The yeast U2A''/U2B complex is required for pre-spliceosome formation.

Human U2 snRNP contains two specific proteins, U2A' and U2B", that interact with U2 snRNA stem-loop IV. In Saccharomyces cerevisiae, only the counterpart of human U2B", Yib9p, has been identified. Database searches revealed a gene potentially coding for a protein with striking similarities to human U2A', henceforth called LEA1 (looks exceptionally like U2A'). We demonstrate that Lea1p is a specific component of the yeast U2 snRNP. In addition, we show that Lea1p interacts directly with Yib9p. In vivo association of Lea1p with U2 snRNA requires Yib9p. Reciprocally, Yib9p binds to the U2 snRNA only in the presence of Lea1p in vivo, even though it has been previously shown to associate on its own with the U2 snRNA stem-loop IV in vitro. Strains lacking LEA1 and/or YIB9 grow slowly, are temperature sensitive and contain reduced levels of U2 snRNA. Pre-mRNA splicing is strongly impaired in these cells. In vitro studies demonstrate that spliceosome assembly is blocked prior to addition of U2 snRNP. This phenotype can be rescued partially, but specifically, by addition of the corresponding recombinant protein(s). This demonstrates a specific role for the yeast U2 snRNP specific proteins during formation of the pre-spliceosome.     

A weak interaction between the U2A'' protein and U2 snRNA helps to stabilize their complex with the U2B" protein.

The U2 snRNP complex contains two specific proteins, U2B" and U2A'. We have analysed the interaction of U2A' with U2B" and with U2 RNA. U2A' can form an weak but detectable RNA-protein complex with U2 RNA and a stable protein complex with U2B". This protein-protein complex binds efficiently and specifically to U2 RNA. Binding experiments with mutant forms of U2A' shows that the region of U2A' essential for binding to U2B" is extensive, being located between amino acid position 1-164. The behaviour of the wild type U2A' protein, and in particular of a mutant version of the protein in which amino acids 3, 4 and 5 are mutated, suggests that U2A' forms a weak interaction with U2 RNA which helps to stabilize the U2A'-U2B"-U2 RNA complex. Mutants of U2 RNA were used to localize the region of U2 RNA important for interaction with U2A'. The results show that U2A' interacts with the stem of hairpin IV.     

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.     

Nuclear exchange of the U1 and U2 snRNP-specific proteins

The snRNP particles include a set of common core snRNP proteins and snRNP specific proteins. In rodent cells the common core proteins are the B, D, D', E, F and G proteins in a suggested stoichiometry of B2D'2D2EFG. The additional U1- and U2-specific proteins are the 70-kD, A and C proteins and the A' and B" proteins, respectively. Previous cell fractionation and kinetic analysis demonstrated the snRNP core proteins are stored in the cytoplasm in large partially assembled snRNA-free intermediates that assemble with newly synthesized snRNAs during their transient appearance in the cytoplasm (Sauterer, R. A., R. J. Feeney, and G. W. Zieve. 1988. Exp. Cell Res. 176:344-359). This report investigates the assembly and intracellular distribution of the U1 and U2 snRNP-specific proteins. Cell enucleation and aqueous cell fractionation are used to prepare nuclear and cytoplasmic fractions and the U1- and U2-specific proteins are identified by isotopic labeling and immunoprecipitation or by immunoblotting with specific autoimmune antisera. The A, C, and A' proteins are found both assembled into mature nuclear snRNP particles and in unassembled pools in the nucleus that exchange with the assembled snRNP particles. The unassembled proteins leak from isolated nuclei prepared by detergent extraction. The unassembled A' protein sediments at 4S-6S in structures that may be multimers. The 70-kD and B" proteins are fully assembled with snRNP particles which do not leak from isolated nuclei. The kinetic studies suggest that the B" protein assembles with the U2 particle in the cytoplasm before it enters the nucleus.     

Target discrimination by RNA-binding proteins: role of the ancillary protein U2A' and a critical leucine residue in differentiating the RNA-binding specificity of spliceosomal proteins U1A and U2B".

