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In order to determine whether there is a general difference in the splicing mechanism of animal and plant pre-mRNAs, we cloned part of the gene for the small subunit of the ribulose 1,5-bisphosphate carboxylase containing both introns into the SP64 vector. RNA was synthesized with SP6 polymerase and used as substrate for in vitro processing in a HeLa cell nuclear splicing extract. Analyses of the processed RNA demonstrate that both introns of the plant pre-mRNA are efficiently removed in an ordered fashion yielding a faithfully ligated mRNA. Two branch points were identified for intron A and three for intron B. The branched nucleotides are adenosine residues in all cases and are located within a distance from the 3' splice site found to be crucial for lariat formation in animal pre-mRNAs. The implications of these results are discussed in light of our previous observation, that a functional pre-mRNA of the human growth hormone gene was not processed in plant tissue in vivo.

Genomic clones containing the closely related genes for human growth hormone (hGH) and chorionic somatomammotropin (hCS) were obtained from genomic bacteriophage lambda and cosmid libraries. The entire GH/CS chromosomal locus was reconstructed utilizing overlapping restriction fragments characterized from the isolated clones. The hGH/hCS locus contains two GH genes and three CS genes spanning 48 kb of DNA in the order: 5'-(hGH-1/hCS-5/hCS-1/hGH-2/hCS-2)-3', confirming analysis of cosmid clones obtained from a different human library (Barsh et al., 1983). To complete the characterization of the hCS genes, the nucleotide sequence of the hCS-5 gene was determined. Sequence analysis revealed a mutation of the 5' splice site at the exon II-intron B boundary, suggesting that the hCS-5 gene is a pseudogene. The nucleotide sequence of an allelic variant of the hCS-2 gene was determined and found to contain a single amino acid substitution and the deletion of a single codon. The hGH/hCS gene locus was further characterized by the localization of at least 27 Alu-type repetitive sequences and identification of three unique sequences in the vicinity of several hGH and hCS genes which define the probable breakpoints of the evolutionary duplication units. These data, combined with the nucleotide sequences of all five GH and CS genes, indicate that the hGH/hCS gene locus has evolved by duplication mechanisms. Evidence for the occurrence of at least one gene conversion event involving the hCS-1 gene precursor and the hCS-2 gene was found, indicating that the hGH/hCS gene locus has evolved by concerted mechanisms. The structure of the hCS genes is discussed in light of recent studies of CS genes from other mammalian species.

To study the sequence of events during processing of primary RNA transcripts and to gain more insight into the mechanism of splice site selection, the in vitro processing of a 2.5 kb human growth hormone (hGH) pre-mRNA containing four introns and an alternative 3' splice site for intron B was analysed. In order to process the hGH pre-mRNA the preparation of the HeLa cell nuclear extract had to be modified, indicating differences in factor requirement for processing this pre-mRNA. After an unusual long lag phase of one hour splicing intermediates begin to accumulate. Intron A and D are removed with correct ligation of exons 1/2 and 4/5. Most splice sites are used--albeit with variable efficiencies--except the splice sites surrounding exon 3 and the 3' alternative splice site within exon 3; as a consequence "exon skipping" events take place. Using a pre-mRNA containing only intron B neither the 5' nor the 3' splice site is cleaved, indicating that the 3' splice site of intron B is not recognized. The results show that splice sites can differ considerably in their requirement for splicing factors.

AcPhe-tRNAPhe from yeast can be photocross-linked to poly(U) on Escherichia coli ribosomes. The photoreaction occurs at the wybutine base situated next to the 3' side of the anticodon. The kinetics and efficiency of crosslinking of AcPhe-Phe-tRNA are the same at both the acceptor site and the peptidyl site. Therefore, the orientation of wybutine with respect to the mRNA is similar in both the pretranslocational and posttranslocational states. AcPhe-Phe-tRNA crosslinked at the acceptor site can still be translocated to the peptidyl site, demonstrating that tRNA and mRNA are transported together. The experiments support a model of translocation in which the conformation of the anticodon loop of tRNA is similar in both the peptidyl site and the acceptor site.

3-(4'-Benzoylphenyl)propionyl[3H] Phe-tRNA bound to the peptidyl site of the ribosome is photo-crosslinked exclusively to 23S RNA on irradiation at 320 nm. The site of reaction has been identified both by hybridization and primer-extension experiments as uridine-2584 and uridine-2585, located within the central loop of domain V according to the secondary structure model of 23S RNA. The fact that the covalently crosslinked tRNA retains its ability to form a peptide bond, together with the proximity of this site to the position of several mutations leading to chloramphenicol or erythromycin resistance strongly argue that this region of the 23S-like rRNAs is an integral component of the peptidyl transferase site. On the basis of these results, and from comparative analysis of the 16 available large subunit rRNA sequences, we propose a model for the functional organization of the peptidyl transferase site involving interaction of domains II and V of 23S rRNA.

