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 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.     

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.     

A general approach for identification of RNA-protein cross-linking sites within native human spliceosomal small nuclear ribonucleoproteins (snRNPs). Analysis of RNA-protein contacts in native U1 and U4/U6.U5 snRNPs

We describe a novel approach to identify RNA-protein cross-linking sites within native small nuclear ribonucleoprotein (snRNP) particles from HeLa cells. It combines immunoprecipitation of the UV-irradiated particles under semi-denaturing conditions with primer extension analysis of the cross-linked RNA moiety. In a feasibility study, we initially identified the exact cross-linking sites of the U1 70-kDa (70K) protein in stem-loop I of U1 small nuclear RNA (snRNA) within purified U1 snRNPs and then confirmed the results by a large-scale preparation that allowed N-terminal sequencing and matrix-assisted laser desorption ionization mass spectrometry of purified cross-linked peptide-oligonucleotide complexes. We identified Tyr(112) and Leu(175) within the RNA-binding domain of the U1 70K protein to be cross-linked to G(28) and U(30) in stem-loop I, respectively. We further applied our immunoprecipitation approach to HeLa U5 snRNP, as part of purified 25 S U4/U6.U5 tri-snRNPs. Cross-linking sites between the U5-specific 220-kDa protein (human homologue of Prp8p) and the U5 snRNA were located at multiple nucleotides within the highly conserved loop 1 and at one site in internal loop 1 of U5 snRNA. The cross-linking of four adjacent nucleotides indicates an extended interaction surface between loop 1 and the 220-kDa protein. In summary, our approach provides a rapid method for identification of RNA-protein contact sites within native snRNP particles as well as other ribonucleoprotein particles.     

Developmental expression of fibrillarin and U3 snRNA in Xenopus laevis.

Fibrillarin is one of the protein components that together with U3 snRNA constitute the U3 snRNP, a small nuclear ribonucleoprotein particle involved in ribosomal RNA processing in eucaryotic cells. Using an antifibrillarin antiserum for protein detection and a fibrillarin cDNA and a synthetic oligonucleotide complementary to U3 snRNA as hybridization probes, the expression of these two components has been studied during Xenopus development. Fibrillarin mRNA is accumulated early in oogenesis, like many other messengers, and translated during oocyte growth. Fibrillarin protein is thus progressively accumulated throughout oogenesis to be assembled with U3 snRNA and used for ribosome production in the amplified nucleoli. After fertilization, the amount of U3 snRNA decreases while the maternally accumulated fibrillarin mRNA is maintained and utilized to produce more protein. After the mid-blastula transition, stored fibrillarin is assembled with newly synthesized U3 snRNA and becomes localized in the prenucleolar bodies and reforming nucleoli.     

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.     

The low-abundance U11 and U12 small nuclear ribonucleoproteins (snRNPs) interact to form a two-snRNP complex.

A novel small nuclear ribonucleoprotein (snRNP) complex containing both U11 and U12 RNAs has been identified in HeLa cell extracts. This U11/U12 snRNP complex can be visualized on glycerol gradients, on native polyacrylamide gels, and by selection with antisense 2'-O-methyl oligoribonucleotides. RNase H-mediated degradation of the U12 snRNA confirmed a direct interaction between the U11 and U12 snRNPs. This snRNP complex is the first to be identified involving low-abundance snRNPs. Selection of the U11/U12 snRNP complex is sensitive to high salt, suggestive of a protein-mediated interaction. Secondary structure analyses revealed several regions of the U11 snRNP accessible for interaction with other RNAs or proteins but no detectable difference between the accessibility of these regions in the U11 monoparticle compared with the U11/U12 snRNP complex. There are also several accessible single-stranded regions in the U12 snRNP, and oligonucleotide-directed RNase H digestion identified nucleotides 28 to 36 of U12 as containing sequences required for the U11/U12 interaction. Both the U12 snRNP and the U11/U12 snRNP complex can be disrupted without altering the cleavage/polyadenylation activity of a nuclear extract.     

Nuclear import of U snRNPs requires importin beta.

