snRNP Sm proteins share two evolutionarily conserved sequence motifs which are involved in Sm protein-protein interactions.

The spliceosomal small nuclear ribonucleoproteins (snRNPs) U1, U2, U4/U6 and U5 share eight proteins B', B, D1, D2, D3, E, F and G which form the structural core of the snRNPs. This class of common proteins plays an essential role in the biogenesis of the snRNPs. In addition, these proteins represent the major targets for the so-called anti-Sm auto-antibodies which are diagnostic for systemic lupus erythematosus (SLE). We have characterized the proteins F and G from HeLa cells by cDNA cloning, and, thus, all human Sm protein sequences are now available for comparison. Similar to the D, B/B' and E proteins, the F and G proteins do not possess any of the known RNA binding motifs, suggesting that other types of RNA-protein interactions occur in the snRNP core. Strikingly, the eight human Sm proteins possess mutual homology in two regions, 32 and 14 amino acids long, that we term Sm motifs 1 and 2. The Sm motifs are evolutionarily highly conserved in all of the putative homologues of the human Sm proteins identified in the data base. These results suggest that the Sm proteins may have arisen from a single common ancestor. Several hypothetical proteins, mainly of plant origin, that clearly contain the conserved Sm motifs but exhibit only comparatively low overall homology to one of the human Sm proteins, were identified in the data base. This suggests that the Sm motifs may also be shared by non-spliceosomal proteins. Further, we provide experimental evidence that the Sm motifs are involved, at least in part, in Sm protein-protein interactions. Specifically, we show by co-immunoprecipitation analyses of in vitro translated B' and D3 that the Sm motifs are essential for complex formation between B' and D3. Our finding that the Sm proteins share conserved sequence motifs may help to explain the frequent occurrence in patient sera of anti-Sm antibodies that cross-react with multiple Sm proteins and may ultimately further our understanding of how the snRNPs act as auto-antigens and immunogens in SLE.

Analysis of genomic clones of the murine U1RNA-associated 70-kDa protein reveals a high evolutionary conservation of the protein between human and mouse.

We have isolated and characterised two overlapping lambda EMBL3 clones carrying sequences of the gene for the murine U1RNA-associated 70-kDa protein. The two clones cover around 23 kb of the 70-kDa protein gene including its 3' end. Southern blot hybridisation revealed the existence of a single copy of the 70-kDa protein gene in the mouse genome. The 23-kb-long portion of the 70-kDa protein gene is divided into eight exons. While most of the exons are quite small and are widely scattered throughout the DNA sequence, the last one consists of about 830 bp and encodes 226 amino acids of the 70-kDa protein, including the C-terminus. The predicted amino acid sequence of the region of the 70-kDa protein encoded by the genomic clones reveals high conservation of structure when it is compared with the sequence of the human 70-kDa protein. Interestingly, all deletions, additions and substitutions are localised exclusively within the C-terminus of the protein, accounting for a 5'-3' polarity with respect to protein conservation. Moreover, the analysis of the genomic sequences predicts the existence of multiple subclasses of mRNAs that may arise by alternative pre-mRNA splicing. A 72-bp alternative exon harboring an in-frame termination codon was also found in the mouse 70-kDa gene and shows, surprisingly, 100% nucleotide identity to its human counterpart.

At least three distinct B cell epitopes reside in the C-terminal half of La protein, as determined by a recombinant DNA approach.

The La antigen is a nuclear protein that is one of the major target antigens of autoantibodies found in the sera of patients with primary Sjögren's syndrome. The formation of such autoantibodies is therefore likely to reflect the basic immunopathogenesis of this disorder. A recombinant DNA strategy has been used to examine the La protein for sequences that encode autoimmunizing B cell epitopes. We have isolated and characterized a 1.2-kb-long cDNA from a human liver cDNA library encoding a region of the La protein; this region contains 296 amino acids, including the C terminus. A sub-library of recombinant DNA in the expression vector pEX was made from portions of the La cDNA. Individual fusion proteins were tested by immunoblotting and enzyme-linked immunosorbent assay for their ability to react with anti-La autoantibodies contained in sera from patients with Sjögren's syndrome. In this way, we have identified at least three distinct epitopes in the C-terminal half of the La protein. Every anti-La serum tested contained antibodies against all three of the antigenic regions identified. Furthermore, most of the sera display similar ratios between the titers of antibodies with the three kinds of specificity. Our data suggest that the production of anti-La autoantibodies may be antigen driven.

