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.     

In vitro nuclear import of snRNPs: cytosolic factors mediate m3G-cap dependence of U1 and U2 snRNP transport.

We have established an in vitro snRNP nuclear import system using digitonin permeabilized somatic cells supplemented with cytosolic extracts. As model karyophiles we used digoxygenin labelled U1 snRNPs or fluorescein labelled U2 snRNPs. In vitro nuclear import of snRNPs is inhibited by anti-pore component antibodies, consistent with transport occurring through nuclear pores. This import requires ATP, cytosolic factors and a nuclear localization signal (NLS). SnRNP nuclear accumulation is saturable and distinct from protein transport. Nuclear import of snRNPs, in permeabilized NRK cells supplemented with somatic cell cytosol, requires the same NLS structures as those identified in micro-injected mammalian cells. In contrast to the situation in Xenopus oocytes, the m3G-cap is not required for in vitro nuclear import of U1 and U2 snRNPs in somatic cells. Instead, assembly of the Sm-core domain is both necessary and sufficient to mediate snRNP nuclear targeting. Interestingly, when the in vitro system was provided with cytosol from Xenopus oocytes instead of somatic cells, U1 and U2 snRNP nuclear import was provided with cytosol from Xenopus oocytes instead of somatic cells, U1 and U2 snRNP nuclear import was m3G-cap dependent. These results indicate that soluble cytosolic factors mediate the differential m3G-cap dependence of U1 and U2 snRNP nuclear import in somatic cells and oocytes. We also demonstrate the existence of a soluble cytosolic factor whose interaction with the U2 snRNP m3G-cap is both saturable and essential for U2 snRNP nuclear import in Xenopus oocytes.     

A new U6 small nuclear ribonucleoprotein-specific protein conserved between cis- and trans-splicing systems.

Spliceosomal U6 small nuclear RNA (snRNA) plays a central role in the pre-mRNA splicing mechanism and is highly conserved throughout evolution. Previously, a sequence element essential for both capping and cytoplasmic-nuclear transport of U6 snRNA was mapped in the 5'-terminal domain of U6 snRNA. We have identified a protein in cytoplasmic extracts of mammalian and Trypanosoma brucei cells that binds specifically to this U6 snRNA element. Competition studies with mutant and heterologous RNAs demonstrated the conserved binding specificity of the mammalian and trypanosomal proteins. The in vitro capping analysis of mutant U6 snRNAs indicated that protein binding is required but not sufficient for capping of U6 snRNA by a gamma-monomethyl phosphate. Through RNA affinity purification of mammalian small nuclear ribonucleoproteins (snRNPs), we detected this protein also in nuclear extract as a new specific component of the U6 snRNP but surprisingly not of the U4/U6 or the U4/U5/U6 multi-snRNP. These results suggest that the U6-specific protein is involved in U6 snRNA maturation and transport and may therefore be functionally related to the Sm proteins of the other spliceosomal snRNPs.     

Monospecific antibodies reveal details of U2 snRNP structure and interaction between U1 and U2 snRNPs.

Monospecific antibodies directed against several U small nuclear ribonucleoprotein (U snRNP) particle proteins were affinity purified from a patient's anti-(U1,U2)RNP serum. These were used to demonstrate that: (i) proteins equivalent to the mammalian U2 snRNP-specific A' and B" proteins are present in Xenopus laevis oocytes; (ii) both proteins A' and B" have the same structural requirements for binding to U2 snRNA; (iii) proteins B, B' and D have the same structural requirement for binding to U2 snRNA; (iv) using very high specific activity RNA probes it is possible to detect a fraction of either U1 or U2 snRNA precipitable by antibodies directed against proteins specific for the other U snRNP, indicating an interaction between U1 and U2 snRNPs. The structural requirements of this interaction were studied for the U2 snRNP. All changes made to U2 snRNA or snRNP structure resulted in loss of the interaction with U1 snRNP.     