Macromolecules that are imported into the nucleus can be divided into classes according to their nuclear import signals. The best characterized class consists of proteins which carry a basic nuclear localization signal (NLS), whose transport requires the importin alpha/beta heterodimer. U snRNP import depends on both the trimethylguanosine cap of the snRNA and a signal formed when the Sm core proteins bind the RNA. Here, factor requirements for U snRNP nuclear import are studied using an in vitro system. Depletion of importin alpha, the importin subunit that binds the NLS, is found to stimulate rather than inhibit U snRNP import. This stimulation is shown to be due to a common requirement for importin beta in both U snRNP and NLS protein import. Saturation of importin beta-mediated transport with the importin beta-binding domain of importin alpha blocks U snRNP import both in vitro and in vivo. Immunodepletion of importin beta inhibits both NLS-mediated and U snRNP import. While the former requires re-addition of both importin alpha and importin beta, re-addition of importin beta alone to immunodepleted extracts was sufficient to restore efficient U snRNP import. Thus importin beta is required for U snRNP import, and it functions in this process without the NLS-specific importin alpha.     

In vitro assembly of U1 snRNPs.

An efficient system for the in vitro assembly of U1 snRNPs is described. RNA-protein interactions in a series of U1 snRNA mutants assembled both in vivo and in vitro were studied in order to verify the accuracy of the system. Two discrete protein binding sites are defined by immunoprecipitation with antibodies against different protein components of the U1 snRNP and a newly developed protein sequestering assay. The U1 snRNP-specific proteins 70K and A require only the 5'-most stem-loop structure of U1 snRNA for binding, the common U snRNP proteins require the conserved Sm binding site (AUnG). Interactions between these two groups of proteins are detected. These results are combined to derive a model of the U1 snRNP structure. The potential use of the in vitro system in the functional analysis of U1 snRNP proteins is discussed.     

U2 snRNA-protein contacts in purified human 17S U2 snRNPs and in spliceosomal A and B complexes

The 17S U2 snRNP plays an essential role in branch point selection and catalysis during pre-mRNA splicing. Much remains to be learned about the molecular architecture of the U2 snRNP, including which proteins contact the functionally important 5' end of the U2 snRNA. Here, RNA-protein interactions within immunoaffinity-purified human 17S U2 snRNPs were analyzed by lead(II)-induced RNA cleavage and UV cross-linking. Contacts between the U2 snRNA and SF3a60, SF3b49, SF3b14a/p14 and SmG and SmB were detected. SF3b49 appears to make multiple contacts, interacting with the 5' end of U2 and nucleotides in loops I and IIb. SF3a60 also contacted different regions of the U2 snRNA, including the base of stem-loop I and a bulge in stem-loop III. Consistent with it contacting the pre-mRNA branch point adenosine, SF3b14a/p14 interacted with the U2 snRNA near the region that base pairs with the branch point sequence. A comparison of U2 cross-linking patterns obtained with 17S U2 snRNP versus purified spliceosomal A and B complexes revealed that RNA-protein interactions with stem-loop I and the branch site-interacting region of U2 are dynamic. These studies provide important insights into the molecular architecture of 17S U2 snRNPs and reveal U2 snRNP remodeling events during spliceosome assembly.     

All small nuclear RNAs (snRNAs) of the [U4/U6.U5] Tri-snRNP localize to nucleoli; Identification of the nucleolar localization element of U6 snRNA

Previously, we showed that spliceosomal U6 small nuclear RNA (snRNA) transiently passes through the nucleolus. Herein, we report that all individual snRNAs of the [U4/U6.U5] tri-snRNP localize to nucleoli, demonstrated by fluorescence microscopy of nucleolar preparations after injection of fluorescein-labeled snRNA into Xenopus oocyte nuclei. Nucleolar localization of U6 is independent from [U4/U6] snRNP formation since sites of direct interaction of U6 snRNA with U4 snRNA are not nucleolar localization elements. Among all regions in U6, the only one required for nucleolar localization is its 3' end, which associates with the La protein and subsequently during maturation of U6 is bound by Lsm proteins. This 3'-nucleolar localization element of U6 is both essential and sufficient for nucleolar localization and also required for localization to Cajal bodies. Conversion of the 3' hydroxyl of U6 snRNA to a 3' phosphate prevents association with the La protein but does not affect U6 localization to nucleoli or Cajal bodies.     

