Polypeptide components of Drosophila small nuclear ribonucleoprotein particles.

In eukaryotes splicing of pre-mRNAs is mediated by the spliceosome, a dynamic complex of small nuclear ribonucleoprotein particles (snRNPs) that associate transiently during spliceosome assembly and the splicing reaction. We have purified snRNPs from nuclear extracts of Drosophila cells by affinity chromatography with an antibody specific for the trimethylguanosine (m3G) cap structure of snRNAs U1-U5. The polypeptide components of Drosophila snRNPs have been characterized and shown to consist of a number of proteins shared by all the snRNPs, and some proteins which appear to be specific to individual snRNP particles. On the basis of their apparent molecular weight and antigenicity many of these common and particle specific Drosophila snRNP proteins are remarkably conserved between Drosophila and human spliceosomes. By probing western blots of the Drosophila snRNP polypeptides with a number of antisera raised against human snRNP proteins, Drosophila polypeptides equivalent to many of the HeLa snRNP-common proteins have been identified, as well as candidates for a number of U1, U2 and U5-specific proteins.     

The C-terminal domain of coilin interacts with Sm proteins and U snRNPs

Coilin is the signature protein of the Cajal body (CB), a nuclear suborganelle involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs). Newly imported Sm-class snRNPs are thought to traffic through CBs before proceeding to their final nuclear destinations. Loss of coilin function in mice leads to significant viability and fertility problems. Coilin interacts directly with the spinal muscular atrophy (SMA) protein via dimethylarginine residues in its C-terminal domain. Although coilin hypomethylation results in delocalization of survival of motor neurons (SMN) from CBs, high concentrations of snRNPs remain within these structures. Thus, CBs appear to be involved in snRNP maturation, but factors that tether snRNPs to CBs have not been described. In this report, we demonstrate that the coilin C-terminal domain binds directly to various Sm and Lsm proteins via their Sm motifs. We show that the region of coilin responsible for this binding activity is separable from that which binds to SMN. Interestingly, U2, U4, U5, and U6 snRNPs interact with the coilin C-terminal domain in a glutathione S-transferase (GST)-pulldown assay, whereas U1 and U7 snRNPs do not. Thus, the ability to interact with free Sm (and Lsm) proteins as well as with intact snRNPs, indicates that coilin and CBs may facilitate the modification of newly formed snRNPs, the regeneration of ‘mature’ snRNPs, or the reclamation of unassembled snRNP components.     

Ongoing U snRNP biogenesis is required for the integrity of Cajal bodies

Cajal bodies (CBs) have been implicated in the nuclear phase of the biogenesis of spliceosomal U small nuclear ribonucleoproteins (U snRNPs). Here, we have investigated the distribution of the CB marker protein coilin, U snRNPs, and proteins present in C/D box small nucleolar (sno)RNPs in cells depleted of hTGS1, SMN, or PHAX. Knockdown of any of these three proteins by RNAi interferes with U snRNP maturation before the reentry of U snRNA Sm cores into the nucleus. Strikingly, CBs are lost in the absence of hTGS1, SMN, or PHAX and coilin is dispersed in the nucleoplasm into numerous small foci. This indicates that the integrity of canonical CBs is dependent on ongoing U snRNP biogenesis. Spliceosomal U snRNPs show no detectable concentration in nuclear foci and do not colocalize with coilin in cells lacking hTGS1, SMN, or PHAX. In contrast, C/D box snoRNP components concentrate into nuclear foci that partially colocalize with coilin after inhibition of U snRNP maturation. We demonstrate by siRNA-mediated depletion that coilin is required for the condensation of U snRNPs, but not C/D box snoRNP components, into nucleoplasmic foci, and also for merging these factors into canonical CBs. Altogether, our data suggest that CBs have a modular structure with distinct domains for spliceosomal U snRNPs and snoRNPs.     