Photo-reactive 3-(4'-benzoylphenyl)propionyl-Phe-tRNA bound to the A- or the P-site was crosslinked to 23S RNA upon irradiation at 320 nm. The sites of reaction were identified as U-2584 and U-2585 at the A-site and A-2451 and C-2452 at the P-site. Minor crosslinks from both sites were observed at nucleotides A-2503 to U-2506. All sites identified lie in close proximity according to the secondary structure model and constitute part of the highly conserved loop region of domain V. Antibiotics known to inhibit peptidyl transferase activity had a pronounced effect on photo-crosslinking. In addition, tetracycline was also shown to photo-crosslink to this region. These experiments permit a dissection of the peptidyl transferase region on the 23S RNA into two distinct areas for the A- and P-site.

The rat growth hormone gene was isolated on a cloned 11.4-kilobase EcoRI-generated DNA fragment from a bacteriophage "library" of chromosomal DNA. The structural gene sequence, approximately 2.1 kilobases long, was identified by hybridization to the corresponding cloned rat growth hormone cDNA and shown to contain four intervening sequences. The complete primary structure of the gene and the 5' and 3'-flanking regions was determined. The mosaic structure of exons and introns can be related to the different biological activities of growth hormone and to the evolution from ancestral sequences of a gene that was the precursor to the growth hormone and the related prolactin and placental lactogen (chorionic somatomammotropin) genes. The largest intron was found to contain a dispersed repetitive DNA sequence flanked by perfect 18-base pair direct repeats. The mobility of sequences of this kind could play a role in the observed variation of intron sizes and in rearrangements of mammalian genes.

To study whether mammalian RNA processing signals function in plants, we have constructed a chimaeric gene in which the complete human growth hormone (hGH) gene is flanked by DNA fragments containing the promoter and polyadenylation site of the nopaline synthase gene. The hGH gene used contains four introns and an additional 440 bp downstream from the hGH poly(A) addition site. The transcription of this chimaeric gene was studied following its introduction into sunflower and tobacco cells using a Ti plasmid vector. Analysis of poly(A)(+) RNA isolated from the transformed tumor tissue demonstrated the following: (1) a single polyadenylated transcript, 2700 bp in length, was transcribed from the chimaeric gene; (2) the transcription was initiated at the published start site of the nopaline synthase gene; (3) the hGH polyadenylation site was not used for processing of the 3' end; only the poly(A) addition site of the nopaline synthase gene was recognized, (4) no splicing of the hGH introns could be detected. We also demonstrate that the hGH pre-mRNA isolated from plant cells can be spliced in a HeLa cell nuclear extract, indicating that the hGH pre-mRNA was functional. These results show that processing signals of the hGH pre-mRNA are not recognized in these plant cells.

Upon irradiation, 3-[4-benzoylphenyl]propionyl-PhetRNA bound to the P-site of poly(U)-primed ribosomes is exclusively cross-linked to 23S RNA. It is shown that the photoreaction only occurs with pyrimidine nucleotides. The site of the cross-link is located within an 11S RNA fragment, which comprises the 1100 nucleotides at the 3′-end of 23S RNA. The cross-linked Phe tRNA derivative is still functionally active in peptide bond formation. The site labelled on the 11S fragment is therefore an integral part of the peptidyltransferase centre.

Photoreactive 3-(4’-benzoylphenyl)propionyl-Phe-tRNA was bound specifically to the P-site or the A-site of the E. coli ribosome. Photoreaction led to high yield crosslinking between the aminoacyl-terminus of Phe-tRNA and 23S RNA. The site of reaction was identified by hybridization and by taking advantage of the fact that reverse transcriptase stops one base before a modified nucleotide. The nucleotides labeled at the P-site were identified as A-2451 and C-2452. A-site specific labeling occurred at U-2584 and U-2585. Although distant in the primary sequence, these residues lie in close proximity within the central loop of domain V according to the secondary structure model of 23S RNA. Only antibiotics known to act at the peptidyl transferase site strongly inhibit the photoaffinity reaction, thus providing further evidence for the specificity of the labeling. The demonstration of specific, high yield crosslinks at the level of 23S RNA strongly supports the involvement of an RNA-catalyzed activity in the peptidyl transferase reaction.