RNA-protein organization of U1, U5 and U4-U6 small nuclear ribonucleoproteins in HeLa cells

Small nuclear ribonucleoproteins (snRNPs) containing U1 and U5 snRNAs from HeLa cells have been fractionated using a combination of isopycnic centrifugation in cesium chloride and ion-exchange chromatography on DEAE-Sepharose. The procedure is based on the extreme stability conferred upon snRNPs by Mg2+ enabling them to withstand the very high ionic strength that prevails in cesium chloride. U1 snRNP prepared by this method contains all nine major proteins (68K, A, B, B', C, D, E, F, G) corresponding to those previously identified by immunoprecipitation and is therefore precipitable by anti-RNP and anti-Sm antibodies. U5 snRNP purified in this way contains the common D to G proteins and is also enriched in a 25 X 10(3) Mr protein that may be U5 snRNP-specific. The core-resistant U5 snRNA sequence (nucleotide 84 to 3' OH) covered by D to G proteins is extended by only six nucleotides. A similar situation is seen in U4-U6 snRNP, which we have obtained in a sufficiently pure form to examine protected sequences. However, the core-resistant sequence of U4 (nucleotide 116 to 3' OH) in U4-U6 snRNP is extended by 37 nucleotides, suggesting that the protein composition of this particle could be more complex than that of U5 snRNP. The ribonucleoprotein organization of snRNPs is summarized and discussed in view of our current knowledge on snRNA sequences protected by proteins.     

Detection of intranuclear clusters of Sm antigens with monoclonal anti-Sm antibodies by immunoelectron microscopy

It has been shown that small nuclear RNA (snRNA) species U1, U2, U4, U5, and U6 are found in the nucleus in the form of small nuclear ribonucleoprotein particles (snRNPs), and that anti-Sm antibodies react with snRNP polypeptides, which are associated with all five snRNAs. We report here a novel intranuclear complex, denoted “Sm cluster,” detected by immunostaining with monoclonal anti-Sm antibodies in HeLa cells.     

In vitro reconstitution of mammalian U2 and U5 snRNPs active in splicing: Sm proteins are functionally interchangeable and are essential for the formation of functional U2 and U5 snRNPs.

An in vitro reconstitution/splicing complementation system has been developed which has allowed the investigation of the role of mammalian U2 and U5 snRNP components in splicing. U2 or U5 snRNP cores are first reconstituted from purified native snRNP core proteins and snRNA in the absence of cellular extract and are subsequently added to splicing extracts depleted of either U2 or U5 snRNP. When snRNPs reconstituted with HeLa U2 or U5 snRNA were added to U2- or U5-depleted nuclear extract, splicing was complemented. Addition of naked snRNA, on the other hand, did not restore splicing, demonstrating that the core proteins are essential for both U2 and U5 snRNP functions in splicing. Hybrid U2 or U5 snRNPs, reconstituted with core proteins isolated from U1 or U2 snRNPs, were equally active in splicing complementation, indicating that the snRNP core proteins are functionally interchangeable. U5 snRNPs reconstituted from in vitro transcribed U5 snRNA restored splicing to a level identical to that observed with particles reconstituted from authentic HeLa U5 snRNA. In contrast, splicing could not be restored to U2-depleted extract by the addition of snRNPs reconstituted from synthetic U2 snRNA, suggesting that U2 snRNA base modifications are essential for U2 snRNP function.     

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.     

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.     

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.     

Direct binding of small nuclear ribonucleoprotein G to the Sm site of small nuclear RNA. Ultraviolet light cross-linking of protein G to the AAU stretch within the Sm site (AAUUUGUGG) of U1 small nuclear ribonucleoprotein reconstituted in vitro.

The major small nuclear ribonucleoproteins (snRNPs) U1, U2, U5 and U4/U6 participate in the splicing of pre-mRNA. U1, U2, U4 and U5 RNAs share a highly conserved sequence motif PuA(U)nGPu, termed the Sm site, which is normally flanked by two hairpin loops. The Sm site provides the major binding site for the group of common proteins, B', B, D1, D2, D3, E, F and G, which are shared by the spliceosomal snRNPs. We have investigated the ability of common snRNP proteins to recognize the Sm site of snRNA by using ultraviolet light-induced RNA-protein cross-linking within U1 snRNP particles. The U1 snRNP particles, reconstituted in vitro, contained U1 snRNA labelled with 32P. Cross-linking of protein to this U1 snRNA occurred only in the presence of the single-stranded stretch of snRNA that makes up the conserved Sm site. Characterization of the cross-linked protein by one and two-dimensional gel electrophoresis indicated that snRNP protein G had become cross-linked to the U1 snRNA. This was confirmed by specific immunoprecipitation of the cross-linked RNA-protein complex with an anti-G antiserum. The cross-link was located on the U1 snRNA by fingerprint analysis with RNases T1 and A; this demonstrated that the protein G has been cross-linked to the AAU stretch within the 5'-terminal half of the Sm site (AAUUUGUGG). These results suggest that the snRNP protein G may be involved in the direct recognition of the Sm site.     

