A novel MECA3 region in human 3p21.3 harboring putative tumor suppressor genes and oncogenes.

BACKGROUND: Human chromosome arm 3p is often affected in various epithelial tumors, and several tumor suppressor genes were recently identified in this region. The most affected is 3p21 region that is 50-100% rearranged in more than 30 types of malignancies, mostly in epithelial cancers: lung, breast, ovarian, cervical, kidney, head and neck, nasopharyngeal, colon etc. These cancers are responsible for 90% of cancer deaths. AIM: To perform the detailed analysis of 3p (especially 3p21 region) to discover novel potential oncogenes and/or tumor suppressors. METHODS: To find novel "hot spots" and genes involved in major cancers, dense 3p microsatellite markers (altogether 24 ) were allelotyped in four epithelial carcinomas (272 patients in total): breast (BC), renal cell (RCC), non-small cell lung (NSCLC) and epithelial ovarian (EOC) cancers. RESULTS: As a main result, a novel region, frequently affected in BC, RCC, NSCLC and EOC was localized between markers D3S2409 and D3S3667 in the 3p21.3. This region (MECA3, major epithelial cancers affected region No. 3) covers numerous UniGene clusters, including genes involved in vital cell functions and carcinogenesis (e.g. MST1, MSTR1/RON, GPX1 and RHOA). The homozygous deletions were detected in the GPX1 in RCC (12%, 6 of 50 cases) and BC (1 of 37 cases). At the same time, amplifications and multiplications within the RHOA putative oncogene were identified in BC and RCC. CONCLUSIONS: The data suggest that genes with potential oncogenic features are located in the close proximity to putative tumor suppressor gene(s) (TSG(s)) in the MECA3. Multiplication of the RHOA was not reported before. Significant correlation of allelic alterations in the, AP20, MECA3 and LUCA regions with tumor progression was found for some common histological tumor subtypes (e.g. clear cell RCC, and serous EOC).

Human chromosome 3: integration of 60 NotI clones into a physical and gene map.

Sequence tagged sites generated for 60 NotI clones (NotI-STSs) from human chromosome 3-specific NotI-jumping and NotI-linking libraries were physically located using PCR screening of a radiation hybrid (RH) GeneBridge4 panel. The NotI map of chromosome 3 was generated using these RH-mapping data and those obtained earlier by FISH and sequencing of the corresponding NotI clones. The sequences of the NotI clones showed significant homologies with known genes and/or ESTs for 58 NotI-STSs (97%). These 58 NotI clones displayed 91-100% identity to 54 genes and 23 cDNA/EST clones. One known and two hypothetical protein-coding genes were localized for the first time and nine cDNA clones (unknown genes) were also carefully mapped only in this work. Three newly mapped genes are histone gene H1X (NR1-BK20C) and genes for hypothetical proteins THC1032178 and THC1024604 (NL1-243).

Translation termination in eukaryotes: from simplicity to complexity.

The last forty years of molecular biology and biochemistry were enormously rich in discoveries, which were not foreseen even by the most eminent scientists of that time. The findings of 1993-2000 profoundly enlarged our views on translation termination, clearly showing that our previous understanding was enormously oversimplified. Now the structural basis is created for much better insight into functions of termination factors. The story of translation termination in eukaryotes could be taken as an illustration of a general trend of molecular biology: "From simplicity to complexity". However, genuine knowledge requires that after this stage a third phase has to be reached, which is "From complexity to clarity". This has not yet been achieved in translation termination and therefore makes this topic quite attractive for researchers.

Structural rearrangements and insertions of dispersed elements in pericentromeric alpha satellites occur preferably at kinkable DNA sites.

