a paralogue, SMN2, whose exon 7 is predominantly skipped, but the limited amount of functional, full-length SMN protein expressed from SMN2 cannot fully compensate for a lack of SMN1. SMN is important for the biogenesis of spliceosomal small nuclear ribonucleoprotein particles, but downstream splicing targets involved in pathogenesis remain elusive. There is no effective SMA treatment, but SMN restoration in spinal cord motor neurons is thought to be necessary and sufficient. Non-central nervous system (CNS) pathologies, including cardiovascular defects, were recently reported in severe SMA mouse models and patients, reflecting autonomic dysfunction or direct effects in cardiac tissues. Here we compared systemic versus CNS restoration of SMN in a severe mouse model. We used an antisense oligonucleotide (ASO), ASO-10-27, that effectively corrects SMN2 splicing and restores SMN expression in motor neurons after intracerebroventricular injection. Systemic administration of ASO-10-27 to neonates robustly rescued severe SMA mice, much more effectively than intracerebroventricular administration; subcutaneous injections extended the median lifespan by 25 fold. Furthermore, neonatal SMA mice had decreased hepatic Igfals expression, leading to a pronounced reduction in circulating insulin-like growth factor 1 (IGF1), and ASO-10-27 treatment restored IGF1 to normal levels. These results suggest that the liver is important in SMA pathogenesis, underscoring the importance of SMN in peripheral tissues, and demonstrate the efficacy of a promising drug candidate.

A mutation linked to retinitis pigmentosa in HPRP31 causes protein instability and impairs its interactions with spliceosomal snRNPs

The AD29 mutation in HPRP31 belongs to a series of mutations that were initially linked with the autosomal dominant disorder retinitis pigmentosa (RP) type 11. The HPRP31 gene encodes the hPrp31 protein that specifically associates with spliceosomal small nuclear ribonucleoprotein particles (snRNPs). Despite intensive research, it is still unclear how the AD29 (Ala216Pro) mutation causes RP. In this study, we report that the expression of this mutant protein affects cell proliferation and alters the structure of nuclear Cajal bodies that are connected with snRNP metabolism. Interestingly, these effects can be reversed by the over-expression of the hPrp6 protein, a binding partner of hPrp31. Although Ala216 is not contained within the U4 or U5 snRNP interacting domains, we present several lines of evidence that demonstrate that the association between the AD29 mutant and snRNPs in the cell nucleus is significantly reduced. Finally, we show that the stability of the AD29 mutant is severely affected resulting in its rapid degradation. Taken together, our results indicate that the Ala216Pro mutation destabilizes the hPrp31 protein structure in turn reducing its interaction with snRNP binding partners and leading to its rapid degradation. These findings significantly impact our understanding of the molecular mechanisms underlying RP and suggest that the insufficiency of the functional hPrp31 protein combined with the potential cytotoxicity associated with the expression the AD29 mutant are at least partially causative of the RP phenotype.

The assembly of a spliceosomal small nuclear ribonucleoprotein particle

The U1, U2, U4, U5 and U6 small nuclear ribonucleoprotein particles (snRNPs) are essential elements of the spliceosome, the enzyme that catalyzes the excision of introns and the ligation of exons to form a mature mRNA. Since their discovery over a quarter century ago, the structure, assembly and function of spliceosomal snRNPs have been extensively studied. Accordingly, the functions of splicing snRNPs and the role of various nuclear organelles, such as Cajal bodies (CBs), in their nuclear maturation phase have already been excellently reviewed elsewhere. The aim of this review is, then, to briefly outline the structure of snRNPs and to synthesize new and exciting developments in the snRNP biogenesis pathways.

Promiscuous Modification of the Nuclear Poly(A)-binding Protein by Multiple Protein-arginine Methyltransferases Does Not Affect the Aggregation Behavior