Purification of snRNPs U1, U2, U4, U5 and U6 with 2,2,7-trimethylguanosine-specific antibody and definition of their constituent proteins reacting with anti-Sm and anti-(U1)RNP antisera

Small nuclear ribonucleoprotein particles (snRNPs) of the U-snRNP class from Ehrlich ascites tumor cells were purified in a one-step procedure by affinity chromatography with antibodies specific for 2,2,7-trimethylguanosine (m23.2.7G), which is part of the 5'-terminal cap structure of snRNAs U1-U5. Antibody-bound snRNPs are desorbed from the affinity column by elution with excess nucleoside m23.2.7G; this guarantees maintenance of their native structure. The snRNPs U1, U2, U4, U5 and U6 can be recovered quantitatively from nuclear extracts by this procedure. Co-isolation of U6 snRNP must be due to interactions between this and other snRNPs, as anti-m23.2.7G antibodies do not react with deproteinized U6 snRNA. We have so far defined nine proteins of approximate mol. wts. 10 000, 12 000, 13 000, 16 000, 21 000, 28 000, 32 000, 34 000 and 75 000. Purified snRNPs react with anti-(U1)RNP and with anti-Sm antisera from patients with mixed connective tissue disease and from MRL/l mice. As determined by the protein blotting technique, six of the snRNP polypeptides, characterized by apparent mol. wts. 13 000, 16 000, 21 000, 28 000, 34 000 and 75 000, bear antigenic determinants for one or the other of the above autoantibody classes. This suggests strongly that the U-snRNPs produced by the procedure described here are indeed representative of the snRNPs in the cell. With highly purified snRNPs available, investigation of possible enzymic functions of the particles may now be undertaken.     

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.     

Human U1 and U2 small nuclear ribonucleoproteins contain common and unique polypeptides.

Immunoprecipitation of human small nuclear ribonucleoproteins (snRNPs) containing the small nuclear RNAs U1, U2, U4, U5, and U6 with two antibodies produced in certain patients suffering from systemic lupus erythematosus was used to identify the polypeptides present on human U1 and U2 snRNPs. U1 and U2 snRNPs contain both common and unique polypeptides; visualization of the differences was possible through the use of non-methionine protein labeling and partial fractionation of snRNP populations. To facilitate comparisons with results from other laboratories, we have designated the snRNP polypeptides by their molecular weights. Four small polypeptides, P8, P9, P10, and P12, of 8,000 to 12,000 daltons, are each present in equal amounts on both U1 and U2 snRNPs. U1 snRNPs also contain a unique 30,000-dalton polypeptide, P30, whereas U2 snRNPs contain a unique 27,000-dalton, methionine-deficient polypeptide, P27. A closely migrating pair of polypeptides, P23 and P22, of 23,000 and 21,500 daltons, respectively, is present on both snRNPs; U2 snRNPs are enriched in the former, and U1 snRNPs are enriched in the latter.     

Nucleo-cytoplasmic transport of U snRNPs: definition of a nuclear location signal in the Sm core domain that binds a transport receptor independently of the m3G cap.

We have investigated the nuclear transport of U1 and U5 snRNPs by microinjection studies in oocytes from Xenopus laevis using snRNP particles prepared by reconstitution in vitro. Competition studies with snRNPs showed that the Sm core domain of U1 snRNPs contains a nuclear location signal that acts independently of the m3G cap. The transport of U1 snRNP can be blocked by saturation with competitor U1 snRNPs or by U5 snRNPs, which indicates that the signals on the respective Sm core domains interact with the same transport receptors. Further, by using a minimal U1 snRNP particle reconstituted in vitro and containing only the Sm core RNP domain and lacking stem-loops I to III of U1 RNA, we show that this is targeted actively to the nucleus, in spite of the absence of the m3G cap. This indicates that under certain conditions the NLS in the Sm core domain not only is an essential, but may also be a sufficient condition for nuclear targeting. We propose that the RNA structure of a given snRNP particle determines at least in part whether the particle's m3G cap is required for nuclear transport or can be dispensed with.     

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.     