Centromeric region of human chromosome 21 comprises two long alphoid DNA arrays: the well homogenized and CENP-B box-rich alpha21-I and the alpha21-II, containing a set of less homogenized and CENP-B box-poor subfamilies located closer to the short arm of the chromosome. Continuous alphoid fragment of 100 monomers bordering the non-satellite sequences in human chromosome 21 was mapped to the pericentromeric short arm region by fluorescence in situ hybridization (alpha21-II locus). The alphoid sequence contained several rearrangements including five large deletions within monomers and insertions of three truncated L1 elements. No binding sites for centromeric protein CENP-B were found. We analyzed sequences with alphoid/non-alphoid junctions selectively screened from current databases and revealed various rearrangements disrupting the regular tandem alphoid structure, namely, deletions, duplications, inversions, expansions of short oligonucleotide motifs and insertions of different dispersed elements. The detailed analysis of more than 1100 alphoid monomers from junction regions showed that the vast majority of structural alterations and joinings with non-alphoid DNAs occur in alpha satellite families lacking CENP-B boxes. Most analyzed events were found in sequences located toward the edges of the centromeric alphoid arrays. Different dispersed elements were inserted into alphoid DNA at kinkable dinucleotides (TG, CA or TA) situated between pyrimidine/purine tracks. DNA rearrangements resulting from different processes such as recombination and replication occur at kinkable DNA sites alike insertions but irrespectively of the occurrence of pyrimidine/purine tracks. It seems that kinkable dinucleotides TG, CA and TA are part of recognition signals for many proteins involved in recombination, replication, and insertional events. Alphoid DNA is a good model for studying these processes.

Suppression of eukaryotic translation termination by selected RNAs.

Using selection-amplification, we have isolated RNAs with affinity for translation termination factors eRF1 and eRF1.eRF3 complex. Individual RNAs not only bind, but inhibit eRF1-mediated release of a model nascent chain from eukaryotic ribosomes. There is also significant but weaker inhibition of eRF1-stimulated eRF3 GTPase and eRF3 stimulation of eRF1 release activity. These latter selected RNAs therefore hinder eRF1.eRF3 interactions. Finally, four RNA inhibitors of release suppress a UAG stop codon in mammalian extracts dependent for termination on eRF1 from several metazoan species. These RNAs are therefore new specific inhibitors for the analysis of eukaryotic termination, and potentially a new class of omnipotent termination suppressors with possible therapeutic significance.

Translational termination comes of age.

Translational termination has been a largely ignored aspect of protein synthesis for many years. However, the recent identification of new release-factor genes, the mapping of release-factor functional sites and in vitro reconstitution experiments have provided a deeper understanding of the termination mechanism. In addition, protein-protein interactions among release factors and with other proteins have been revealed. The three-dimensional structures of a prokaryotic ribosome recycling factor and eukaryotic release factor 1 (eRF1) mimic the shape of transfer RNA, indicating that they bind to the same ribosomal site. Post-termination events in bacteria have been clarified, linking termination, ribosomal recycling and translation initiation.

Translation termination in eukaryotes: polypeptide release factor eRF1 is composed of functionally and structurally distinct domains.

Class-1 polypeptide chain release factors (RFs) trigger hydrolysis of peptidyl-tRNA at the ribosomal peptidyl transferase center mediated by one of the three termination codons. In eukaryotes, apart from catalyzing the translation termination reaction, eRF1 binds to and activates another factor, eRF3, which is a ribosome-dependent and eRF1-dependent GTPase. Because peptidyl-tRNA hydrolysis and GTP hydrolysis could be uncoupled in vitro, we suggest that the two main functions of eRF1 are associated with different domains of the eRF1 protein. We show here by deletion analysis that human eRF1 is composed of two physically separated and functionally distinct domains. The "core" domain is fully competent in ribosome binding and termination-codon-dependent peptidyl-tRNA hydrolysis, and encompasses the N-terminal and middle parts of the polypeptide chain. The C-terminal one-third of eRF1 binds to eRF3 in vivo in the absence of the core domain, but both domains are required to activate eRF3 GTPase in the ribosome. The calculated isoelectric points of the core and C domains are 9.74 and 4.23, respectively. This highly uneven charge distribution between the two domains implies that electrostatic interdomain interaction may affect the eRF1 binding to the ribosome and eRF3, its activity in the termination reaction and activation of eRF3 GTPase. The positively charged core of eRF1 may interact with negatively charged rRNA and peptidyl-tRNA phosphate backbones at the ribosomal eRF1 binding site and exhibit RNA-binding ability. The structural and functional dissimilarity of the core and eRF3-binding domains implies that evolutionarily eRF1 originated as a product of gene fusion.

Analysis of NotI linking clones isolated from human chromosome 3 specific libraries.