The spliceosomal proteins U1A and U2B" each use a homologous RRM domain to bind specifically to their respective snRNA targets, U1hpll and U2hpIV, two stem-loops that are similar yet distinct in sequence. Previous studies have shown that binding of U2B" to U2hpIV is facilitated by the ancillary protein U2A', whereas specific binding of U1A to U1hpll requires no cofactor. Here we report that U2A' enables U2B" to distinguish the loop sequence of U2hpIV from that of U1hpll but plays no role in stem sequence discrimination. Although U2A' can also promote heterospecific binding of U1A to U2hpIV, a much higher concentration of the ancillary protein is required due to the approximately 500-fold greater affinity of U2A' for U2B". Additional experiments have identified a single leucine residue in U1A(Leu-44) that is critical for the intrinsic specificity of this protein for the loop sequence of U1 hpll in preference to that of U2hpIV. Our data suggest that most of the difference in RNA-binding specificity between U1A and U2B" can be accounted for by this leucine residue and by the contribution of the ancillary protein U2A' to the specificity of U2B".     

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.     

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.     

The U2B'''' RNP motif as a site of protein-protein interaction.

The U2 snRNP contains two specific proteins, U2B' and U2A'. Neither of these proteins, on its own, is capable of specific interactions with U2 RNA. Here, a complex between U2B' and U2A' that forms in the absence of RNA is identified. Analysis of mutant forms of U2B' shows that the smallest fragment able to bind specifically U2 RNA (amino acids 1-88) is also the minimal region required for complex formation with U2A', and implies that this region must be largely structurally intact for U2A' interaction. Although this truncated U2B' fragment is capable of making specific protein--RNA and protein-protein interactions its structure, as measured by the ability to bind to U2A', appears to depend on the rest of the protein. Hybrids between U2B' and the closely related U1A protein are used to localize U2B' specific amino acids involved in protein-protein interaction. These can be divided into two functional groups. U2A' interaction with U2B' amino acids 37-46 permits binding to U2 RNA whereas interaction with U2B' specific amino acids between positions 14 and 25 reduces non-specific binding to U1 RNA. These two proteins may serve as a general example of how RNA binding may be modulated by protein-protein interaction in the assembly of RNPs, particularly since the region of U2' involved in interaction with U2A' consists mainly of a conserved RNP motif.     

Molecular comparison of monocot and dicot U1 and U2 snRNAs

To elucidate differences between the pre-mRNA splicing components in monocots and dicots, we have cloned and characterized several U1 and U2 snRNA sequence variants expressed in wheat seedling nuclei. Primer extension sequencing on wheat and pea snRNA populations has demonstrated that two 5'-terminal nucleotides found in most other U1 snRNAs are missing/modified in many plant U1 snRNAs. Comparison of the wheat U1 and U2 snRNA variants with their counterparts expressed in pea nuclei has defined regions of structural divergence between monocot and dicot U1 and U2 snRNAs. The U1 and U2 snRNA sequences involved in RNA:RNA interaction with pre-mRNAs are absolutely conserved. Significant differences occur between wheat and pea U1 snRNAs in stem I and II structures implicated in the binding of U1-specific proteins suggesting that the monocot and dicot U1-specific snRNP proteins differ in their binding specificities. Stem III structures, which are required in mammalian systems for splicing complex formation but not for U1-specific protein binding, differ more extensively than stems I, II, or IV. In U2 snRNAs, the sequence differences between these two species are primarily localized in stem III and in stem IV which has been implicated in snRNP protein binding. These differences suggest that monocot and dicot U1 and U2 snRNPs represent distinct entities that may have monocot- and dicot-specific snRNP protein variants associated with each snRNA.     

In vitro reconstitution of U1 and U2 snRNPs from isolated proteins and snRNA

In this paper we describe a method for preparing native, RNA-free, proteins from anti-m₃G purified snRNPs (U1, U2, U4/U6 and U5) and the subsequent quantitative reconstitution of U1 and U2 snRNPs from purified proteins and snRNA. Reconstituted U1 and U2 snRNPs contained the full complement of core proteins, B, B, D1, D2, D3, E, F and G. Both the U1 and U2 reconstituted particles were stable in CsCl gradients and had the expected buoyant density of 1.4 g/cm³. Reconstituted RNP particle formation was not competited by a 50 fold molar excess of tRNA, as determined by gel retardation assays. However, U1 and U2 particle formation was reduced in the presence of an excess of cold U1 or U2 snRNA demonstrating a specific RNA-protein interaction. U1 and U2 snRNPs were also efficiently reconstituted in vitro, utilizing proteins prepared from mono Q purified U1 and U2 snRNPs. This suggests that for the assembly of snRNPs in vitro no auxiliary proteins other than bona fide snRNP proteins appear to be required. The potential of this reconstitution technique for investigating snRNP assembly and snRNA-protein interactions is discussed.Abbreviations PEG Polyethelene glycol - PMSF Phenylmethyl sulfonylfluoride - TP total proteins - mAb monoclonal antibody     

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.     

