U1 Small Nuclear Ribonucleoprotein Particle Research Articles

Differential block of U small nuclear ribonucleoprotein particle interactions during in vitro splicing of adenovirus E1A transcripts containing abnormally short introns.

We have studied the consequences of decreasing the donor site-branch site distance on splicing factor-splice site interactions by analyzing alternative splicing of adenovirus E1A pre-mRNAs in vitro. We show that the proximal 13S donor site has a cis-inhibiting effect on the 9S and 12S mRNA reactions when it is brought too close to the common branch site, suggesting that the factor interactions in the common 3' part of the intron are impaired by the U1 small nuclear ribonucleoprotein particle (snRNP) binding to the displaced 13S donor site. Further analysis of the interactions was carried out by studying complex assembly and the accessibility to micrococcal nuclease digestion of 5'-truncated E1A substrates containing only splice sites for the 13S mRNA reaction. A deletion which brings the donor site- branch site distance to 49 nucleotides, which is just below the minimal functional distance, results in a complete block of the U4-U5-U6 snRNP binding, whereas a deletion 15 nucleotides larger results in a severe inhibition of the formation of the U2 snRNP-containing complexes. Sequence accessibility analyses performed by using the last mini-intron-containing transcript demonstrate that the interactions of U2 snRNP with the branch site are strongly impaired whereas the initial bindings of U1 snRNP to the donor site and of specific factors to the 3' splice site are not significantly modified. Our results strongly suggest that the interaction of U1 snRNP with the donor site of a mini-intron is stable enough in vitro to affect the succession of events leading to U2 snRNP binding with the branch site.

Differential Block of U Small Nuclear Ribonucleoprotein Particle Interactions during In Vitro Splicing of Adenovirus E1A Transcripts Containing Abnormally Short Introns

We have studied the consequences of decreasing the donor site-branch site distance on splicing factor-splice site interactions by analyzing alternative splicing of adenovirus E1A pre-mRNAs in vitro. We show that the proximal 13S donor site has a cis-inhibiting effect on the 9S and 12S mRNA reactions when it is brought too close to the common branch site, suggesting that the factor interactions in the common 3' part of the intron are impaired by the U1 small nuclear ribonucleoprotein particle (snRNP) binding to the displaced 13S donor site. Further analysis of the interactions was carried out by studying complex assembly and the accessibility to micrococcal nuclease digestion of 5'-truncated E1A substrates containing only splice sites for the 13S mRNA reaction. A deletion which brings the donor site- branch site distance to 49 nucleotides, which is just below the minimal functional distance, results in a complete block of the U4-U5-U6 snRNP binding, whereas a deletion 15 nucleotides larger results in a severe inhibition of the formation of the U2 snRNP-containing complexes. Sequence accessibility analyses performed by using the last mini-intron-containing transcript demonstrate that the interactions of U2 snRNP with the branch site are strongly impaired whereas the initial bindings of U1 snRNP to the donor site and of specific factors to the 3' splice site are not significantly modified. Our results strongly suggest that the interaction of U1 snRNP with the donor site of a mini-intron is stable enough in vitro to affect the succession of events leading to U2 snRNP binding with the branch site.

Definition of a C-reactive protein binding determinant on histones

C-reactive protein (CRP) is an acute phase inflammatory protein in man which binds to phosphocholine, chromatin, histones, and the 70-kDa protein of the U1 small nuclear ribonucleoprotein particle in a calcium-dependent, phosphocholine-inhibitable manner. CRP also binds to other proteins including fibronectin. The determinants involved in CRP binding to these diverse proteins have not been identified. The binding of CRP to histones was examined as these proteins are available in large quantity at high purity and subject to protease digestion with well characterized products. Histone H1 was digested with thrombin and trypsin to produce three distinct fragments, N-terminal, central globular, and C-terminal. CRP was shown only to bind to the C-terminal fragment. Binding to histone H2A was also examined. CRP binding was not diminished by cleavage of the C-terminal fragment but was greatly decreased when the central globular region of H2A was tested. Peptides were prepared to be identical to the N- and C-terminal fragments of H2A. The N-terminal (15 amino acid) fragment of H2A blocked CRP-induced precipitation of phosphocholine-coupled bovine serum albumin and histone H2A, whereas the C-terminal fragment showed no inhibition. Thus we have defined the first reported CRP binding determinant on a protein.

