Cloning of the cDNA for U1 small nuclear ribonucleoprotein particle 70K protein from Arabidopsis thaliana.

We cloned and sequenced a plant cDNA that encodes U1 small nuclear ribonucleoprotein (snRNP) 70K protein. The plant U1 snRNP 70K protein cDNA is not full length and lacks the coding region for 68 amino acids in the amino-terminal region as compared to human U1 snRNP 70K protein. Comparison of the deduced amino acid sequence of the plant U1 snRNP 70K protein with the amino acid sequence of animal and yeast U1 snRNP 70K protein showed a high degree of homology. The plant U1 snRNP 70K protein is more closely related to the human counter part than to the yeast 70K protein. The carboxy-terminal half is less well conserved but, like the vertebrate 70K proteins, is rich in charged amino acids. Northern analysis with the RNA isolated from different parts of the plant indicates that the snRNP 70K gene is expressed in all of the parts tested. Southern blotting of genomic DNA using the cDNA indicates that the U1 snRNP 70K protein is coded by a single gene.     

A specific 31-nucleotide domain of U1 RNA directly interacts with the 70K small nuclear ribonucleoprotein component.

We have defined the nucleotide sequence of a protein-binding domain within U1 RNA that specifically recognizes and binds both to a U1 small nuclear ribonucleoprotein component (the 70K protein) and to the previously defined RNA-binding domain of the 70K protein. We have investigated direct interactions between purified U1 RNA and 70K protein by reconstitution in vitro. Thirty-one nucleotides of U1 RNA, corresponding to stem-loop I, were required for this interaction. Nucleotides at the 5' end of U1 RNA that are involved in base pairing with the 5' splice site of pre-mRNA were not required for binding. In contrast to other reports, these findings demonstrate that a specific domain of U1 RNA can bind directly to the 70K protein independently of any other snRNP-associated proteins.     

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.     

C-reactive protein reacts with the U1 small nuclear ribonucleoprotein

C-reactive protein (CRP) was found to produce a small, discrete, speckled fluorescence pattern in the nucleus of HEp-2 cells. Double staining with anti-RNP serum and CRP produced very similar staining patterns. By counterimmunoelectrophoresis CRP was bound to extractable nuclear antigens found in rabbit thymus extract. The reactive components of the extract were only partially sensitive to treatment with RNase. CRP immunoprecipitated the U1 RNA species from [32P]labeled HeLa cells and the protein bands of the Sm/RNP complex from [35S]-methionine-labeled HeLa cells. By blotting, CRP bound to several discrete bands in a calcium-dependent, PC-inhibitable manner. Two of the bands comigrated with the 70K protein band associated with the U1 snRNP, and its major breakdown product. Binding to these bands was inhibited by both EDTA and PC indicating that CRP binds these proteins through the PC-binding site. Binding to the 70K protein of the U1 snRNP was confirmed by reactivity with the recombinant 70K protein in a dot blot. These findings indicate the CRP binds to the U1-RNP snRNP particle. Considering the ability of CRP to inhibit antibody responses to its ligands and its ability to activate C and promote phagocytosis it is suggested that CRP may play a role in the regulation of autoantibody responses to nuclear Ag.     

The plant U1 small nuclear ribonucleoprotein particle 70K protein interacts with two novel serine/arginine-rich proteins.

The U1 small nuclear ribonucleoprotein particle (U1 snRNP) 70K protein (U1-70K), one of the three U1 snRNP-specific proteins, is implicated in basic and alternative splicing of nuclear pre-mRNAs. We have used the Arabidopsis U1-70K in the yeast two-hybrid system to isolate cDNAs encoding proteins that interact with it. This screening has resulted in the isolation of two novel plant serine/arginine-rich (SR) proteins, SRZ-22 and SRZ-21 (SRZ proteins). Neither the N-terminal region nor the arginine-rich C-terminal region of U1-70K alone interact with the SRZ proteins. The interaction of U1-70K with the SRZ proteins is confirmed further in vitro using a blot overlay assay. The plant SRZ proteins are highly similar to each other and contain conserved modular domains unique to different groups of splicing factors in the SR family of proteins. SRZ proteins are similar to human 9G8 splicing factor because they contain a zinc knuckle, precipitate with 65% ammonium sulfate, and cross-react with the 9G8 monoclonal antibody. However, unlike the 9G8 splicing factor, SRZ proteins contain a glycine hinge, a unique feature in other splicing factors (SC35 and ASF/SF2), located between the RNA binding domain and the zinc knuckle. SRZ-22 and SRZ-21 are encoded by two distinct genes and are expressed in all tissues tested with varied levels of expression. Our results suggest that the plant SRZ proteins represent a new group of SR proteins. The interaction of plant U1-70K with the SRZ proteins may account for some differences in pre-mRNA splicing between plants and animals.     