The mammalian nuclear poly(A)-binding protein, PABPN1, carries 13 asymmetrically dimethylated arginine residues in its C-terminal domain. By fractionation of cell extracts, we found that protein-arginine methyltransferases (PRMTs)-1, -3, and -6 are responsible for the modification of PABPN1. Recombinant PRMT1, -3, and -6 also methylated PABPN1. Our data suggest that these enzymes act on their own, and additional polypeptides are not involved in recognizing PABPN1 as a substrate. PRMT1 is the predominant methyltransferase acting on PABPN1. Nevertheless, PABPN1 was almost fully methylated in a Prmt1(-/-) cell line; thus, PRMT3 and -6 suffice for methylation. In contrast to PABPN1, the heterogeneous nuclear ribonucleoprotein (hnRNP) K is selectively methylated only by PRMT1. Efficient methylation of synthetic peptides derived from PABPN1 or hnRNP K suggested that PRMT1, -3, and -6 recognize their substrates by interacting with local amino acid sequences and not with additional domains of the substrates. However, the use of fusion proteins suggested that the inability of PRMT3 and -6 to modify hnRNP K is because of structural masking of the methyl-accepting amino acid sequences by neighboring domains. Mutations leading to intracellular aggregation of PABPN1 cause the disease oculopharyngeal muscular dystrophy. The C-terminal domain containing the methylated arginine residues is known to promote PAPBN1 self-association, and arginine methylation has been reported to inhibit self-association of an orthologous protein. Thus, arginine methylation might be relevant for oculopharyngeal muscular dystrophy. However, in two different types of assays we have been unable to detect any effect of arginine methylation on the aggregation of bovine PABPN1.

Spliceosomal Small Nuclear Ribonucleoprotein Particles Repeatedly Cycle through Cajal Bodies

The Cajal body (CB) is a nuclear structure closely associated with import and biogenesis of small nuclear ribonucleoprotein particles (snRNPs). Here, we tested whether CBs also contain mature snRNPs and whether CB integrity depends on the ongoing snRNP splicing cycle. Sm proteins tagged with photoactivatable and color-maturing variants of fluorescent proteins were used to monitor snRNP behavior in living cells over time; mature snRNPs accumulated in CBs, traveled from one CB to another, and they were not preferentially replaced by newly imported snRNPs. To test whether CB integrity depends on the snRNP splicing cycle, two human orthologues of yeast proteins involved in distinct steps in spliceosome disassembly after splicing, hPrp22 and hNtr1, were depleted by small interfering RNA treatment. Surprisingly, depletion of either protein led to the accumulation of U4/U6 snRNPs in CBs, suggesting that reassembly of the U4/U6.U5 tri-snRNP was delayed. Accordingly, a relative decrease in U5 snRNPs compared with U4/U6 snRNPs was observed in CBs, as well as in nuclear extracts of treated cells. Together, the data show that particular phases of the spliceosome cycle are compartmentalized in living cells, with reassembly of the tri-snRNP occurring in CBs.

Enhancement of U4/U6 Small Nuclear Ribonucleoprotein Particle Association in Cajal Bodies Predicted by Mathematical Modeling

Spliceosomal small nuclear ribonucleoprotein particles (snRNPs) undergo specific assembly steps in Cajal bodies (CBs), nonmembrane-bound compartments within cell nuclei. An example is the U4/U6 di-snRNP, assembled from U4 and U6 monomers. These snRNPs can also assemble in the nucleoplasm when cells lack CBs. Here, we address the hypothesis that snRNP concentration in CBs facilitates assembly, by comparing the predicted rates of U4 and U6 snRNP association in nuclei with and without CBs. This was accomplished by a random walk-and-capture simulation applied to a three-dimensional model of the HeLa cell nucleus, derived from measurements of living cells. Results of the simulations indicated that snRNP capture is optimal when nuclei contain three to four CBs. Interestingly, this is the observed number of CBs in most cells. Microinjection experiments showed that U4 snRNA targeting to CBs was U6 snRNP independent and that snRNA concentration in CBs is approximately 20-fold higher than in nucleoplasm. Finally, combination of the simulation with calculated association rates predicted that the presence of CBs enhances U4 and U6 snRNP association by up to 11-fold, largely owing to this concentration difference. This provides a chemical foundation for the proposal that these and other cellular compartments promote molecular interactions, by increasing the local concentration of individual components.