The yeast PRP8 protein intersacts directly with pre-mRNA

The PRP8 protein of Saccharomyces cerevisiae is required for nuclear pre-mRNA splicing. Previously, immunological procedures demonstrated that PRP8 is a protein component of the U5 small nuclear ribonucleoprotein particle (U5 snRNP), and that PRP8 protein maintains a stable association with the spliceosome during both step 1 and step 2 of the splicing reaction. We have combined immunological analysis with a UV-crosslinking assay to investigate interaction(s) of PRP8 protein with pre-mRNA. We show that PRP8 protein interacts directly with splicing substrate RNA during in vitro splicing reactions. This contact event is splicing-specific in that it is ATP-dependent, and does not occur with mutant RNAs that contain 5' splice site or branchpoint mutations. The use of truncated RNA substrates demonstrated that the assembly of PRP8 protein into splicing complexes is not, by itself, sufficient for the direct interaction with the RNA; PRP8 protein only becomes UV-crosslinked to RNA substrates capable of participating in step 1 of the splicing reaction. We propose that PRP8 protein may play an important structural and/or regulatory role in the spliceosome.

Evidence for base-pairing between mammalian U2 and U6 small nuclear ribonucleoprotein particles.

Intramolecular and intermolecular snRNA cross-links were generated by irradiating HeLa nuclear extract with 365 nm light in the presence of the psoralen derivative AMT. After deproteinization, cross-linked RNAs were resolved by gel electrophoresis and identified as anomalously migrating species by Northern blotting. In addition to the U4/U6 snRNA cross-link, we detected an intermolecular U2/U6 cross-link, as well as several apparently intramolecular U1, U2, and U5 cross-links. Photoreversal of the U2/U6 cross-link with 254 nm irradiation released stoichiometric amounts of U2 and U6 snRNA. To localize the U2/U6 cross-link, the 3' end of U2 in the purified U2/U6 complex was labeled selectively using a novel oligonucleotide "splint" technique. The labeled U2/U6 complex was then subjected to rapid enzymatic RNA sequencing or to targeted digestion of the U2 and U6 components of the complex by RNase H and a panel of complementary oligonucleotides. The U2/U6 cross-link is located upstream of nucleotide 15 in U2 and downstream of nucleotide 85 in U6, suggesting that the phylogenetically conserved base-pairing between these regions (6 consecutive base pairs in human, Drosophila melanogaster, and Caenorhabditis elegans, 7 in Schizosaccharomyces pombe and Trypanasoma brucei, 8 in Pisum sativum, 11 in Saccharomyces cerevisiae) is significant.

Mechanism for cryptic splice site activation during pre-mRNA splicing.

The 5' splice site of a pre-mRNA is recognized by U1 small nuclear ribonucleoprotein particles (snRNP) through base pairing with the 5' end of U1 small nuclear RNA (snRNA). Single-base substitutions within a 9-nucleotide 5'-splice-site sequence can abolish or attenuate use of that site and, in higher eukaryotes, can also activate nearby "cryptic" 5' splice sites. Here we show that the effects of single-base substitutions within a 5' splice site can be completely or partially suppressed by cis mutations that improve the overall complementarity of the site to U1 snRNA. We further show that in the presence of the normal 5' splice site, a cryptic 5' splice site can be activated by increasing its complementarity to U1 snRNA. U1 snRNP binding experiments confirm that cryptic 5' splice sites are activated when their affinity for U1 snRNP approaches that of the authentic 5' splice site. Based upon these results, we propose a spliceosome competition model for 5'-splice-site selection and cryptic 5'-splice-site activation. We discuss our results with regard to the factors involved in 5'-splice-site recognition.