Publisher Summary Oligonucleotide analysis is usually employed for identifying regions of RNA which have been modified in cross-linking experiments. However, the peculiar properties of the nucleotides modified by the aromatic ketone derivative made it impossible to isolate an oligonucleotide of sufficient length in pure form. As an alternative, the method of DNA–RNA hybridization for Southern blot analysis was used. The pattern of hybridizing fragments enabled to localize the site of reaction to a 183-nucleotide sequence. The technique of primer extension as a way of identifying the site of reaction on a RNA molecule is based on the observation that reverse transcriptase stops one nucleotide before encountering a modified base on the RNA template. An aminoacyl-tRNA attached to 23S RNA via a benzophenone–propionic acid residue will act as a barrier for reverse transcriptase, thereby generating cDNA strands of defined length. Stops in reverse transcription caused by regions of high secondary and/or tertiary structure can be corrected for by a control experiment using unmodified 23S RNA as a template.

Our understanding of the structure and function of ribosomal RNA has evolved rapidly during the past few years. Complete primary structures for 16S, 23S, and 5S rRNA (and their analogs) from a wide range of organisms and organelles are now available, as are accurate secondary structure models that are helping us to understand the higher order folding of these molecules (reviewed in Woese et al., 1983; Brimacombe et al., 1983; Ebel et al., 1983; Noller, 1984). Current efforts in this area are focused on understanding tertiary and quaternary structure of rRNA and the nature of its involvement in protein synthesis.

Publisher Summary Several derivatives of aminoacyl-tRNA have been studied as affinity labels for the ribosomal peptidyltransferase center. Attachment of the reactive substituent can be obtained most easily by allowing the amino group of the aminoacyl moiety to react with N-hydroxysuccinimide esters according to the method of Rappaport and Lapidot. For this reason most analogs studied until now are acyl derivatives of aminoacyl-tRNA. One of the main obstacles faced in these studies are the low yields of the affinity labeling reaction usually attained. This problem is particularly serious in the case of photoaffinity labels. The chapter describes the synthesis of aromatic ketone derivatives of aminoacyl-tRNA. It explains the method that has been employed for the synthesis of 3-benzoylpropionyl- and 3- (4-benzoylphenyl) propionyl-Phe-tRNA but can be applied to other systems as well.

Crosslinking between phenylalanyl transfer RNA from brewer's yeast and poly(U) can be obtained when ribosomal complexes are irradiated at > 300 nm; the ribosomal complexes were formed by enzymatic binding of the tRNA. The crosslinked product was isolated on oligo(dA)cellulose columns. The experiments suggest that the photo-crosslinking occurs via the wybutine in the anticodon loop of the tRNA. This system can be used to study interactions between tRNA and messenger RNA on the ribosome. The structure of yeast phenylalanine transfer RNA (tRNAPhe) in the crystalline state has been resolved by X-ray analysis [I - 31. Studies of tRNA in aqueous solution by various physicochemical techniques and by chemical modifications are consistent with the notion that tRNA in solution assumes a configuration similar or identical to that of the structure in the crystal [4-71. Much less is known about the configuration of the tRNA when it is bound to the ribosome. In addition to binding of anticodon and messenger RNA (mRNA), specific nucleotide interactions between the tRNA and the ribosome are believed to occur. The T-$-C sequence in tRNA seems to be involved in the interaction with the ribosome [8,9]. This paper describes photo-crosslinking between tRNA and mRNA on the ribosome. Irradiation of ribosomal complexes containing Phe-tRNAPhe from brewer's yeast with ultraviolet light (> 300 nm) results in crosslinking between the tRNAPhe and poly(U). Crosslinking seems to occur at the wybutine situated next to the 3' side of the anticodon of tRNAPhe. This photo-crosslinking reaction is of potential interest for the study of codonanticodon interactions on the ribosome. Other authors have also observed photoreactivity of the wybutine.

The effects of various concentrations of cadmium on Nicotiana tabacum L. cv. Xanthi suspension cells were examined. Surprisingly, certain concentrations of Cd (100–150 μM) stimulated growth of cell cultures considerably, whereas all other concentrations were inhibitory. Synthesis of DNA was severly affected in a dose-dependent manner by Cd concentrations of 250 μM and higher. In contrast, RNA and protein synthesis were similarly stimulated by 100 μM Cd, thus indicating that enhancement of RNA synthesis was the primary cause for the observed stimulation of cell culture growth. The transient expression of a chimeric chloramphenicol-acetyltransferase gene was similarly affected by Cd. When the effects of other heavy metals (Cu, Zn, Pb, Co, Mn, Al) on these cellular processes were investigated, only Zn showed a comparable stimulation of RNA and protein synthesis, although a tenfold higher concentration of Zn compared with Cd was required. As Zn and Cd are chemically very similar, these results are discussed in view of the well-known role of Zn in the regulation of transcription.

