Monoclonal antibody specific to a subclass of polyproline-Arg motif provides evidence for the presence of an snRNA-free spliceosomal Sm protein complex in vivo: implications for molecular interactions involving proline-rich sequences of Sm B/B' proteins.

The human spliceosomal Sm B/B' proteins are essential for the biogenesis of the snRNP particles. B/B' proteins contain several clusters of the PPPPGM/IR sequence, which occurs within the C-terminus of Sm B/B'. This sequence is very similar to the PPPPPGHR sequence of the cytoplasmic tail of the CD2 receptor and closely resembles the class II of SH3 ligands, suggesting a similarly important role. We report that a monoclonal antibody (3E10) against the PPPPPGHR sequence recognizes spliceosomal Sm B/B' proteins. Proteins that are specifically immunoprecipitated by 3E10 include Sm B, B', D1, D2, D3, E, F, and G. However, unlike Y12 and other anti-Sm immunoprecipitates, 3E10 immunoprecipitates appear to lack the U1 snRNP-specific proteins A and C and U snRNAs. These findings indicate that 3E10 recognizes a subset of Sm protein core and suggest the presence of snRNA-free Sm protein complex(es) in vivo. We propose that the epitope binding for 3E10 may become unaccessible upon interactions of Sm proteins and their subsequent incorporation into the core particles. The Sm proline-rich sequences may have an important role in mediating protein-protein interactions necessary for the proper snRNP core assembly or function, or both. To our knowledge, 3E10 is the first well characterized mAb specific for a subclass of polyproline-arg motif recognizing Sm B/B' and CD2 proteins. 3E10 antibody can be used to further characterize the nature of protein components in the snRNA-free Sm subcore protein complex(es) that are formed during the snRNP core assembly steps.     

Mapping of B cell epitopes on small nuclear ribonucleoproteins that react with human autoantibodies as well as with experimentally-induced mouse monoclonal antibodies

Small nuclear ribonucleoprotein (snRNP) particles are a class of RNA-containing particles in the nucleus of eukaryotic cells. They consist of uridylate-rich small nuclear RNA complexed with several proteins. snRNP particles U1, U2, U4/U6, and U5 all contain a common protein core consisting of proteins B'/B, D, D', E, F, and G. In addition to this core, U1 snRNP particles contain proteins 70K, A, and C, whereas U2 snRNP particles contain proteins A' and B". Almost any of the small nuclear RNA-associated polypeptides is targeted by autoantibodies in the sera from patients with SLE or related connective tissue diseases. We immunized a genetically non-autoimmune mouse with recombinant human B" protein and obtained three mAb reactive with native U2 snRNP particles. Two of these mAb particles cross-reacted with U1 snRNP, 9A9 and 11A1, via epitopes present on the U2 snRNP B" protein as well as on the U1 snRNP-specific A protein. A third mAb 4g3, reacted exclusively with U2 snRNP via a unique epitope on protein B". Two epitopes mapped at the carboxy-terminal region of the B" protein, whereas binding of the third mAb involved both amino- and carboxy-terminal amino acids of the B" protein. Epitope mapping, employing a DNAse I fragment library of the B" cDNA, revealed that the three mAb-reactive sites were discontinuous. Autoantibodies in sera from patients with SLE and other connective tissue diseases competed for binding with the mAb, implying that the mAb define a major autoantibody-reactive region on protein B".     

Small nuclear ribonucleoprotein particle assembly in vivo: demonstration of a 6S RNA-free core precursor and posttranslational modification

The in vivo synthesis and assembly of human small nuclear ribonucleoproteins (snRNPs) have been studied using pulse/chase analysis. Antibodies derived from patients with systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD) recognize distinguishable subsets of pulse-labeled snRNP peptides. These antibodies were used to immunoprecipitate sucrose gradient fractionated pulse-labeled and pulse/chased snRNP proteins. The results indicate that assembly of the U RNA-containing snRNPs is a multistep process involving prior assembly of an RNA-free 6S core particle. This precursor contains snRNP peptides D, E, F, and G, which are common to all the different U RNA-containing particles. Furthermore, a posttranslational modification of one of the U1 snRNP-specific peptides has been observed, and the kinetics of this process indicates that the modification occurs after particle assembly. Functional and structural implications of a protein core for snRNP particles are discussed.     