The U1 small nuclear ribonucleoprotein (snRNP) 70K protein is transported independently of U1 snRNP particles via a nuclear localization signal in the RNA-binding domain.

Expression of the recombinant human U1-70K protein in COS cells resulted in its rapid transport to the nucleus, even when binding to U1 RNA was debilitated. Deletion analysis of the U1-70K protein revealed the existence of two segments of the protein which were independently capable of nuclear localization. One nuclear localization signal (NLS) was mapped within the U1 RNA-binding domain and consists of two typically separated but interdependent elements. The major element of this NLS resides in structural loop 5 between the beta 4 strand and the alpha 2 helix of the folded RNA recognition motif. The C-terminal half of the U1-70K protein which was capable of nuclear entry contains two arginine-rich regions, which suggests the existence of a second NLS. Site-directed mutagenesis of the RNA recognition motif NLS demonstrated that the U1-70K protein can be transported independently of U1 RNA and that its association with the U1 small nuclear ribonucleoprotein particle can occur in the nucleus.     

A three-dimensional model of the U1 small nuclear ribonucleoprotein particle

Most of the pre-mRNAs in the eukaryotic cell are comprised of protein-coding exons and non-protein-coding introns. The introns are removed and the exons are ligated together, or spliced, by a large, macromolecular complex known as the spliceosome. This RNA-protein assembly is made up of five uridine-rich small nuclear RNAs (U1-, U2-, U4-, U5- and U6-snRNA) as well over 300 proteins, which form small nuclear ribonucleoprotein particles (snRNPs). Initial recognition of the 5′ exon/intron splice site is mediated by the U1 snRNP, which is composed of the U1 snRNA as well as at least ten proteins. By combining structural informatics tools with the available biochemical and crystallographic data, we attempted to simulate a complete, three dimensional U1 snRNP from the silk moth, Bombyx mori. Comparison of our model with empirically derived crystal structures and electron micrographs pinpoints both the strengths and weaknesses in the in silico determination of macromolecular complexes. One of the most striking differences between our model and experimentally generated structures is in the positioning of the U1 snRNA stem-loops. This highlights the continuing difficulties in generating reliable, complex RNA structures; however, three-dimensional modeling of individual protein subunits by threading provided models of biological significance and the use of both automated and manual docking strategies generated a complex that closely reflects the assembly found in nature. Yet, without utilizing experimentally-derived contacts to select the most likely docking scenario, ab initio docking would fall short of providing a reliable model. Our work shows that the combination of experimental data with structural informatics tools can result in generation of near-native macromolecular complexes.     

Identification of molecular contacts between the U1 A small nuclear ribonucleoprotein and U1 RNA.

We recently determined the crystal structure of the RNP domain of the U1 small nuclear ribonucleoprotein A and identified Arg and Lys residues involved in U1 RNA binding. These residues are clustered around the two highly conserved segments, RNP1 and RNP2, located in the central two beta strands. We have now studied the U1 RNA binding of mutants where potentially hydrogen bonding residues on the RNA binding surface were replaced by non-hydrogen bonding residues. In the RNP2 segment, the Thr11----Val and Asn15----Val mutations completely abolished, and the Tyr13----Phe and Asn16----Val mutations substantially reduced the U1 RNA binding, suggesting that these residues form hydrogen bonds with the RNA. In the RNP1 segment Arg52----Gln abolished, but Arg52----Lys only slightly affected U1 RNA binding, suggesting that Arg52 may form a salt bridge with phosphates of U1 RNA. Ethylation protection experiments of U1 RNA show that the backbone phosphates of the 3' two-thirds of loop II and the 5' stem are in contact with the U1 A protein. The U1 A protein-U1 RNA binding constant is substantially reduced by A----G and G----A replacements in loop II, but not by C----U or U----C replacements. Based on these biochemical data we propose a structure for the complex between the U1 A ribonucleoprotein and U1 RNA.     

Ser/Arg-rich protein-mediated communication between U1 and U2 small nuclear ribonucleoprotein particles

Previous work demonstrated that U1 small nuclear ribonucleoprotein particle (snRNP), bound to a downstream 5' splice site, can positively influence utilization of an upstream 3' splice site via exon definition in both trans- and cis-splicing systems. Although exon definition results in the enhancement of splicing of an upstream intron, the nature of the factors involved has remained elusive. We assayed the interaction of U1 snRNP as well as the positive effect of a downstream 5' splice site on trans-splicing in nematode extracts containing either inactive (early in development) or active (later in development) serine/arginine-rich splicing factors (SR proteins). We have determined that U1 snRNP interacts with the 5' splice site in the downstream exon even in the absence of active SR proteins. In addition, we determined that U1 snRNP-directed loading of U2 snRNP onto the branch site as well as efficient trans-splicing in these inactive extracts could be rescued upon the addition of active SR proteins. Identical results were obtained when we examined the interaction of U1 snRNP as well as the requirement for SR proteins in communication across a cis-spliced intron. Weakening of the 3' splice site uncovered distinct differences, however, in the ability of U1 snRNP to promote U2 addition, dependent upon its position relative to the branch site. These results demonstrate that SR proteins are required for communication between U1 and U2 snRNPs whether this interaction is across introns or exons.     