Drosophila SNF/D25 combines the functions of the two snRNP proteins U1A and U2B' that are encoded separately in human, potato, and yeast.

The plant and vertebrate snRP proteins U1A and U2B' are structurally closely related, but bind to different U snRNAs. Two additional related snRNP proteins, the yeast U2B' protein and Drosophila SNF/D25 protein, are analyzed here. We show that the previously described yeast open reading frame YIB9w encodes yeast U2B' as judged by the fact that the protein encoded by YIB9w bindsto stem-loop IV of yeast U2 snRNA in vitro and is part of the U2 snRNP in vivo. In contrast to the human U2B' protein, specific binding of yeast U2B' to RNA in vitro can occur in the absence of an accessory U2A' protein. The Drosophila SNF-D25 protein, unlike all other U1A/U2B' proteins studied to date, is shown to be a component of both U1 and U2 snRNPs. In vitro, SNF/D25 binds to U1 snRNA on itsown and to U2 snRNA in the presence of either the human U2A' protein or of Drosophila nuclear extract. Thus, its RNA-binding properties are the sum of those exhibited by human or potato U1A and U2B' proteins. Implications for the role of SNF/D25 in alternative splicing, and for the evolution of the U1A/U2B' protein family, are discussed.     

The 5'' end domain of U2 snRNA is required to establish the interaction of U2 snRNP with U2 auxiliary factor(s) during mammalian spliceosome assembly.

Stable association of U2 snRNP with the branchpoint sequence of mammalian pre-mRNAs requires binding of a non-snRNP protein to the polypyrimidine tract. In order to determine how U2 snRNP contacts this protein, we have used an RNA containing the consensus 5' and the (Py)n-AG 3' splice sites but lacking the branchpoint sequence so as to prevent direct U2 snRNA base pairing to the branchpoint. Different approaches including electrophoretic separation of RNP complexes formed in nuclear extracts, RNase T1 protection immunoprecipitation assays with antibodies against snRNPs and UV cross-linking experiments coupled to immunoprecipitations allowed us to demonstrate that at least three splicing factors contact this RNA at 0 degree C without ATP. As expected, U1 snRNP interacts with the region comprising the 5' splice site. A protein of approximately 65,000 molecular weight recognizes the RNA specifically at the 5' boundary of the polypyrimidine tract. It could be either the U2 auxiliary factor (U2AF) (Zamore and Green (1989) PNAS 86, 9243-9247), the polypyrimidine tract binding protein (pPTB) (Garcia-Blanco et al. (1989) Genes and Dev. 3, 1874-1886) or a mixture of both. U2 snRNP also contacts the RNA in a way depending on p65 binding, thereby further arguing that the latter may correspond to the previously characterized U2AF and pPTB. Cleavage of U2 snRNA sequence by a complementary oligonucleotide and RNase H led us to conclude that the 5' terminus of U2 snRNA is required to ensure the contact between U2 snRNP and p65 bound to the RNA. More importantly, this conclusion can be extended to authentic pre-mRNAs. When we have used a human beta-globin pre-mRNA instead of the above artificial substrate, RNA bound p65 became precipitable by anti-(U2) RNP and anti-Sm antibodies except when the 5' end of U2 snRNA was selectively cleaved.     

A Potential Role for U2AF-SAP 155 Interactions in Recruiting U2 snRNP to the Branch Site

Base pairing between U2 snRNA and the branchpoint sequence (BPS) is essential for pre-mRNA splicing. Because the metazoan BPS is short and highly degenerate, this interaction alone is insufficient for specific binding of U2 snRNP. The splicing factor U2AF binds to the pyrimidine tract at the 3′ splice site in the earliest spliceosomal complex, E, and is essential for U2 snRNP binding in the spliceosomal complex A. We show that the U2 snRNP protein SAP 155 UV cross-links to pre-mRNA on both sides of the BPS in the A complex. SAP 155’s downstream cross-linking site is immediately adjacent to the U2AF binding site, and the two proteins interact directly in protein-protein interaction assays. Using UV cross-linking, together with functional analyses of pre-mRNAs containing duplicated BPSs, we show a direct correlation between BPS selection and UV cross-linking of SAP 155 on both sides of the BPS. Together, our data are consistent with a model in which U2AF binds to the pyrimidine tract in the E complex and then interacts with SAP 155 to recruit U2 snRNP to the BPS.     