We have partially sequenced more than 1000 NotI linking clones isolated from human chromosome 3-specific libraries. Of these clones, 152 were unique chromosome 3-specific clones. The clones were precisely mapped using a combination of fluorescence in situ hybridization (FISH) and hybridization to somatic cell or radiation hybrids. Two- and three-color FISH was used to order the clones that mapped to the same chromosomal region, and in some cases, chromosome jumping was used to resolve ambiguous mapping. When this NotI restriction map was compared with the yeast artificial chromosome (YAC) based chromosome 3 map, significant differences in several chromosome 3 regions were observed. A search of the EMBL nucleotide database with these sequences revealed homologies (90-100%) to more than 100 different genes or expressed sequence tags (ESTs). Many of these homologies were used to map new genes to chromosome 3. These results suggest that sequencing NotI linking clones, and sequencing CpG islands in general, may complement the EST project and aid in the discovery of all human genes by sequencing random cDNAs. This method may also yield information that cannot be obtained by the EST project alone; namely, the identification of the 5' ends of genes, including potential promoter/enhancer regions and other regulatory sequences

Prediction of eukaryotic mRNA translational properties.

MOTIVATION: It is well known that eukaryotic mRNAs are translated at different levels depending on their sequence characteristics. Evaluation of mRNA translatability is of importance in prediction of the gene expression pattern by computer methods and to improve the recognition of mRNAs within cloned nucleotide sequences. It may also be used in biotechnological experiments to optimize the expression of foreign genes in transgenic organisms. RESULTS: The sets of 5' untranslated region characteristics, significantly different between mRNAs encoding abundant and scarce polypeptides, were determined for mammals, dicot plants and monocot plants, and collected in the LEADER_RNA database. Computer tools for the prediction of mRNA translatability are presented. AVAILABILITY: Programs for mRNA translatability prediction are available at http://wwwmgs.bionet.nsc. ru/programs/acts2/mo_mRNA.htm (for monocots), http://wwwmgs.bionet. nsc.ru/programs/acts2/di_mRNA.htm (for dicots) and http://wwwmgs. bionet.nsc.ru/programs/acts2/ma_mRNA.htm (for mammals). The LEADER_RNA database may be accessed at: http://wwwmgs.bionet.nsc. ru/systems/LeaderRNA/.

Mutations in the highly conserved GGQ motif of class 1 polypeptide release factors abolish ability of human eRF1 to trigger peptidyl-tRNA hydrolysis.

Although the primary structures of class 1 polypeptide release factors (RF1 and RF2 in prokaryotes, eRF1 in eukaryotes) are known, the molecular basis by which they function in translational termination remains obscure. Because all class 1 RFs promote a stop-codon-dependent and ribosome-dependent hydrolysis of peptidyl-tRNAs, one may anticipate that this common function relies on a common structural motif(s). We have compared amino acid sequences of the available class 1 RFs and found a novel, common, unique, and strictly conserved GGQ motif that should be in a loop (coil) conformation as deduced by programs predicting protein secondary structure. Site-directed mutagenesis of the human eRF1 as a representative of class 1 RFs shows that substitution of both glycyl residues in this motif, G183 and G184, causes complete inactivation of the protein as a release factor toward all three stop codons, whereas two adjacent amino acid residues, G181 and R182, are functionally nonessential. Inactive human eRF1 mutants compete in release assays with wild-type eRF1 and strongly inhibit their release activity. Mutations of the glycyl residues in this motif do not affect another function, the ability of eRF1 together with the ribosome to induce GTPase activity of human eRF3, a class 2 RF. We assume that the novel highly conserved GGQ motif is implicated directly or indirectly in the activity of class 1 RFs in translation termination.

Structure of the human CpG-island-containing lung Kruppel-like factor (LKLF) gene and its location in chromosome 19p13.11-13 locus.

We have cloned earlier a short human genomic fragment which showed strong similarity with the mouse cDNA encoding lung Kruppel-like zinc finger transcription factor (LKLF), predominantly expressed in mouse developing lung, spleen, and vascular system, which might play a key role in programming the quiescent state of single positive T cells and blood vessel wall morphogenesis. Here we report the successful cloning of the human LKLF cDNA, its genomic structure and chromosomal localization at the 19p13.11-p13.13 locus. The full-length human LKLF cDNA has longer 5'-UTR with higher GC content than mouse cDNA and encodes a predicted protein of 355 amino acids which has three zinc fingers at the C-terminus and a proline-rich N-terminal domain. Human and mouse proteins share 87.3% identity and 90.2% amino acid similarity. The human LKLF gene consists of three exons. From the proximal promoter to the end of the second exon, we have found a CpG island with an average 76% GC content and two regions of unusually high GC density.

Amino acid composition of protein termini are biased in different manners.