Decoding at the ribosomal A site: antibiotics, misreading and energy of aminoacyl-tRNA binding.

The binding of Phe-tRNAPhe at the programmed ribosomal A site has been investigated using antibiotics that influence this binding in different ways. The adhesion of Phe-tRNAPhe, the consumption of GTP and the extent of the peptidyl transfer reaction were monitored. All of the five known misreading-inducing antibiotics that were tested stabilised the binding of Phe-tRNAPhe after its affixture to the A site by EF-Tu with GTP hydrolysis. The stabilisation was sufficient to overcome a single mismatch in the codon-anticodon interaction. Combinations of stabilising and destabilising influences were found to be additive, thus supporting the concepts: (1) that there is a 'correct' binding energy for aminoacyl tRNA in the A site, whose reduction hampers polypeptide synthesis and whose increase makes it inaccurate by by-passing proofreading; and (2) that the different antibiotics affect the bound aminoacyl tRNA at different points.

5' cleavage site in eukaryotic pre-mRNA splicing is determined by the overall 5' splice region, not by the conserved 5' GU.

We have generated all possible single point mutations of the invariant 5' GT of the large beta-globin intron and determined their effect on splicing in vitro. None of the mutants prevented cleavage in the 5' splice region, but many reduced or abolished exon joining. The mutations GT----TT and GT----CT resulted in a shift of the 5' cleavage site on nucleotide upstream; in the case of the mutation GT----TT, this shift was reverted by a second site mutation within the 5' splice region. Our results suggest that the 5' cleavage site is determined not by the conserved GU sequence but by the 5' splice region as a whole, most probably via base-pairing to the 5' end of the U1 snRNA.

Competition between tetracycline and tRNA at both P and A sites of the ribosome of Escherichia coli.

Fluorescence anisotropy studies performed on 6-demethylchlortetracycline, binding to the ribosome of E. coli in competition with tRNA at the P site or at both P and A sites, have provided a quantitative assessment in situ of the interaction of this antibiotic with the A site and have demonstrated that there is also an interaction between tetracycline and the P site.

Effect of mutations at the lariat branch acceptor site on beta-globin pre-mRNA splicing in vitro.

Introns are excised from full-length transcripts (pre-messenger RNAs) of eukaryotic genes in two steps. First, the pre-mRNA is cleaved at the 5' splice site and a branched (lariat) intermediate is formed. Then, cleavage at the 3' splice site and ligation of the two exons leads to the release of the lariat intron. The intron sequence which accepts the 5' end to form the lariat branch is strictly conserved in yeast, but shows more variation in eukaryotes. To investigate the requirements for branch formation in eukaryotes further, we have studied in vitro splicing of a rabbit globin gene intron with mutations of the normal branch-accepting adenosine nucleotide. We conclude that all four nucleotides can serve as branch acceptors, but that A and C are preferred to G and U in lariat formation. Mutation of the normal A to G or U can lead to an A residue one nucleotide upstream of the normal branch site being used instead. Only branches to A or C participate efficiently in the second splicing step.

Sequence requirements for splicing of higher eukaryotic nuclear pre-mRNA.

We determined the effect on splicing of 24 point mutations in the 5' and 3' splice region of the large rabbit beta-globin intron. In vitro, 3' AG mutations drastically reduce 5' cleavage and abolish splicing. In vivo, the same mutations elicit efficient splicing at a cryptic, rather than the correct, 3' splice site. In vitro, mutations at all but 2 positions of the consensus 5' splice region impair correct splicing and promote joining of exon 1 to exon 3. In vivo, the same mutations show no effect, except for those converting 5' GT to AT or GA, which cause accumulation of lariat intermediate in vitro and in vivo. We conclude that the 5' GT need not be conserved for 5' cleavage and that it plays an important role in cleavage and exon joining at the 3' splice site.