The U5 snRNA internal loop 1 is a platform for Brr2, Snu114 and Prp8 protein binding during U5 snRNP assembly

The U5 small nuclear ribonucleoprotein particle (snRNP) forms the heart of the spliceosome which is required for intron removal from pre‐mRNA. The proteins Prp8, Snu114 and Brr2 all assemble with the U5 small nuclear RNA (snRNA) to produce the U5 snRNP. Successful assembly of the U5 snRNP, then incorporation of this snRNP into the U4/U6.U5 tri‐snRNP and the spliceosome, is essential for producing an active spliceosome. We have investigated the requirements for Prp8, Snu114 and Brr2 association with the U5 snRNA to form the U5 snRNP in yeast. Mutations were constructed in the highly conserved loop 1 and internal loop 1 (IL1) of the U5 snRNA and their function assessed in vivo. The influence of these U5 mutations on association of Prp8, Snu114 and Brr2 with the U5 snRNA were then determined. U5 snRNA loop 1 and both sides of IL1 in U5 were important for association of Prp8, Snu114 and Brr2 with the U5 snRNA. Mutations in the 3′ side of U5 IL1 resulted in the greatest reduction of Prp8, Snu114 and Brr2 association with the U5 snRNA. Genetic screening of brr2 and U5 snRNA mutants revealed synthetic lethal interactions between alleles in Brr2 and the 3′ side of U5 snRNA IL1 which reflects reduced association between Brr2 and U5 IL1. We propose that the U5 snRNA IL1 is a platform for protein binding and is required for Prp8, Brr2 and Snu114 association with the U5 snRNA to form the U5 snRNP. J. Cell. Biochem. 114: 2770–2784, 2013. © 2013 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals Inc.     

Sm and Sm-like proteins belong to a large family: identification of proteins of the U6 as well as the U1, U2, U4 and U5 snRNPs.

Several small nuclear RNAs (snRNAs), including the spliceosomal U1, U2, U4 and U5 snRNAs, are associated with Sm proteins. These eight small proteins form a heteromeric complex that binds to snRNAs and plays a major role in small nuclear ribonucleoprotein (snRNP) biogenesis and transport. These proteins are also a major target for autoantibodies in the human disease systemic lupus erythematosus. By sequence comparison I have shown that all the known Sm proteins share a common structural motif which might explain their immunological cross-reactivity. Database searches using this motif uncovered a large number of Sm-like proteins from plants, animals and fungi. These proteins have been grouped in at least 13 different subfamilies. Genes encoding divergent yeast members were cloned and used to produce tagged fusion proteins. Some of these proteins are canonical Sm proteins as they associate with the yeast U1, U2, U4/U6 and U5 snRNAs. Surprisingly, one Sm-like protein was found to be a component of the U6 snRNP. These findings have implications for the structure of the Sm protein complex, spliceosomal snRNP evolution, snRNA transport and modification as well as the involvement of Sm proteins in systemic lupus erythematosus.     

Functional analysis of the sea urchin U7 small nuclear RNA.

U7 small nuclear RNA (snRNA) is an essential component of the RNA-processing machinery which generates the 3' end of mature histone mRNA in the sea urchin. The U7 small nuclear ribonucleoprotein particle (snRNP) is classified as a member of the Sm-type U snRNP family by virtue of its recognition by both anti-trimethylguanosine and anti-Sm antibodies. We analyzed the function-structure relationship of the U7 snRNP by mutagenesis experiments. These suggested that the U7 snRNP of the sea urchin is composed of three important domains. The first domain encompasses the 5'-terminal sequences, up to about nucleotides 7, which are accessible to micrococcal nuclease, while the remainder of the RNA is highly protected and hence presumably bound by proteins. This region contains the sequence complementarities between the U7 snRNA and the histone pre-mRNA which have previously been shown to be required for 3' processing (F. Schaufele, G. M. Gilmartin, W. Bannwarth, and M. L. Birnstiel, Nature [London] 323:777-781, 1986). Nucleotides 9 to 20 constitute a second domain which includes sequences for Sm protein binding. The complementarities between the U7 snRNA sequences in this region and the terminal palindrome of the histone mRNA appear to be fortuitous and play only a secondary, if any, role in 3' processing. The third domain is composed of the terminal palindrome of U7 snRNA, the secondary structure of which must be maintained for the U7 snRNP to function, but its sequence can be drastically altered without any observable effect on snRNP assembly or 3' processing.     

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.     

The association of the U1-specific 70K and C proteins with U1 snRNPs is mediated in part by common U snRNP proteins.