Spliceosomal U snRNP core assembly: Sm proteins assemble onto an Sm site RNA nonanucleotide in a specific and thermodynamically stable manner.

The association of Sm proteins with U small nuclear RNA (snRNA) requires the single-stranded Sm site (PuAU(4-6)GPu) but also is influenced by nonconserved flanking RNA structural elements. Here we demonstrate that a nonameric Sm site RNA oligonucleotide sufficed for sequence-specific assembly of a minimal core ribonucleoprotein (RNP), which contained all seven Sm proteins. The minimal core RNP displayed several conserved biochemical features of native U snRNP core particles, including a similar morphology in electron micrographs. This minimal system allowed us to study in detail the RNA requirements for Sm protein-Sm site interactions as well as the kinetics of core RNP assembly. In addition to the uridine bases, the 2' hydroxyl moieties were important for stable RNP formation, indicating that both the sugar backbone and the bases are intimately involved in RNA-protein interactions. Moreover, our data imply that an initial phase of core RNP assembly is mediated by a high affinity of the Sm proteins for the single-stranded uridine tract but that the presence of the conserved adenosine (PuAU.) is essential to commit the RNP particle to thermodynamic stability. Comparison of intact U4 and U5 snRNAs with the Sm site oligonucleotide in core RNP assembly revealed that the regions flanking the Sm site within the U snRNAs facilitate the kinetics of core RNP assembly by increasing the rate of Sm protein association and by decreasing the activation energy.     

A minimal spliceosomal complex A recognizes the branch site and polypyrimidine tract.

The association of U2 snRNP with the pre-mRNA branch region is a critical step in the assembly of spliceosomal complexes. We describe an assembly process that reveals both minimal requirements for formation of a U2 snRNP-substrate RNA complex, here designated the Amin complex, and specific interactions with the branch site adenosine. The substrate is a minimal RNA oligonucleotide, containing only a branch sequence and polypyrimidine tract. Interactions at the branch site adenosine and requirements for polypyrimidine tract-binding proteins for the Amin complex are the same as those of authentic prespliceosome complex A. Surprisingly, Amin complex formation does not require U1 snRNP or ATP, suggesting that these factors are not necessary for stable binding of U2 snRNP per se, but rather are necessary for accessibility of components on longer RNA substrates. Furthermore, there is an ATP-dependent activity that releases or destabilizes U2 snRNP from branch sequences. The simplicity of the Amin complex will facilitate a detailed understanding of the assembly of prespliceosomes.     

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.     

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.     

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.     

Functional analysis of SNF, the Drosophila U1A/U2B" homolog: identification of dispensable and indispensable motifs for both snRNP assembly and function in vivo

In Drosophila, the spliceosomal protein SNF fulfills the functions of two vertebrate proteins, U1 snRNP-UlA and U2 snRNP-U2B". The structure and sequence of SNF, U1A, and U2B" are nearly identical with two RNA recognition motifs (RRM) separated by a short linker region, yet they have different RNA-binding properties: U1A binds U1 snRNA, U2B" binds U2 snRNA, and SNF binds both snRNAs. Structure/function studies on the human proteins have identified motifs in the N-terminal RRM that are critical for RNA-binding specificity but have failed to identify a function for the C-terminal RRM. Interestingly, SNF is chimeric in these motifs, suggesting a basis for its dual specificity. Here, we test the importance of these motifs by introducing site-directed mutations in the snf coding region and examining the effects of these mutations on assembly into the snRNP and on snf function in vivo. We found that an N-terminal RRM mutant protein predicted to eliminate RNA binding still assembles into snRNPs and is capable of rescuing snf's lethal phenotype only if the normally dispensable C-terminal RRM is present. We also found that the mixed motif in the "RNA-specificity" domain is necessary for SNF's dual function whereas the mixed motif in the U2A'-protein-binding region is not. Finally, we demonstrate that animals carrying a snf mutation that converts SNF from a bifunctional protein to a U1 snRNP-specific protein are viable. This unexpected result suggests that SNF's presence within the U2 snRNP is not essential for splicing.     