Structure and Expression of the Drosophila melanogaster Gene for the U1 Small Nuclear Ribonucleoprotein Particle 70K Protein

A genomic clone encoding the Drosophila U1 small nuclear ribonucleoprotein particle 70K protein was isolated by hybridization with a human U1 small nuclear ribonucleoprotein particle 70K protein cDNA. Southern blot and in situ hybridizations showed that this U1 70K gene is unique in the Drosophila genome, residing at cytological position 27D1,2. Polyadenylated transcripts of 1.9 and 3.1 kilobases were observed. While the 1.9-kilobase mRNA is always more abundant, the ratio of these two transcripts is developmentally regulated. Analysis of cDNA and genomic sequences indicated that these two RNAs encode an identical protein with a predicted molecular weight of 52,879. Comparison of the U1 70K proteins predicted from Drosophila, human, and Xenopus cDNAs revealed 68% amino acid identity in the most amino-terminal 214 amino acids, which include a sequence motif common to many proteins which bind RNA. The carboxy-terminal half is less well conserved but is highly charged and contains distinctive arginine-rich regions in all three species. These arginine-rich regions contain stretches of arginine-serine dipeptides like those found in transformer, transformer-2, and suppressor-of-white-apricot proteins, all of which have been identified as regulators of mRNA splicing in Drosophila melanogaster.

Structure and expression of the Drosophila melanogaster gene for the U1 small nuclear ribonucleoprotein particle 70K protein.

A genomic clone encoding the Drosophila U1 small nuclear ribonucleoprotein particle 70K protein was isolated by hybridization with a human U1 small nuclear ribonucleoprotein particle 70K protein cDNA. Southern blot and in situ hybridizations showed that this U1 70K gene is unique in the Drosophila genome, residing at cytological position 27D1,2. Polyadenylated transcripts of 1.9 and 3.1 kilobases were observed. While the 1.9-kilobase mRNA is always more abundant, the ratio of these two transcripts is developmentally regulated. Analysis of cDNA and genomic sequences indicated that these two RNAs encode an identical protein with a predicted molecular weight of 52,879. Comparison of the U1 70K proteins predicted from Drosophila, human, and Xenopus cDNAs revealed 68% amino acid identity in the most amino-terminal 214 amino acids, which include a sequence motif common to many proteins which bind RNA. The carboxy-terminal half is less well conserved but is highly charged and contains distinctive arginine-rich regions in all three species. These arginine-rich regions contain stretches of arginine-serine dipeptides like those found in transformer, transformer-2, and suppressor-of-white-apricot proteins, all of which have been identified as regulators of mRNA splicing in Drosophila melanogaster.

Affinity purification of spliceosomes reveals that the precursor RNA processing protein PRP8, a protein in the U5 small nuclear ribonucleoprotein particle, is a component of yeast spliceosomes.

Nuclear pre-mRNA splicing in Saccharomyces cerevisiae, as in higher eukaryotes, occurs in large RNA-protein complexes called spliceosomes. The small nuclear RNA components, U1, U2, U4, U5, and U6, have been extensively studied; however, very little is known about the protein components of yeast spliceosomes. Here we use antibodies against the precursor RNA processing protein PRP8, a protein component of the U5 small nuclear ribonucleoprotein particle, to detect its association with spliceosomes throughout the splicing reaction and in a post-splicing complex containing the excised intron. In addition, an indirect immunological approach has been developed that confirms the presence of precursor RNA processing protein PRP8 in isolated spliceosomes. This method has possible general application for the analysis of ribonucleoprotein particle complexes.

RNA processing and ribonucleoprotein assembly studied in vivo by RNA transfection.