An exhaustive statistical analysis of the amino acid sequences at the carboxyl (C) and amino (N) termini of proteins and of coding nucleic acid sequences at the 5' side of the stop codons was undertaken. At the N ends, Met and Ala residues are over-represented at the first (+1) position whereas at positions 2 and 5 Thr is preferred. These peculiarities at N-termini are most probably related to the mechanism of initiation of translation (for Met) and to the mechanisms governing the life-span of proteins via regulation of their degradation (for Ala and Thr). We assume that the C-terminal bias facilitates fixation of the C ends on the protein globule by a preference for charged and Cys residues. The terminal biases, a novel feature of protein structure, have to be taken into account when molecular evolution, three-dimensional structure, initiation and termination of translation, protein folding and life-span are concerned. In addition, the bias of protein termini composition is an important feature which should be considered in protein engineering experiments.

Termination of translation in eukaryotes: new results and new hypotheses.

Important new results obtained in studies of prokaryotic and eukaryotic translation termination during 1994-1998 are reviewed. Properties of the newly discovered factors RF3, eRF1, and eRF3 are described. Similarity and difference between prokaryotic and eukaryotic systems of translation termination and recent models of molecular mechanisms of protein synthesis at the termination stage are discussed. Hypotheses concerning the biological role of eRF3 are formulated and discussed.

C-terminal domains of human translation termination factors eRF1 and eRF3 mediate their in vivo interaction.

At the termination step of protein synthesis, hydrolysis of the peptidyl-tRNA is jointly catalysed at the ribosome by the termination codon and the polypeptide release factor (eRF1 in eukaryotes). eRF1 forms in vivo and in vitro a stable complex with release factor eRF3, an eRF1-dependent and ribosome-dependent GTPase. The role of the eRF1-eRF3 complex in translation remains unclear. We have undertaken a systematic analysis of the interactions between the human eRF1 and eRF3 employing a yeast two-hybrid assay. We show that the N-terminal parts of eRF1 (positions 1-280) and of eRF3 (positions 1477) are either not involved or non-essential for binding. Two regions in each factor are critical for mutual binding: positions 478-530 and 628-637 of eRF3 and positions 281-305 and 411-415 of eRF1. The GTP binding domain of eRF3 is not involved in complex formation with eRF1. The GILRY pentamer (positions 411-415) conserved in eukaryotes and archaebacteria is critical for eRF1's ability to stimulate eRF3 GTPase. The human eRF1 lacking 22 C-terminal amino acids remains active as a release factor and promotes an eRF3 GTPase activity whereas C-terminally truncated eRF3 is inactive as a GTPase.

Translation termination and yeast prions.

Protein biosynthesis is the final step in the transfer of genetic information in the cell. In turn, its last step is the release of a nascent polypeptide from the ribosome. Therefore, termination of translation may be considered (if we do not take into account protein post-translational modification and folding) as a final step of the transition from genotype to phenotype through the classic DNA--RNA--protein pathway. In a narrow sense, termination of translation is the hydrolytic cleavage of peptidyl-tRNA into free tRNA and completed polypeptide chain carrying all the information encoded in the corresponding mRNA and DNA. Then the completed protein molecule is released from the ribosome and the ribosome dissociates into its components (subunits, factors, mRNA, tRNA, etc. ). After the synthesis is completed, the polypeptide chain is folded either cotranslationally or by an additional specialized mechanism, depending on the nature of the protein, organism, and other factors. This issue of Biochemistry (Moscow) highlights from various points of view the problem of translation termination, excluding protein folding. Yeast termination factors with prion-like properties are also considered.

Functional expression of eukaryotic polypeptide chain release factors 1 and 3 by means of baculovirus/insect cells and complex formation between the factors.

Translation termination in eukaryotes is governed by termination codons in mRNA and two release factors, eRF1 and eRF3. In this work, human eRF1 and eRF3 have been produced in insect cells using a recombinant baculovirus expression system for the corresponding human cDNAs. Purification of eRF1 has led to a homogeneous 50-kDa protein active in promoting ribosome-dependent and termination-codon-dependent hydrolysis of formylmethionyl-tRNAf(Met). Purification of eRF3 yielded a full-length protein and shorter polypeptides. Microsequencing of the N-terminus of the shortest form detected a site of proteolytic cleavage between Arg91 and Gly92, probably due to exposed region(s) hypersensitive to proteolysis. The mixture of full-length and truncated forms of eRF3 as well as bacterially expressed eRF3 lacking 138 N-terminal amino acids (eRF3Cp) are active as an eRF1-dependent and ribosome-dependent GTPase and in stimulating the GTP-dependent release activity of eRF1. Complex formation between eRF1 and eRF3Cp was demonstrated by affinity and gel-filtration chromatographies and by native-gel electrophoresis. An abnormal electrophoretic mobility observed for eRF1 as compared with the complex points to a significant conformational change of either eRF1 or both factors in the complex. Co-expression of both factors in baculovirus-infected insect cells and a yeast two-hybrid assay were applied to monitor complex formation in vivo. In yeast cells, both eRF1 and eRF3 are either in a monomeric or in a heterodimeric but not in a homodimeric state.