The snRNP core assembly pathway: identification of stable core protein heteromeric complexes and an snRNP subcore particle in vitro.

Stable association of the eight common Sm proteins with U1, U2, U4 or U5 snRNA to produce a spliceosomal snRNP core structure is required for snRNP biogenesis, including cap hypermethylation and nuclear transport. Here, the assembly of snRNP core particles was investigated in vitro using both native HeLa and in vitro generated Sm proteins. Several RNA-free, heteromeric protein complexes were identified, including E.F.G, B/B'.D3 and D1.D2.E.F.G. While the E.F.G complex alone did not stably bind to U1 snRNA, these proteins together with D1 and D2 were necessary and sufficient to form a stable U1 snRNP subcore particle. The subcore could be chased into a core particle by the subsequent addition of the B/B'.D3 protein complex even in the presence of free competitor U1 snRNA. Trimethylation of U1 snRNA's 5' cap, while not observed for the subcore, occurred in the stepwise-assembled U1 snRNP core particle, providing evidence for the involvement of the B/B' and D3 proteins in the hypermethylation reaction. Taken together, these results suggest that the various protein heterooligomers, as well as the snRNP subcore particle, are functional intermediates in the snRNP core assembly pathway.     

NUFIP and the HSP90/R2TP chaperone bind the SMN complex and facilitate assembly of U4-specific proteins

The Sm proteins are loaded on snRNAs by the SMN complex, but how snRNP-specific proteins are assembled remains poorly characterized. U4 snRNP and box C/D snoRNPs have structural similarities. They both contain the 15.5K and proteins with NOP domains (PRP31 for U4, NOP56/58 for snoRNPs). Biogenesis of box C/D snoRNPs involves NUFIP and the HSP90/R2TP chaperone system and here, we explore the function of this machinery in U4 RNP assembly. We show that yeast Prp31 interacts with several components of the NUFIP/R2TP machinery, and that these interactions are separable from each other. In human cells, PRP31 mutants that fail to stably associate with U4 snRNA still interact with components of the NUFIP/R2TP system, indicating that these interactions precede binding of PRP31 to U4 snRNA. Knock-down of NUFIP leads to mislocalization of PRP31 and decreased association with U4. Moreover, NUFIP is associated with the SMN complex through direct interactions with Gemin3 and Gemin6. Altogether, our data suggest a model in which the NUFIP/R2TP system is connected with the SMN complex and facilitates assembly of U4 snRNP-specific proteins.     

U1 snRNP is mislocalized in ALS patient fibroblasts bearing NLS mutations in FUS and is required for motor neuron outgrowth in zebrafish

Mutations in FUS cause amyotrophic lateral sclerosis (ALS), but the molecular pathways leading to neurodegeneration remain obscure. We previously found that U1 snRNP is the most abundant FUS interactor. Here, we report that components of the U1 snRNP core particle (Sm proteins and U1 snRNA), but not the mature U1 snRNP-specific proteins (U1-70K, U1A and U1C), co-mislocalize with FUS to the cytoplasm in ALS patient fibroblasts harboring mutations in the FUS nuclear localization signal (NLS). Similar results were obtained in HeLa cells expressing the ALS-causing FUS R495X NLS mutation, and mislocalization of Sm proteins is RRM-dependent. Moreover, as observed with FUS, knockdown of any of the U1 snRNP-specific proteins results in a dramatic loss of SMN-containing Gems. Significantly, knockdown of U1 snRNP in zebrafish results in motor axon truncations, a phenotype also observed with FUS, SMN and TDP-43 knockdowns. Our observations linking U1 snRNP to ALS patient cells with FUS mutations, SMN-containing Gems, and motor neurons indicate that U1 snRNP is a component of a molecular pathway associated with motor neuron disease. Linking an essential canonical splicing factor (U1 snRNP) to this pathway provides strong new evidence that splicing defects may be involved in pathogenesis and that this pathway is a potential therapeutic target.     