We present a method for studying RNA processing and ribonucleoprotein assembly in vivo, by using RNA synthesized in vitro. SP6-transcribed 32P-labeled U2 small nuclear RNA precursor molecules were introduced into cultured human 293 cells by calcium phosphate-mediated uptake, as in standard DNA transfection experiments. RNase protection mapping demonstrated that the introduced pre-U2 RNA underwent accurate 3' end processing. The introduced U2 RNA was assembled into ribonucleoprotein particles that reacted with an antibody specific for proteins known to be associated with the U2 small nuclear ribonucleoprotein particle. The 3' end-processed, ribonucleoprotein-assembled U2 RNA accumulated in the nuclear fraction. When pre-U2 RNA with a 7-methylguanosine group at the 5' end was introduced into cells, it underwent conversion to a 2,2,7-trimethylguanosine cap structure, a characteristic feature of the U-small nuclear RNAs. Pre-U2 RNA introduced with an adenosine cap (Ap-ppG) also underwent processing, small nuclear ribonucleoprotein assembly, and nuclear accumulation, establishing that a methylated guanosine cap structure is not required for these steps in U2 small nuclear ribonucleoprotein biosynthesis. Beyond its demonstrated usefulness in the study of small nuclear ribonucleoprotein biosynthesis, RNA transfection may be of general applicability to the investigation of eukaryotic RNA processing in vivo and may also offer opportunities for introducing therapeutically targeted RNAs (ribozymes or antisense RNA) into cells.

A U1 Small Nuclear Ribonucleoprotein Particle with Altered Specificity Induces Alternative Splicing of an Adenovirus E1A mRNA Precursor

We have altered the specificity of U1 small nuclear RNA by replacing its 5' splice site recognition sequence (nucleotides 3 to 11) with sequences complementary to other regions of either the adenovirus E1A or the rabbit beta-globin mRNA precursor. We then used a HeLa cell transient expression assay to test whether such altered U1 small nuclear ribonucleoprotein particles (snRNPs) could interfere with splicing of the targeted mRNA precursors. The altered U1 snRNPs were able to cause novel splicing of the E1A mRNA precursor, minor changes in the ratio of E1A 12 to 13S mRNAs, and modest nuclear accumulation of beta-globin mRNA precursors with either one of the two introns removed. Most of the altered U1 snRNPs did not affect the level of mature cytoplasmic mRNA significantly, but in one case an altered U1 snRNP (alpha 1) whose intended target was located downstream from the adenovirus E1A 12S 5' splice site was able to reduce the level of cytoplasmic 12S mRNA by approximately 60% and that of 13S mRNA by 90%. This alpha 1 snRNP induced an additional E1A splice, resulting in the appearance of 10 and 11S E1A mRNAs normally found only late in adenovirus infection. Thus, a trans-acting factor can induce alternative splicing. Surprisingly, the effects of alpha 1 on E1A splicing were not abolished by deleting the intended target sequence on the mRNA precursor.

A U1 small nuclear ribonucleoprotein particle with altered specificity induces alternative splicing of an adenovirus E1A mRNA precursor.

We have altered the specificity of U1 small nuclear RNA by replacing its 5' splice site recognition sequence (nucleotides 3 to 11) with sequences complementary to other regions of either the adenovirus E1A or the rabbit beta-globin mRNA precursor. We then used a HeLa cell transient expression assay to test whether such altered U1 small nuclear ribonucleoprotein particles (snRNPs) could interfere with splicing of the targeted mRNA precursors. The altered U1 snRNPs were able to cause novel splicing of the E1A mRNA precursor, minor changes in the ratio of E1A 12 to 13S mRNAs, and modest nuclear accumulation of beta-globin mRNA precursors with either one of the two introns removed. Most of the altered U1 snRNPs did not affect the level of mature cytoplasmic mRNA significantly, but in one case an altered U1 snRNP (alpha 1) whose intended target was located downstream from the adenovirus E1A 12S 5' splice site was able to reduce the level of cytoplasmic 12S mRNA by approximately 60% and that of 13S mRNA by 90%. This alpha 1 snRNP induced an additional E1A splice, resulting in the appearance of 10 and 11S E1A mRNAs normally found only late in adenovirus infection. Thus, a trans-acting factor can induce alternative splicing. Surprisingly, the effects of alpha 1 on E1A splicing were not abolished by deleting the intended target sequence on the mRNA precursor.

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.

U1 Small Nuclear Ribonucleoprotein Particle-Specific Proteins Interact with the First and Second Stem-Loops of Ul 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.

Small nuclear RNA-associated proteins are immunologically related as revealed by mapping of autoimmune reactive B-cell epitopes.