Autoantibodies to the U2 small nuclear ribonucleoprotein in a patient with scleroderma-polymyositis overlap syndrome

Autoantibodies directed against the U2 small nuclear ribonucleoprotein (snRNP) have been found in the serum of a patient with scleroderma-polymyositis overlap syndrome. This specificity, called anti-(U2)-RNP, is distinct from all previously described autoantibodies, including those that precipitate related snRNPs: anti-Sm antibodies, which react with the entire set of U1, U2, U4, U5, and U6 snRNPs, and anti-(U1)RNP antibodies, which recognize only U1 snRNPs. From HeLa cell extracts, anti-(U2)RNP immunoprecipitates predominantly one 32P-labeled RNA species, identified as U2 small nuclear RNA, and six [35S]methionine-labeled protein bands, A' (Mr = 32,000), B (Mr = 28,000), D (Mr = 16,000), E (Mr = 13,000), F (Mr = 12,000), and G (Mr = 11,000). Protein blot analysis reveals that the A' protein carries (U2)RNP antigenic determinant(s) and therefore represents a polypeptide unique to the U2 snRNP; the B protein associated with U2 snRNPs may also be unique. Like U1 and the other Sm snRNPs, U2 snRNPs occupy a nuclear, non-nucleolar location and are antigenically conserved from insects to man. An antibody specific for the U2 snRNP will be useful in deciphering the function of this particle.     

An abundant U6 snRNP found in germ cells and embryos of Xenopus laevis.

The particle state of U snRNPs was analyzed in oocytes, eggs, embryos and testes from Xenopus laevis. In each case both the relative abundance and the composition of some U snRNPs were found to differ from that of somatic cells. U2 and U6 snRNPs were the most prominent U snRNPs in germ cells and early embryos. In particular, the concentration of U6 snRNA was 10-20 times higher than that of U4 snRNA. Most of the U6 snRNA was not associated with U4 snRNA and migrated on sucrose gradients as a U6 snRNP. The structure of this novel U snRNP was analyzed. A single protein of 50 kd was copurified with U6 snRNPs by a combination of gradient fractionation, immunodepletion with anti-Sm antibodies and immunoprecipitation with anti-6-methyl adenosine antibodies. Although the U6 snRNP did not contain Sm proteins it migrated into the nucleus when U6 snRNA was injected into the cytoplasm of oocytes. Two U6 snRNA elements have been identified. The first is essential for nuclear migration in oocytes, but not for the formation of U4/6 snRNPs in vitro and might be the binding site of a U6-specific protein. The second element was required for interaction with U4 snRNPs but not for nuclear targeting.     

Trimethylguanosine nucleoside inhibits cross-linking between Snurportin 1 and m3G-CAPPED U1 snRNA

Macromolecular nuclear import is an energy-and signal-dependent process. The best characterized type of nuclear import consists of proteins carrying the classical NLS that is mediated by the heterodimeric receptor importin alpha/beta. Spliceosomal snRNPs U1, U2, U4, and U5 nuclear import depend both on the 5' terminal m3G (trimethylguanosine) cap structure of the U snRNA and the Sm core domain. Snurportin 1 recognizes the m3G-cap structure of m3G-capped U snRNPs. In this report, we show how a synthesized trimethylguanosine nucleoside affects the binding of Snurportin 1 to m3G-capped U1 snRNA in a UV-cross-linking assay. The data indicated that TMG nucleoside is an essential component required in the recognition by Snurportin 1, thus suggesting that interaction of Snurportin 1 with U1 snRNA is not strictly dependent on the presence of the whole cap structure, but rather on the presence of the TMG nucleoside structure. These results indicate that the free nucleoside TMG could be a candidate to be an inhibitor of the interaction between Snurportin 1 and U snRNAs. We also show the behavior of free TMG nucleoside in in vitro U snRNPs nuclear import.     

Structural requirements for the function of a yeast chromosomal replicator

We have investigated the role of small nuclear ribonucleoprotein particles (snRNPs) in the in vitro splicing of messenger RNA precursors by a variety of procedures. Removal of the U-type snRNPs from the nuclear extracts of HeLa cells with protein A-Sepharose-coupled human autoimmune antibodies leads to complete loss of splicing activity. The inhibition of splicing can be prevented by saturating the coupled antibodies with purified nucleoplasmic U snRNPs prior to incubation with nuclear extract. We further demonstrate that an intact 5' terminus of U1 snRNA is required for the functioning of U1 snRNP in the splicing reaction. Antibodies directed against the trimethylated cap structure of the U snRNAs inhibit splicing. Upon removal of the first eight nucleotides of the U1 snRNA in the particles by site-directed hydrolysis with ribonuclease H in the presence of a synthetic complementary oligodeoxynucleotide splicing is completely abolished. These results are in strong support of current models suggesting that a base-pairing interaction between the 5' terminus of the U1 snRNA and the 5' splice site of a mRNA precursor is a prerequisite for proper splicing.     