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

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

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.     

In vivo detection of snRNP-rich organelles in the nuclei of mammalian cells.

The in vivo distribution of snRNPs has been analysed by microinjecting fluorochrome-labelled antisense probes into the nuclei of live HeLa and 3T3 cells. Probes for U2 and U5 snRNAs specifically label the same discrete nuclear foci while a probe for U1 snRNA shows widespread nucleoplasmic labelling, excluding nucleoli, in addition to labelling foci. A probe for U3 snRNA specifically labels nucleoli. These in vivo data confirm that mammalian cells have nuclear foci which contain spliceosomal snRNPs. Co-localization studies, both in vivo and in situ, demonstrate that the spliceosomal snRNAs are present in the same nuclear foci. These foci are also stained by antibodies which recognize snRNP proteins, m3G-cap structures and the splicing factor U2AF but are not stained by anti-SC-35 or anti-La antibodies. U1 snRNP and the splicing factor U2AF closely co-localize in the nucleus, both before and after actinomycin D treatment, suggesting that they may both be part of the same complex in vivo.     

The trimethylguanosine cap structure of U1 snRNA is a component of a bipartite nuclear targeting signal

The ability of series of U1 snRNAs and U6 snRNAs to migrate into the nucleus of Xenopus oocytes after injection into the cytoplasm was analyzed. The U snRNAs were made either by injecting U snRNA genes into the nucleus of oocytes or, synthetically, by T7 RNA polymerase, incorporating a variety of cap structures. The results indicate that nuclear targeting of U1 snRNA requires both a trimethylguanosine cap structure and binding of at least one common U snRNP protein. Using synthetic U6 snRNAs, it is further demonstrated that the trimethylguanosine cap structure can act in nuclear targeting in the absence of the common U snRNP proteins. These results imply that U snRNP nuclear targeting signals are of a modular nature.     

RNA structure and RNA-protein interactions in purified yeast U6 snRNPs

The U6 small nuclear RNA (snRNA) undergoes major conformational changes during the assembly of the spliceosome and catalysis of splicing. It associates with the specific protein Prp24p, and a set of seven LSm2p-8p proteins, to form the U6 small nuclear ribonucleoprotein (snRNP). These proteins have been proposed to act as RNA chaperones that stimulate pairing of U6 with U4 snRNA to form the intermolecular stem I and stem II of the U4/U6 duplex, whose formation is essential for spliceosomal function. However, the mechanism whereby Prp24p and the LSm complex facilitate U4/U6 base-pairing, as well as the exact binding site(s) of Prp24p in the native U6 snRNP, are not well understood. Here, we have investigated the secondary structure of the U6 snRNA in purified U6 snRNPs and compared it with its naked form. Using RNA structure-probing techniques, we demonstrate that within the U6 snRNP a large internal region of the U6 snRNA is unpaired and protected from chemical modification by bound Prp24p. Several of these U6 nucleotides are available for base-pairing interaction, as only their sugar backbone is contacted by Prp24p. Thus, Prp24p can present them to the U4 snRNA and facilitate formation of U4/U6 stem I. We show that the 3' stem-loop is not bound strongly by U6 proteins in native particles. However, when compared to the 3' stem-loop in the naked U6 snRNA, it has a more open conformation, which would facilitate formation of stem II with the U4 snRNA. Our data suggest that the combined association of Prp24p and the LSm complex confers upon U6 nucleotides a conformation favourable for U4/U6 base-pairing. Interestingly, we find that the open structure of the yeast U6 snRNA in native snRNPs can also be adopted by human U6 and U6atac snRNAs.