Autoantibodies from a patient with systemic lupus erythematosus, which recognize U1 and U2 small nuclear ribonucleoprotein particles (snRNPs), were used to map B-cell autoepitopes on the U1 snRNP-specific A protein. This protein contains two regions that are highly similar to regions in the U2 snRNP-specific B" protein. A site termed epitope 2 maps in one such region and was found to react with antibodies cross-reactive between A and B". A second site, epitope 1, is situated in a proline-rich region that shows no homology with B". This epitope can bind three different autoantibodies with distinct specificities. Epitope 1-affinity-purified antibodies from different patients react with either (i) the A protein exclusively; (ii) proteins A, B'/B, a synthetic peptide for part of the N polypeptide, and an unidentified protein with a molecular mass of 50 kDa; or (iii) proteins A, B'/B, C, and the N-derived peptide. Comparison of the primary structures of proteins B'/B, N, and C reveals multiple epitope 1-like sequences in all of them. The possibility that these repeating regions act as immunogens in patients with autoimmune disease is discussed.

Electron microscopy of U1 small nuclear ribonucleoprotein particles: shape of the particle and position of the 5′ RNA terminus.

Small nuclear ribonucleoprotein particles U1 (U1 snRNPs) from HeLa cells were purified to homogeneity and examined in the electron microscope by negative staining with the double carbon film technique. A survey of several hundred views of the U1 snRNP particles revealed a main body about 8 nm in diameter with two characteristic protuberances 4-7 nm long and 3-4 nm wide. Anti-m3G antibodies were then used to define the site of the m3G base in the 5' cap of the U1 snRNA, close to the site where base-pairing occurs with the 5' splice sequence of pre-mRNA. The m3G base is located on the body of the U1 snRNP, close to one of the protuberances and approximately opposite the other.

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.

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.

Reconstituted U1 small nuclear ribonucleoprotein complex restores 5′ splice site cleavage activity

Functional reconstitution of U1 small nuclear ribonucleoprotein particle (U1 snRNP) was performed using in vitro transcribed U1 snRNA. Hela cell nuclear extract was depleted of its constituent snRNPs by centrifugation at 100,000 × g. The supernatant was devoid of snRNAs and lacked cleavage activity in splicing reactions using in vitro transcribed β-globin pre-mRNA as substrate. The resulting pellet which contained the snRNAs, retained 5′ splice site cleavage activity in a similar splicing reaction. Supplementation of the inactive supernatant fraction with in vitro transcribed U1 snRNA, partially restored 5′ splice site cleavage activity thereby demonstrating the specific requirement of U1 snRNP in the initial stage of pre-mRNA splicing.

The U2 small nuclear ribonucleoprotein particle as an autoantigen. Analysis with sera from patients with overlap syndromes.

We identified eight patients whose sera contained autoantibodies to the U2 small nuclear ribonucleoprotein (snRNP), an RNA protein particle involved in the splicing of newly transcribed messenger RNA. Each of these patients had an overlap syndrome that included features of either systemic lupus erythematosus (SLE), scleroderma, and/or polymyositis. We then used these sera to characterize the autoantigenic polypeptides of the U1 and U2 snRNP particles. In immunoblots, all sera contained antibodies to the B" polypeptide of the U2 snRNP. A subset of these sera that more effectively immunoprecipitated the native U2 particle contained an additional antibody system that recognized the A' polypeptide of this snRNP. Antibodies eluted from the B" protein bound the A polypeptide of the U1 snRNP and vice versa. Moreover, antibodies to the B" polypeptide were accompanied by antibodies to the 68K and C polypeptides of the U1 snRNP. Finally, the A' and B" polypeptides remained physically associated after the U2 particle was cleaved with RNase. Thus these sera contain multiple autoantibody systems that, at one level, target two physically associated antigenic polypeptides of the U2 particle and, at another, target two snRNP particles which are associated during the splicing of premessenger RNA. These linked autoantibody sets provide further evidence that intact macromolecular structures are targeted by the immune response in SLE and related diseases.