U2 as well as U1 small nuclear ribonucleoproteins are involved in premessenger RNA splicing

Two different experimental approaches have provided evidence that both U2 and U1 snRNPs function in pre-mRNA splicing. When the U2 snRNPs in a nuclear extract are selectively degraded using ribonuclease H and either of two deoxyoligonucleotides complementary to U2 RNA, splicing activity is abolished. Mixing an extract in which U2 has been degraded with one in which U1 has been degraded recovers activity. Use of anti-(U2)RNP autoantibodies demonstrates that U2 snRNPs associate with the precursor RNA during in vitro splicing. At 60 min, but not at 0 min, into the reaction intron fragments that include the branch-point sequence are immunoprecipitated by anti-(U2)RNP. At all times, U1 snRNPs bind the 5' splice site of the pre-mRNA. Possible interactions of the U2 snRNP with the U1 snRNP and with the pre-mRNA during splicing are considered.     

A split luciferase-based reporter for detection of a cellular macromolecular complex

The spliceosome is a highly dynamic macromolecular ribonucleoprotein (RNP) machine that catalyzes pre-mRNA splicing by assembling U1, U2, U4, U5, and U6 small nuclear RNPs (snRNPs). To process large numbers of introns with a limited number of snRNPs, synthesis and recycling of snRNPs must be maintained within an appropriate range to avoid their shortage. However, the mechanism that maintains cellular snRNP levels is unknown. Molecules that modulate cellular snRNP levels may help to define this mechanism but are not available. Therefore, the goal of the current study was to develop a reporter for snRNP levels using split luciferase based on proteomic analysis of snRNPs. We constructed an expression library of a luciferase fragment fused to core components of U5 snRNP and used it to isolate pre-mRNA processing factor 6 (PRPF6) and small nuclear ribonucleoprotein 40 kDa (U5-40K) that specifically reconstitute luciferase activity in the U5 snRNP complex. Here we show that this reporter detects the effects of small molecules on the levels of the U5 snRNP reporter protein complex. Our approach provides an alternative assay to discover small molecules targeting a macromolecular complex when the structure of the complex is not precisely identified.     

Evidence from complementation assays in vitro that U5 snRNP is required for both steps of mRNA splicing.

We have established an in vitro complementation system that has allowed us to investigate the role of individual purified snRNPs in the splicing of pre-mRNA molecules. For the preparation of snRNP-depleted nuclear extracts we have first removed the majority of endogenous snRNPs from the nuclear extracts by one passage over an anti-m3G column and then degraded the remaining snRNPs with micrococcal nuclease. The mixture of snRNPs U1, U2, U4/U6 and U5, obtained by anti-m3G immuno-affinity chromatography, was functionally active and able to restore the splicing of snRNP-depleted nuclear extracts. Mono-Q chromatography was used for further fractionation of the snRNPs U1-U6. This produced three fractions that were highly enriched in snRNPs U1 and U2, U5 and U4/U6 respectively. Conditions were found where addition of the [U1, U2] and the U4/U6 snRNP fractions to the snRNP-depleted nuclear extracts gave rise to the formation of splice intermediates in the absence of any 3' cleavage/exon 1-exon 2 product formation. Only when purified 20S U5 snRNPs were added did both steps of the splicing reaction occur efficiently. Our data suggest that U5 snRNP is absolutely required for the second step of splicing and is needed further for efficient initiation of the splicing reaction. The requirement for U5 snRNPs for splicing was corroborated by glycerol gradient sedimentation analysis of the respective reconstituted pre-mRNP complexes. Stable and efficient formation of 50-60S spliceosomes was observed only in the presence of all snRNPs.     