The U1 small nuclear ribonucleoprotein particle (snRNP)-specific 70K and A proteins are known to bind directly to stem-loops of the U1 snRNA, whereas the U1-C protein does not bind to naked U1 snRNA, but depends on other U1 snRNP protein components for its association. Focusing on the U1-70K and U1-C proteins, protein-protein interactions contributing to the association of these particle-specific proteins with the U1 snRNP were studied. Immunoprecipitation of complexes formed after incubation of naked U1 snRNA or purified U1 snRNPs lacking their specific proteins (core U1 snRNP) with in vitro translated U1-C protein, revealed that both common snRNP proteins and the U1-70K protein are required for the association of U1-C with the U1 snRNP. Binding studies with various in vitro translated U1-70K mutants demonstrated that the U1-70K N-terminal domain is necessary and sufficient for the interaction of U1-C with core U1 snRNPs. Surprisingly, several N-terminal fragments of the U1-70K protein, which lacked the U1-70K RNP-80 motif and did not bind naked U1 RNA, associated stably with core U1 snRNPs. This suggests that a new U1-70K binding site is generated upon association of common U1 snRNP proteins with U1 RNA. The interaction between the N-terminal domain of U1-70K and the core RNP domain was specific for the U1 snRNP; stable binding was not observed with core U2 or U5 snRNPs, suggesting essential structural differences among snRNP core domains. Evidence for direct protein-protein interactions between U1-specific proteins and common snRNP proteins was supported by chemical crosslinking experiments using purified U1 snRNPs. Individual crosslinks between the U1-70K and the common D2 or B'/B protein, as well as between U1-C and B'/B, were detected. A model for the assembly of U1 snRNP is presented in which the complex of common proteins on the RNA backbone functions as a platform for the association of the U1-specific proteins.     

5'-terminal caps of snRNAs are accessible for reaction with 2,2,7-trimethylguanosine-specific antibody in intact snRNPs

Immune precipitation assays with antibodies specific for 2,2,7-trimethylguanosine (m2,2,7(3)G) have been used to study the accessibility of the 5'-terminal m2,2,7(3)G-containing caps of eucaryotic small nuclear RNAs (snRNAs) either as naked RNAs or in intact small nuclear ribonucleoprotein (snRNPs). The antibody selectively precipitates snRNA species U1a, U1b, U2, U4, and U5 from total deproteinized RNA isolated from Ehrlich ascites cells. Binding by the antibody occurs via the m2,2,7(3)G moiety of the snRNAs' caps, since complex formation with the antibody can be completely abolished by excess nucleoside m2,2,7(3)G. The specificity of the antibody is further demonstrated by the complete absence of reaction with deproteinized snRNA species U6, the 5' terminus of which does not contain m2,2,7(3)G. Most importantly, the cap structures of the snRNAs U1a, U1b, U2, U4, and U5 are also accessible for anti-m2,2,7(3)G IgGs when intact snRNPs are reacted with the antibody. In this case, snRNP species U6 is coprecipitated, suggesting that there are intermolecular interactions between this and other snRNPs. Our data demonstrate that the 5'-terminal regions of the above snRNAs are not protected by the snRNP proteins. This finding is of special interest for snRNP species U1, and is discussed in terms of a model which proposes that the 5'-terminal region of U1 participates in the proper alignment of splice junctions in eucaryotic pre-mRNAs (Lerner, M. R., Boyle, J.A., Mount, S.M., Wolin, S.L., and Steitz, J. A. (1980) Nature (Lond.) 283, 220-224).     

Interactions between highly conserved U2 small nuclear RNA structures and Prp5p, Prp9p, Prp11p, and Prp21p proteins are required to ensure integrity of the U2 small nuclear ribonucleoprotein in Saccharomyces cerevisiae.

Binding of U2 small nuclear ribonucleoprotein (snRNP) to the pre-mRNA is an early and important step in spliceosome assembly. We searched for evidence of cooperative function between yeast U2 small nuclear RNA (snRNA) and several genetically identified splicing (Prp) proteins required for the first chemical step of splicing, using the phenotype of synthetic lethality. We constructed yeast strains with pairwise combinations of 28 different U2 alleles with 10 prp mutations and found lethal double-mutant combinations with prp5, -9, -11, and -21 but not with prp3, -4, -8, or -19. Many U2 mutations in highly conserved or invariant RNA structures show no phenotype in a wild-type PRP background but render mutant prp strains inviable, suggesting that the conserved but dispensable U2 elements are essential for efficient cooperative function with specific Prp proteins. Mutant U2 snRNA fails to accumulate in synthetic lethal strains, demonstrating that interaction between U2 RNA and these four Prp proteins contributes to U2 snRNP assembly or stability. Three of the proteins (Prp9p, Prp11p, and Prp21p) are associated with each other and pre-mRNA in U2-dependent splicing complexes in vitro and bind specifically to synthetic U2 snRNA added to crude splicing extracts depleted of endogenous U2 snRNPs. Taken together, the results suggest that Prp9p, -11p, and -21p are U2 snRNP proteins that interact with a structured region including U2 stem loop IIa and mediate the association of the U2 snRNP with pre-mRNA.