Conserved amino acid residues within and outside of the N-terminal ribonucleoprotein motif of U1A small nuclear ribonucleoprotein involved in U1 RNA binding

By the use of hybrids between a U1 small nuclear ribonucleoprotein (snRNP: U1A) and a U2 snRNP (U2B") we have identified regions containing 29 U1A-specific amino acid residues scattered throughout the 117 N-terminal residues of the protein, which are involved in binding to U1 RNA. The U1A-specific amino acid residues have been arbitrarily divided into seven contiguous groups. None of these groups is sufficient for U1 binding when transferred singly into the U2B" context, and none of the groups is essential for U1 binding in U1A. Several different combinations of two or more groups can, however, confer the ability to bind U1 RNA to U2B", suggesting that most or all of the U1A-specific amino acid residues contribute incrementally to the strength of the specific binding interaction. Further evidence for the importance of the U1A-specific amino acid residues, some of which lie outside the region previously shown to be sufficient for U1 RNA binding, is obtained by comparison of the sequence of human and Xenopus laevis U1A cDNAs. These are extremely similar (94.4% identical) between amino acid residues 7 and 114 but much less conserved immediately upstream and downstream from this region.     

Loop I of U1 small nuclear RNA is the only essential RNA sequence for binding of specific U1 small nuclear ribonucleoprotein particle proteins.

The binding of the U1 small nuclear ribonucleoprotein (snRNP)-specific proteins C, A, and 70K to U1 small nuclear RNA (snRNA) was analyzed. Assembly of U1 snRNAs from bean and soybean and a set of mutant Xenopus U1 snRNAs into U1 snRNPs in Xenopus egg extracts was studied. The ability to bind proteins was analyzed by immunoprecipitation with monospecific antibodies and by a protein-sequestering assay. The only sequence essential for binding of the U1-specific proteins was the conserved loop sequence in the 5' hairpin of U1. Further analysis suggested that protein C binds directly to the loop and that the assembly of proteins A and 70K into the RNP requires mainly protein-protein interactions. Protein C apparently recognizes a specific RNA sequence rather than a secondary structural element in the RNA.     

U1 small nuclear ribonucleoprotein particle-specific proteins interact with the first and second stem-loops of U1 RNA, with the A protein binding directly to the RNA independently of the 70K and Sm proteins.

The U1 small nuclear ribonucleoprotein particle (U1 snRNP), a cofactor in pre-mRNA splicing, contains three proteins, termed 70K, A, and C, that are not present in the other spliceosome-associated snRNPs. We studied the binding of the A and C proteins to U1 RNA, using a U1 snRNP reconstitution system and an antibody-induced nuclease protection technique. Antibodies that reacted with the A and C proteins induced nuclease protection of the first two stem-loops of U1 RNA in reconstituted U1 snRNP. Detailed analysis of the antibody-induced nuclease protection patterns indicated the existence of relatively long-range protein-protein interactions in the U1 snRNP, with the 5' end of U1 RNA and its associated specific proteins interacting with proteins bound to the Sm domain near the 3' end. UV cross-linking experiments in conjunction with an A-protein-specific antibody demonstrated that the A protein bound directly to the U1 RNA rather than assembling in the U1 snRNP exclusively via protein-protein interactions. This conclusion was supported by additional experiments revealing that the A protein could bind to U1 RNA in the absence of bound 70K and Sm core proteins.     

T cell immunity in connective tissue disease patients targets the RNA binding domain of the U1-70kDa small nuclear ribonucleoprotein

Although the T cell dependence of autoimmune responses in connective tissue diseases has been well established, limited information exists regarding the T cell targeting of self Ags in humans. To characterize the T cell response to a connective tissue disease-associated autoantigen, this study generated T cell clones from patients using a set of peptides encompassing the entire linear sequence of the 70-kDa subunit of U1 snRNP (U1-70kDa) small nuclear ribonucleoprotein. Despite the ability of U1-70kDa to undergo multiple forms of Ag modification that have been correlated with distinct clinical disease phenotypes, a remarkably limited and consistent pattern of T cell targeting of U1-70kDa was observed. All tested T cell clones generated against U1-70kDa were specific for epitopes within the RNA binding domain (RBD) of the protein. High avidity binding of the RBD with U1-RNA was preserved with the disease-associated modified forms of U1-70kDa tested. The high avidity interaction between the U1-RBD on the polypeptide and U1-RNA may be critical in immune targeting of this region in autoimmunity. The T cell autoimmune response to U1-70kDa appears to have less diversity than is seen in the humoral response; and therefore, may be a favorable target for therapeutic intervention.     