SR proteins are a family of splicing factors important for splice site recognition and spliceosome assembly. Their ability to bind to RNA and to interact with proteins as well identifies them as important players in splice site choice and alternative splicing. Plants possess twice as many SR proteins as animals, and some of the subfamilies are plant specific. Arabidopsis SR proteins are involved in different aspects of plant growth and development as well as in responses to environmental cues. The plant-specific subfamilies have been shown to be regulated by alternative splicing events, which are highly conserved in evolution. The tight regulation of splicing factors by alternative splicing might allow coordinated responses of their target genes.

The proteins in a living cell are synthesized on a large bipartite ribonucleoprotein complex termed the ribosome. The peptidyl transferase, which polymerizes amino acids to yield peptides, is localized on the large subunit. Biochemical investigations over the past 35 years have led to the hypothesis that rRNA has a major role in all ribosomal functions. The recent high resolution X-ray structures of the ribosomal subunits clearly demonstrated that peptidyl transfer is an RNA-mediated process. As all ribosomal activities are dependent on bivalent metal ions, as is the case for most ribozymes, we investigated metal-ion-binding sites in rRNA by metal-ion-cleavage reactions. Some cleavage sites are near active sites and are evolutionarily highly conserved. The structure of the active site is flexible and undergoes changes during translocation and activation of the ribosome. Using modified P-site substrates, we showed that the 2′-OH group of the terminal adenosine is important for peptidyl transfer. These substrates were also used to investigate the metal ion dependency of the peptidyl transferase reaction.

Intron A of the human growth hormone gene does not contain an A residue within 56 nucleotides preceding the 3' splice site. The analysis of the excised intron lariat revealed a C residue 28 nucleotides upstream from the 3' splice site as the major branch acceptor nucleotide. Two additional minor branched nucleotides were identified as U residues at positions -22 and -36. An adenosine substitution at position -22 results in lariat formation solely to this nucleotide. Therefore, C and U residues can function efficiently as natural branch acceptors, but an A residue is preferred if available in the proper region. In addition, the data strongly reinforce the importance of the distance constraint for lariat formation. To explain selection of the branch acceptor nucleotide, potential base-pairing interactions of branch point sequences with the U2 RNA are discussed.

The recently described anti-A2/RA33 autoantibodies occur in 20-40% of patients with RA, SLE, and mixed connective tissue disease (MCTD). They are directed to the A2 protein of the heterogeneous nuclear ribonucleoprotein complex (hnRNP-A2), an abundant nuclear protein associated with the spliceosome. The NH2-terminal half of the antigen contains two conserved RNA binding domains whereas its COOH-terminal part is extremely glycine-rich. The aim of this study was to characterize the autoepitopes of hnRNP-A2 and to investigate the effects of anti-A2/RA33 autoantibodies on possible functions of the antigen. Using bacterially expressed fragments, two major discontinuous epitopes were identified. One containing the complete second RNA binding domain was recognized by the majority of patients with RA and SLE but not by patients with MCTD. The second epitope contained sequences of both RNA binding domains and was preferentially targeted by patients with MCTD. When the RNA binding properties of the antigen were investigated, oligoribonucleotides containing the sequence motif r(UUAG) were found to bind to a site closely adjacent or overlapping with the epitope targeted by autoantibodies from patients with RA and SLE. Moreover, anti-A2/RA33 autoantibodies from patients with RA or SLE, but not from patients with MCTD, inhibited binding of RNA. Thus, anti-A2/RA33 autoantibodies recognize conformation-dependent epitopes located in a functionally important region of the antigen. Furthermore, the specific recognition of an epitope by MCTD patients may be used as another argument in favor of considering MCTD a distinct connective tissue disease.

The sequence of one of the two major expressed human chorionic somatomammotropin genes (hCS-1) was determined. The hCS-1 gene and the human growth hormone gene (hGH-1) share 92% nucleotide sequence homology in their 5'- and 3'-flanking regions, introns and exons. This finding, in addition to the existence of multiple closely linked hGH and hCS genes, suggests that these genes are evolving by concerted mechanisms. S1 nuclease, hybridization, and primer extension analysis of placental poly(A+) RNA demonstrated the presence of two functional initiation sites within the hCS-1 and/or the hCS-2 gene(s). The majority (about 95%) of the transcripts initiate 30 nucleotides downstream from the TATAAA sequence, and about 5% of the transcripts initiate 30 nucleotides downstream from a CATAAA sequence located 55 nucleotides 5' to the TATAAA sequence. The presence of two functional promoter elements as well as direct repeated sequences flanking the TATAAA sequence and exon I of the hCS genes are consistent with the hypothesis (Cooke, N. E., and Baxter, J. D. (1982) Nature (Lond.) 297, 603-606) that an important regulatory element may have been inserted into the gene in a separate evolutionary event. An analysis of other direct repeats, internal homology, and homology between other growth hormone and chorionic somatomammotropin genes offers a more extensive conceptualization of how this gene family evolved.