A doughnut-shaped heteromer of human Sm-like proteins binds to the 3'-end of U6 snRNA, thereby facilitating U4/U6 duplex formation in vitro.

We describe the isolation and molecular characterization of seven distinct proteins present in human [U4/U6.U5] tri-snRNPs. These proteins exhibit clear homology to the Sm proteins and are thus denoted LSm (like Sm) proteins. Purified LSm proteins form a heteromer that is stable even in the absence of RNA and exhibits a doughnut shape under the electron microscope, with striking similarity to the Sm core RNP structure. The purified LSm heteromer binds specifically to U6 snRNA, requiring the 3'-terminal U-tract for complex formation. The 3'-end of U6 snRNA was also co-precipitated with LSm proteins after digestion of isolated tri-snRNPs with RNaseT(1). Importantly, the LSm proteins did not bind to the U-rich Sm sites of intact U1, U2, U4 or U5 snRNAs, indicating that they can only interact with a 3'-terminal U-tract. Finally, we show that the LSm proteins facilitate the formation of U4/U6 RNA duplices in vitro, suggesting that the LSm proteins may play a role in U4/U6 snRNP formation.     

Role of Cajal Bodies and Nucleolus in the Maturation of the U1 snRNP in Arabidopsis

Background

The biogenesis of spliceosomal snRNPs takes place in both the cytoplasm where Sm core proteins are added and snRNAs are modified at the 5′ and 3′ termini and in the nucleus where snRNP-specific proteins associate. U1 snRNP consists of U1 snRNA, seven Sm proteins and three snRNP-specific proteins, U1-70K, U1A, and U1C. It has been shown previously that after import to the nucleus U2 and U4/U6 snRNP-specific proteins first appear in Cajal bodies (CB) and then in splicing speckles. In addition, in cells grown under normal conditions U2, U4, U5, and U6 snRNAs/snRNPs are abundant in CBs. Therefore, it has been proposed that the final assembly of these spliceosomal snRNPs takes place in this nuclear compartment. In contrast, U1 snRNA in both animal and plant cells has rarely been found in this nuclear compartment.

Methodology/Principal Findings

Here, we analysed the subnuclear distribution of Arabidopsis U1 snRNP-specific proteins fused to GFP or mRFP in transiently transformed Arabidopsis protoplasts. Irrespective of the tag used, U1-70K was exclusively found in the nucleus, whereas U1A and U1C were equally distributed between the nucleus and the cytoplasm. In the nucleus all three proteins localised to CBs and nucleoli although to different extent. Interestingly, we also found that the appearance of the three proteins in nuclear speckles differ significantly. U1-70K was mostly found in speckles whereas U1A and U1C in ∼90% of cells showed diffuse nucleoplasmic in combination with CBs and nucleolar localisation.

Conclusions/Significance

Our data indicate that CBs and nucleolus are involved in the maturation of U1 snRNP. Differences in nuclear accumulation and distribution between U1-70K and U1A and U1C proteins may indicate that either U1-70K or U1A and U1C associate with, or is/are involved, in other nuclear processes apart from pre-mRNA splicing.     

Cytoplasmic assembly of snRNP particles from stored proteins and newly transcribed snRNA's in L929 mouse fibroblasts

Newly synthesized snRNAs appear transiently in the cytoplasm where they assemble into ribonucleoprotein particles, the snRNP particles, before returning permanently to the interphase nucleus. In this report, bona fide cytoplasmic fractions, prepared by cell enucleation, are used for a quantitative analysis of snRNP assembly in growing mouse fibroblasts. The half-lives and abundances of the snRNP precursors in the cytoplasm and the rates of snRNP assembly are calculated in L929 cells. With the exception of U6, the major snRNAs are stable RNA species; U1 is almost totally stable while U2 has a half-life of about two cell cycles. In contrast, the majority of newly synthesized U6 decays with a half-life of about 15 h. The relative abundances of the newly synthesized snRNA species U1, U2, U3, U4 and U6 in the cytoplasm are determined by Northern hybridization using cloned probes and are approximately 2% of their nuclear abundance. The half-lives of the two major snRNA precursors in the cytoplasm (U1 and U2) are approximately 20 min as determined by labeling to steady state. The relative abundance of the snRNP B protein in the cytoplasm is determined by Western blotting with the Sm class of autoantibodies and is approximately 25% of the nuclear abundance. Kinetic studies, using the Sm antiserum to immunoprecipitate the methionine-labeled snRNP proteins, suggest that the B protein has a half-life of 90 to 120 min in the cytoplasm. These data are discussed and suggest that there is a large pool of more stable snRNP proteins in the cytoplasm available for assembly with the less abundant but more rapidly turning-over snRNAs.     