The Cajal body: a meeting place for spliceosomal snRNPs in the nuclear maze

Spliceosomal small nuclear ribonucleoprotein particles (snRNPs) are essential pre-mRNA splicing factors that consist of small nuclear RNAs (snRNAs) complexed with specific sets of proteins. A considerable body of evidence has established that snRNP assembly is accomplished after snRNA synthesis in the nucleus through a series of steps involving cytoplasmic and nuclear phases. Recent work indicates that snRNPs transiently localize to the Cajal body (CB), a nonmembrane-bound inclusion present in the nuclei of most cells, for the final steps in snRNP maturation, including snRNA base modification, U4/U6 snRNA annealing, and snRNA-protein assembly. Here, we review these findings that suggest a crucial role for CBs in the spliceosome cycle in which production of new snRNPs--and perhaps regenerated snRNPs after splicing--is promoted by the concentration of substrates in this previously mysterious subnuclear organelle. These insights allow us to speculate on the role of nuclear bodies in regulating the dynamics of RNP assembly to maintain a functional pool of factors available for key steps in gene expression.

Tudor Domains Bind Symmetrical Dimethylated Arginines

The Tudor domain is an approximately 60-amino acid structure motif in search of a function. Herein we show that the Tudor domains of the spinal muscular atrophy gene product SMN, the splicing factor 30 kDa (SPF30), and the Tudor domain-containing 3 (TDRD3) proteins interacted with arginine-glycine-rich motifs in a methylarginine-dependent manner. The Tudor domains also associated with methylarginine-containing cellular proteins, providing evidence that methylated arginines represent physiological ligands for this protein module. In addition, we report that spliceosomal small nuclear ribonucleoprotein particles core Sm proteins accumulated in the cytoplasm when arginine methylation was inhibited with adenosine dialdehyde or in the presence of an excessive amount of unmethylated arginine-glycine-rich peptides. These data provide in vivo evidence in support of a role for arginine methylation in the proper assembly and localization of spliceosomal Sm proteins.

Detection of snRNP assembly intermediates in Cajal bodies by fluorescence resonance energy transfer.

Spliceosomal small nuclear ribonucleoprotein particles (snRNPs) are required for pre-mRNA splicing throughout the nucleoplasm, yet snRNPs also concentrate in Cajal bodies (CBs). To address a proposed role of CBs in snRNP assembly, we have used fluorescence resonance energy transfer (FRET) microscopy to investigate the subnuclear distribution of specific snRNP intermediates. Two distinct complexes containing the protein SART3 (p110), required for U4/U6 snRNP assembly, were localized: SART3•U6 snRNP and SART3•U4/U6 snRNP. These complexes segregated to different nuclear compartments, with SART3•U6 snRNPs exclusively in the nucleoplasm and SART3•U4/U6 snRNPs preferentially in CBs. Mutant cells lacking the CB-specific protein coilin and consequently lacking CBs exhibited increased nucleoplasmic levels of SART3•U4/U6 snRNP complexes. Reconstitution of CBs in these cells by expression of exogenous coilin restored accumulation of SART3•U4/U6 snRNP in CBs. Thus, while some U4/U6 snRNP assembly can occur in the nucleoplasm, these data provide evidence that SART3•U6 snRNPs form in the nucleoplasm and translocate to CBs where U4/U6 snRNP assembly occurs.

SnRNAs contain specific SMN-binding domains that are essential for snRNP assembly.

To serve in its function as an assembly machine for spliceosomal small nuclear ribonucleoprotein particles (snRNPs), the survival of motor neurons (SMN) protein complex binds directly to the Sm proteins and the U snRNAs. A specific domain unique to U1 snRNA, stem-loop 1 (SL1), is required for SMN complex binding and U1 snRNP Sm core assembly. Here, we show that each of the major spliceosomal U snRNAs (U2, U4, and U5), as well as the minor splicing pathway U11 snRNA, contains a domain to which the SMN complex binds directly and with remarkable affinity (low nanomolar concentration). The SMN-binding domains of the U snRNAs do not have any significant nucleotide sequence similarity yet they compete for binding to the SMN complex in a manner that suggests the presence of at least two binding sites. Furthermore, the SMN complex-binding domain and the Sm site are both necessary and sufficient for Sm core assembly and their relative positions are critical for snRNP assembly. These findings indicate that the SMN complex stringently scrutinizes RNAs for specific structural features that are not obvious from the sequence of the RNAs but are required for their identification as bona fide snRNAs. It is likely that this surveillance capacity of the SMN complex ensures assembly of Sm cores on the correct RNAs only and prevents illicit, potentially deleterious, assembly of Sm cores on random RNAs.

Sequence-specific interaction of U1 snRNA with the SMN complex.