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.     

Direct, sequence-specific binding of the human U1-70K ribonucleoprotein antigen protein to loop I of U1 small nuclear RNA.

We have studied the interaction of two of the U1 small nuclear ribonucleoprotein (snRNP)-specific proteins, U1-70K and U1-A, with U1 small nuclear RNA (snRNA). The U1-70K protein is a U1-specific RNA-binding protein. Deletion and mutation analyses of a beta-galactosidase/U1-70K partial fusion protein indicated that the central portion of the protein, including the RNP sequence domain, is both necessary and sufficient for specific U1 snRNA binding in vitro. The highly conserved eight-amino-acid RNP consensus sequence was found to be essential for binding. Deletion and mutation analyses of U1 snRNA showed that both the U1-70K fusion protein and the native HeLa U1-70K protein bound directly to loop I of U1 snRNA. Binding was sequence specific, requiring 8 of the 10 bases in the loop. The U1-A snRNP protein also interacted specifically with U1 snRNA, principally with stem-loop II.     

Hierarchical,clustered protein interactions with U4/U6 snRNA: a biochemical role for U4/U6 proteins

During activation of the spliceosome, the U4/U6 snRNA duplex is dissociated, releasing U6 for subsequent base pairing with U2 snRNA. Proteins that directly bind the U4/U6 interaction domain potentially could mediate these structural changes. We thus investigated binding of the human U4/U6-specific proteins, 15.5K, 61K and the 20/60/90K protein complex, to U4/U6 snRNA in vitro. We demonstrate that protein 15.5K is a nucleation factor for U4/U6 snRNP assembly, mediating the interaction of 61K and 20/60/90K with U4/U6 snRNA. A similar hierarchical assembly pathway is observed for the U4atac/U6atac snRNP. In addition, we show that protein 61K directly contacts the 5' portion of U4 snRNA via a novel RNA-binding domain. Furthermore, the 20/60/90K heteromer requires stem II but not stem I of the U4/U6 duplex for binding, and this interaction involves a direct contact between protein 90K and U6. This uneven clustering of the U4/U6 snRNP-specific proteins on U4/U6 snRNA is consistent with a sequential dissociation of the U4/U6 duplex prior to spliceosome catalysis.     

Molecular dynamics simulation of the human U2B" protein complex with U2 snRNA hairpin IV in aqueous solution.

A 2200-ps molecular dynamics (MD) simulation of the U2 snRNA hairpin IV/U2B" complex was performed in aqueous solution using the particle mesh Ewald method to consider long-range electrostatic interactions. To investigate the interaction and recognition process between the RNA and protein, the free energy contributions resulting from individual amino acids of the protein component of the RNA/protein complex were calculated using the recently developed glycine-scanning method. The results revealed that the loop region of the U2 snRNA hairpin IV interacted mainly with three regions of the U2B" protein: 1) beta 1-helix A, 2) beta 2-beta 3, and 3) beta 4-helix C. U2 snRNA hairpin IV bound U2B" in a similar orientation as that previously described for U1 snRNA with the U1A' protein; however, the details of the interaction differed in several aspects. In particular, beta 1-helix A and beta 4-helix C in U2B" were not observed to interact with RNA in the U1A' protein complex. Most of the polar and charged residues in the interacting regions had larger mutant free energies than the nonpolar residues, indicating that electrostatic interactions were important for stabilizing the RNA/protein complex. The interaction was further stabilized by a network of hydrogen bonds and salt bridges formed between RNA and protein that was maintained throughout the MD trajectory. In addition to the direct interactions between RNA and the protein, solvent-mediated interactions also contributed significantly to complex stability. A detailed analysis of the ordered water molecules in the hydration of the RNA/protein complex revealed that bridged water molecules reside at the interface of RNA and protein as long as 2100 ps in the 2200-ps trajectory. At least 20 bridged water molecules, on average, contributed to the instantaneous stability of the RNA/protein complex. The stabilizing interaction energy due to bridging water molecules was obtained from ab initio Hartree-Fock and density functional theory calculations.