A 69-kD protein that associates reversibly with the Sm core domain of several spliceosomal snRNP species

The biogenesis of the spliceosomal small nuclear ribonucleoproteins (snRNPs) U1, U2, U4, and U5 involves: (a) migration of the snRNA molecules from the nucleus to the cytoplasm; (b) assembly of a group of common proteins (Sm proteins) and their binding to a region on the snRNAs called the Sm-binding site; and (c) translocation of the RNP back to the nucleus. A first prerequisite for understanding the assembly pathway and nuclear transport of the snRNPs in more detail is the knowledge of all the snRNP proteins that play essential roles in these processes. We have recently observed a previously undetected 69- kD protein in 12S U1 snRNPs isolated from HeLa nuclear extracts under non-denaturing conditions that is clearly distinct from the U1-70K protein. The following evidence indicates that the 69-kD protein is a common, rather than a U1-specific, protein, possibly associating with the snRNP core particles by protein-protein interaction. (a) Antibodies raised against the 69-kD protein, which did not cross-react with any of the Sm proteins B'-G, precipitated not only U1 snRNPs, but also the other spliceosomal snRNPs U2, U4/U6 and U5, albeit to a lower extent. (b) U1, U2, and U5 core RNP particles reconstituted in vitro contain the 69-kD protein. (c) Xenopus laevis oocytes contain an immunologically related homologue of the human 69-kD protein. When U1 snRNA as well as a mutant U1 snRNA, that can bind the Sm core proteins but lacks the capacity to bind the U1-specific proteins 70K, A, and C, were injected into Xenopus oocytes to allow assembly in vivo, they were recognized by antibodies specific against the 69-kD protein in the ooplasm and in the nucleus. The 69-kD protein is under-represented, if present at all, in purified 17S U2 and in 25S [U4/U6.U5] tri-snRNPs, isolated from HeLa nuclear extracts. Our results are consistent with the working hypothesis that this protein may either play a role in the cytoplasmic assembly of the core domain of the snRNPs and/or in the nuclear transport of the snRNPs. After transport of the snRNPs into the nucleus, it may dissociate from the particles as for example in the case of the 17S U2 or the 25S [U4/U6.U5] tri-snRNP, which bind more than 10 different snRNP specific proteins each in the nucleus.     

Sm core variation in spliceosomal small nuclear ribonucleoproteins from Trypanosoma brucei

Messenger RNA processing in trypanosomes by cis and trans splicing requires spliceosomal small nuclear ribonucleoproteins (snRNPs) U1, U2, U4/U6, and U5, as well as the spliced leader (SL) RNP. As in other eukaryotes, these RNPs share a core structure of seven Sm polypeptides. Here, we report that the identity of the Sm protein constituents varies between spliceosomal snRNPs: specifically, two of the canonical Sm proteins, SmB and SmD3, are replaced in the U2 snRNP by two novel, U2 snRNP-specific Sm proteins, Sm15K and Sm16.5K. We present a model for the variant Sm core in the U2 snRNP, based on tandem affinity purification-tagging and in vitro protein-protein interaction assays. Using in vitro reconstitutions with canonical and U2-specific Sm cores, we show that the exchange of two Sm subunits determines discrimination between individual Sm sites. In sum, we have demonstrated that the heteroheptameric Sm core structure varies between spliceosomal snRNPs, and that modulation of the Sm core composition mediates the recognition of small nuclear RNA-specific Sm sites.     

Intranuclear binding kinetics and mobility of single native U1 snRNP particles in living cells

Uridine-rich small nuclear ribonucleoproteins (U snRNPs) are splicing factors, which are diffusely distributed in the nucleoplasm and also concentrated in nuclear speckles. Fluorescently labeled, native U1 snRNPs were microinjected into the cytoplasm of living HeLa cells. After nuclear import single U1 snRNPs could be visualized and tracked at a spatial precision of 30 nm at a frame rate of 200 Hz employing a custom-built microscope with single-molecule sensitivity. The single-particle tracks revealed that most U1 snRNPs were bound to specific intranuclear sites, many of those presumably representing pre-mRNA splicing sites. The dissociation kinetics from these sites showed a multiexponential decay behavior on time scales ranging from milliseconds to seconds, reflecting the involvement of U1 snRNPs in numerous distinct interactions. The average dwell times for U1 snRNPs bound at sites within the nucleoplasm did not differ significantly from those in speckles, indicating that similar processes occur in both compartments. Mobile U1 snRNPs moved with diffusion constants in the range from 0.5 to 8 μm²/s. These values were consistent with uncomplexed U1 snRNPs diffusing at a viscosity of 5 cPoise and U1 snRNPs moving in a largely restricted manner, and U1 snRNPs contained in large supramolecular assemblies such as spliceosomes or supraspliceosomes.