Functional organization of the Sm core in the crystal structure of human U1 snRNP

U1 small nuclear ribonucleoprotein (snRNP) recognizes the 5′‐splice site early during spliceosome assembly. It represents a prototype spliceosomal subunit containing a paradigmatic Sm core RNP. The crystal structure of human U1 snRNP obtained from natively purified material by in situ limited proteolysis at 4.4 Å resolution reveals how the seven Sm proteins, each recognize one nucleotide of the Sm site RNA using their Sm1 and Sm2 motifs. Proteins D1 and D2 guide the snRNA into and out of the Sm ring, and proteins F and E mediate a direct interaction between the Sm site termini. Terminal extensions of proteins D1, D2 and B/B′, and extended internal loops in D2 and B/B′ support a four‐way RNA junction and a 3′‐terminal stem‐loop on opposite sides of the Sm core RNP, respectively. On a higher organizational level, the core RNP presents multiple attachment sites for the U1‐specific 70K protein. The intricate, multi‐layered interplay of proteins and RNA rationalizes the hierarchical assembly of U snRNPs in vitro and in vivo.     

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.     

The determinants for Sm protein binding to Xenopus U1 and U5 snRNAs are complex and non-identical.

The Sm binding sites of different spliceosomal U small nuclear RNAs (snRNAs), the RNA structural elements required for interaction with common snRNP proteins, have been considered to be similar or identical. Here we show that this is not the case. Instead, structural and sequence features unique to U1 or U5 snRNAs that contribute to common protein binding are identified. The determinants of Sm protein binding in both RNAs are complex, consisting in U5 of minimally two and in U1 of minimally four separate structural elements. Even the most conserved features of the two RNAs, single-stranded regions whose generalized sequence is PuA(U)nGPu, are not functionally interchangeable in protein binding. At least one of the newly defined RNA elements functions in assembly with the common proteins, but is not required for their stable binding thereafter. U1, but not U5, snRNP requires a trimethyl guanosine cap structure for its transport to the nucleus. This is not a consequence of the differences in common snRNP binding to the two RNAs, but is due to structural features of U1 RNA that do not contribute to Sm protein binding.     

The Methylosome, a 20S Complex Containing JBP1 and pICln, Produces Dimethylarginine-Modified Sm Proteins

snRNPs, integral components of the pre-mRNA splicing machinery, consist of seven Sm proteins which assemble in the cytoplasm as a ring structure on the snRNAs U1, U2, U4, and U5. The survival motor neuron (SMN) protein, the spinal muscular atrophy disease gene product, is crucial for snRNP core particle assembly in vivo. SMN binds preferentially and directly to the symmetrical dimethylarginine (sDMA)-modified arginine- and glycine-rich (RG-rich) domains of SmD1 and SmD3. We found that the unmodified, but not the sDMA-modified, RG domains of SmD1 and SmD3 associate with a 20S methyltransferase complex, termed the methylosome, that contains the methyltransferase JBP1 and a JBP1-interacting protein, pICln. JBP1 binds SmD1 and SmD3 via their RG domains, while pICln binds the Sm domains. JBP1 produces sDMAs in the RG domain-containing Sm proteins. We further demonstrate the existence of a 6S complex that contains pICln, SmD1, and SmD3 but not JBP1. SmD3 from the methylosome, but not that from the 6S complex, can be transferred to the SMN complex in vitro. Together with previous results, these data indicate that methylation of Sm proteins by the methylosome directs Sm proteins to the SMN complex for assembly into snRNP core particles and suggest that the methylosome can regulate snRNP assembly.