Gel electrophoretic isolation of splicing complexes containing U1 small nuclear ribonucleoprotein particles.

Assembly of splicing precursor RNAs into ribonucleoprotein particle (RNP) complexes during incubation in in vitro splicing extracts was monitored by a new system of RNP gel electrophoresis. The temporal pattern of assembly observed by our system was identical to that obtained by other gel and gradient methodologies. In contrast to the results obtained by other systems, however, we observed requirements of U1 small nuclear RNPs (snRNPs) and 5' splice junction sequences for formation of specific complexes and retention of U1 snRNPs within gel-fractionated complexes. Single-intron substrate RNAs rapidly assembled into slow-migrating complexes. The first specific complex (A) appeared within a minute of incubation and required ATP, 5' and 3' precursor RNA consensus sequences, and intact U1 and U2 RNAs for formation. A second complex (B) containing precursor RNA appeared after 15 min of incubation. Lariat-exon 2 and exon 1 intermediates first appeared in this complex, operationally defining it as the active spliceosome. U4 RNA was required for appearance of complex B. Released lariat first appeared in a complex of intermediate mobility (A') and subsequently in rapidly migrating diffuse complexes. Ligated product RNA was observed only in fast-migrating complexes. U1 snRNPs were detected as components of gel-isolated complexes. Radiolabeled RNA within the A and B complexes was immunoprecipitated by U1-specific antibodies under gel-loading conditions and from gel-isolated complexes. Therefore, the RNP antigen remained associated with assembled complexes during gel electrophoresis. In addition, 5' splice junction sequences within gel-isolated A and B complexes were inaccessible to RNase H cleavage in the presence of a complementary oligonucleotide. Therefore, nuclear factors that bind 5' splice junctions also remained associated with 5' splice junctions under our gel conditions.     

Evolutionary conservation of the U7 small nuclear ribonucleoprotein in Drosophila melanogaster

The U7 snRNP involved in histone RNA 3' end processing is related to but biochemically distinct from spliceosomal snRNPs. In vertebrates, the Sm core structure assembling around the noncanonical Sm-binding sequence of U7 snRNA contains only five of the seven standard Sm proteins. The missing Sm D1 and D2 subunits are replaced by U7-specific Sm-like proteins Lsm10 and Lsm11, at least the latter of which is important for histone RNA processing. So far, it was unknown if this special U7 snRNP composition is conserved in invertebrates. Here we describe several putative invertebrate Lsm10 and Lsm11 orthologs that display low but clear sequence similarity to their vertebrate counterparts. Immunoprecipitation studies in Drosophila S2 cells indicate that the Drosophila Lsm10 and Lsm11 orthologs (dLsm10 and dLsm11) associate with each other and with Sm B, but not with Sm D1 and D2. Moreover, dLsm11 associates with the recently characterized Drosophila U7 snRNA and, indirectly, with histone H3 pre-mRNA. Furthermore, dLsm10 and dLsm11 can assemble into U7 snRNPs in mammalian cells. These experiments demonstrate a strong evolutionary conservation of the unique U7 snRNP composition, despite a high degree of primary sequence divergence of its constituents. Therefore, Drosophila appears to be a suitable system for further genetic studies of the cell biology of U7 snRNPs.     

U1-small nuclear ribonucleoprotein activates the NLRP3 inflammasome in human monocytes

The NOD-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome is a caspase-1-containing cytosolic protein complex that is essential for processing and secretion of IL-1β. The U1-small nuclear ribonucleoprotein (U1-snRNP) that includes U1-small nuclear RNA is a highly conserved intranuclear molecular complex involved in splicing pre-mRNA. Abs against this self nuclear molecule are characteristically found in autoimmune diseases like systemic lupus erythematosus, suggesting a potential role of U1-snRNP in autoimmunity. Although endogenous DNA and microbial nucleic acids are known to activate the inflammasomes, it is unknown whether endogenous RNA-containing U1-snRNP could activate this molecular complex. In this study, we show that U1-snRNP activates the NLRP3 inflammasome in CD14(+) human monocytes dependently of anti-U1-snRNP Abs, leading to IL-1β production. Reactive oxygen species and K(+) efflux were responsible for this activation. Knocking down the NLRP3 or inhibiting caspase-1 or TLR7/8 pathway decreased IL-1β production from monocytes treated with U1-snRNP in the presence of anti-U1-snRNP Abs. Our findings indicate that endogenous RNA-containing U1-snRNP could be a signal that activates the NLRP3 inflammasome in autoimmune diseases like systemic lupus erythematosus where anti-U1-snRNP Abs are present.