The survival of motor neurons (SMN) protein complex functions in the biogenesis of spliceosomal small nuclear ribonucleoprotein particles (snRNPs) and prob ably other RNPs. All spliceosomal snRNPs have a common core of seven Sm proteins. To mediate the assembly of snRNPs, the SMN complex must be able to bring together Sm proteins with U snRNAs. We showed previously that SMN and other components of the SMN complex interact directly with several Sm proteins. Here, we show that the SMN complex also interacts specifically with U1 snRNA. The stem--loop 1 domain of U1 (SL1) is necessary and sufficient for SMN complex binding in vivo and in vitro. Substitution of three nucleotides in the SL1 loop (SL1A3) abolishes SMN interaction, and the corresponding U1 snRNA (U1A3) is impaired in U1 snRNP biogenesis. Microinjection of excess SL1 but not SL1A3 into Xenopus oocytes inhibits SMN complex binding to U1 snRNA and U1 snRNP assembly. These findings indicate that SMN complex interaction with SL1 is sequence-specific and critical for U1 snRNP biogenesis, further supporting the direct role of the SMN complex in RNP biogenesis.

Structure and assembly of the spliceosomal small nuclear ribonucleoprotein particles

The spliceosome is a macromolecular assembly that carries out the excision of introns from nuclear pre-mRNAs. It consists of four large RNA-protein complexes, called the U1, U2, U4/U6 and U5 small nuclear ribonucleoproteins (snRNPs), and many protein factors. Crystal structures of seven protein components and fragments of the U1 and U2 small nuclear RNAs have been determined in the form of RNA-protein and protein-protein complexes. Together with electron microscopy studies of the snRNPs, these structures have begun to provide important insights into the architecture of the snRNPs and the mechanisms of RNA-protein and protein-protein recognition.

Structural/functional properties of a mammalian multi-component structure containing all major spliceosomal small nuclear ribonucleoprotein particles.

An approx. 40 S multi-component structure, consisting of all major spliceosomal small nuclear ribonucleoprotein particles (snRNP) (U1, U2, U4/U6 and U5) in stable association with a large number of polypeptides, mainly in the range 50-210 kDa, has been reported to exist within rat liver nuclear extracts [Guialis, Moraitou, Patrinou-Georgoula and Dangli (1991) Nucleic Acids Res. 19, 287-296]. Using a new polyclonal antibody recognizing a 63 kDa protein component of the complex, this multi-snRNP assembly was detected within rat liver nuclear extracts as efficiently as with the antibody for the U2 snRNP-specific B' polypeptide. The 63 kDa protein was found to correspond to the 66 kDa subunit of the splicing factor SF3a, a known integral component of the HeLa 17 S U2 snRNP. Anti-2,2,7-trimethylguanosine affinity chromatography was an easy and efficient way of purifying the multi-snRNP complex from rat liver 40 S heterogeneous nuclear ribonucleoprotein particle (hnRNP)-containing sucrose gradient fractions. By subsequent glycerol-gradient sedimentation, all known snRNP forms active in RNA splicing were identified among its constituents. A complex structurally similar to the rat multi-snRNP was also identified in HeLa nuclear extracts. Preservation of hnRNP-snRNP interactions was observed within HeLa 40 S fractions. Moreover, these fractions were capable of restoring splicing activity when applied in reconstitution studies to supplement a micrococcal nuclease-treated splicing extract.

A U1/U4/U5 snRNP complex induced by a 2'-O-methyl-oligoribonucleotide complementary to U5 snRNA.

Nuclear messenger RNA splicing involves multiple interactions between the five spliceosomal small nuclear ribonucleoprotein particles (snRNPs) U1, U2, U4, U5, and U6 and numerous spliceosomal proteins. Here it is shown that binding of a 2'-O-methyl-oligoribonucleotide complementary to U5 small nuclear RNA (snRNA) nucleotides 68 to 88 (BU5Ae) disrupts the initial U4/U5/U6 tri-snRNP complex, enhances the U2/U6 interaction, and induces a Ul/U4/U5 snRNP complex. The Ul/U4/U5 snRNP complex interacts specifically with an RNA oligonucleotide containing the 5' splice site sequence and may therefore represent a transitional stage in the displacement of U1 from the 5' splice site by U5 snRNP.