Nonconsensus branch-site sequences in the in vitro splicing of transcripts of mutant rabbit beta-globin genes.

Mutants of the rabbit beta-globin gene lacking the natural site of branch formation in the second intervening sequence have been analyzed for in vitro splicing activity. RNAs transcribed from these mutants were spliced, via lariat formation, at a reduced rate compared to wild-type RNA. The sites of branch formation were mapped by direct RNA analysis and primer-extension analysis. The sequences at the branch sites in the three mutants examined did not conform to the previously determined consensus sequence, nor were the 5' splice sites and branch sites complementary.

Decoding at the ribosomal A site. The effect of a defined codon-anticodon mismatch upon the behavior of bound aminoacyl transfer RNA.

Ribosomes from Escherichia coli were programmed by being allowed to bind a molecule of tRNAMetf or fMet-tRNAMetf and the hexanucleotide messenger AUGN1N2N3. The interaction of the ternary complex [EF-Tu X GTP X Phe-tRNAPhe] with the A site (containing the codon N1N2N3) was then studied by measuring the extent of (i) the binding of Phe-tRNAPhe to the ribosome, (ii) the hydrolysis of GTP, and (iii) the formation of the dipeptide fMet-Phe. By variation of N1,N2, and N3, a defined degree and position of mismatch could be obtained; the correct A-site codon UUU was compared with the incorrect codons CUU, UCU, GUU, and UUG. Each single-point alteration led to catalytic hydrolysis of GTP and to a strong reduction in the amounts of Phe-tRNAPhe binding and of dipeptide formation. The observations were explicable qualitatively by a hypothesis according to which the behavior of the bound aa-tRNA, after hydrolysis of GTP and before peptidyl transfer, is determined principally by the energy of binding of the aminoacyl-tRNA to the A site. This binding in turn was found to depend upon both the nature and the position of the mismatch. The results further suggest a steric interplay between the 3' (acceptor) end of the A-site tRNA and the second and third positions of the anticodon, so that a mismatch at one of these positions can impair directly the interaction between the aminoacylated 3' end and the ribosome and can thus reduce the rate of peptide bond formation and contribute to the overall fidelity of the elongation cycle.

Recognition of termination codon by release factor in the presence of a tRNA-occupied A site. Evidence for flexibility in accommodation of the release factor on the ribosome.

Recognition of the termination codon by release factor was studied with 70 S ribosomes containing initiator tRNA at the P site and an amino acid-specifying codon as well as the corresponding cognate tRNA at the A site. In vitro termination was not excluded when either the A site was occupied with an amino acid-specifying codon or the termination codon was displaced from the A site codon position by spacer nucleotides between UAA and the P site bound AUG. The release factor may have flexibility in its codon recognizing domain to be able to adjust to this enforced change in the position of the termination codon on the ribosome without loss of functional specificity. Deacylated tRNA bound at the A site of 70 S ribosomes did not interfere with stoichiometric UAA-directed release factor binding to these particles. In contrast, a ternary complex (aminoacyl-tRNA elongation factor-Tu . GTP) abolished the binding of the release factor while aminoacyl-tRNA inhibited it only weakly. This suggests that release factor and elongation factor-Tu have overlapping binding sites but the A site binding domains of the release factor and tRNA are exclusive.

Effect of codon shortening and the antibiotics viomycin and sparsomycin upon the behaviour of bound aminoacyl-tRNA. Decoding at the ribosomal A site.

70 S ribosomes were programmed with initiator tRNA and messenger oligonucleotides AUG(U)n and AUG(C)n, where n = 1, 2 or 3. The binding of the ternary complexes [Phe-tRNA X EF-Tu X GTP] and [Pro-tRNA X EF-Tu X GTP] to the programmed ribosomes was studied. If codon-anticodon interaction is restricted to only one basepair, the ternary complex leaves the ribosome before GTP hydrolysis. Two basepairs allow hydrolysis of GTP, but the aminoacyl-tRNA dissociates and is recycled, resulting in wastage of GTP. Three basepairs result in apparently stable binding of aminoacyl-tRNA to the ribosome. The antibiotic sparsomycin weakens the binding by an amount roughly equivalent to one messenger base, while viomycin has the reverse effect.