Diadenosine oligophosphates (Ap(n)A), a novel class of signalling molecules?

The diadenosine oligophosphates (Ap(n)A) were discovered in the mid-sixties in the course of studies on aminoacyl-tRNA synthetases (aaRS). Now, more than 30 years later, about 300 papers have been published around these substances in attempt to decipher their role in cells. Recently, Ap(n)A have emerged as intracellular and extracellular signalling molecules implicated in the maintenance and regulation of vital cellular functions and become considered as second messengers. Great variety of physiological and pathological effects in mammalian cells was found to be associated with alterations of Ap(n)A levels (n from 2 to 6) and Ap3A/Ap4A ratio. Cell differentiation and apoptosis have substantial and opposite effects on Ap3A/Ap4A ratio in cultured cells. A human Ap3A hydrolase, Fhit, appeared to be involved in protection of cells against tumourigenesis. Ap3A is synthesised by mammalian u synthetase (TrpRS) which in contrast to most other aaRS is unable to synthesise Ap4A and is an interferon-inducible protein. Moreover, Ap3A appeared to be a preferred substrate for 2-5A synthetase, also interferon-inducible, priming the synthesis of 2' adenylated derivatives of Ap3A, which in turn may serve as substrates of Fhit. Tumour suppressor activity of Fhit is assumed to be associated with involvement of the Fhit.Ap3A complex in cytokine signalling pathway(s) controlling cell proliferation. The Ap(n)A family is potentially a novel class of signal-transducing molecules whose functions are yet to be determined.

Eukaryotic mRNAs encoding abundant and scarce proteins are statistically dissimilar in many structural features.

It is well known that non-coding mRNA sequences are dissimilar in many structural features. For individual mRNAs correlations were found for some of these features and their translational efficiency. However, no systematic statistical analysis was undertaken to relate protein abundance and structural characteristics of mRNA encoding the given protein. We have demonstrated that structural and contextual features of eukaryotic mRNAs encoding high- and low-abundant proteins differ in the 5' untranslated regions (UTR). Statistically, 5' UTRs of low-expression mRNAs are longer, their guanine plus cytosine content is higher, they have a less optimal context of the translation initiation codons of the main open reading frames and contain more frequently upstream AUG than 5' UTRs of high-expression mRNAs. Apart from the differences in 5' UTRs, high-expression mRNAs contain stronger termination signals. Structural features of low- and high-expression mRNAs are likely to contribute to the yield of their protein products.

Transfer RNA(Phe) isoacceptors possess non-identical set of identity elements at high and low Mg2+ concentration.

Primary structures of phage T5- and Escherichia coli-encoded tRNA(Phe) are distinct at four out of 11 positions known as identity elements for E. coli phenylalanyl-tRNA synthetase (FRS). In order to reveal structural requirements for FRS recognition, aminoacylation of wild-type phage T5 tRNA(Phe) gene transcript and mutants containing substitutions of the identity elements at positions 20, 34, 35 and 36 was compared with E. coli tRNA(Phe) gene transcript. The wild-type phage T5 transcript can be aminoacylated with the same catalytic efficiency as the E. coli counterpart. However, the maximal aminoacylation rate for T5 and E. coli transcripts was reached at different Mg2+ concentrations: 4 and 15 mM, respectively. Aminoacylation assays with tRNA(Phe) mutants revealed that (i) phage transcripts with the substituted anticodon bases at positions 35 and 36 were efficient substrates for aminoacylation at 15 mM Mg2+ but not at optimal 4 mM Mg2+; (ii) any change of G34 in phage transcripts dramatically decreased the aminoacylation efficiency at both 4 and 15 mM Mg2+ whereas G34A mutation in the E. coli transcript exhibits virtually no influence on aminoacylation rate at 15 mM Mg2+; (iii) substitution of A20 with U in the phage transcript caused no significant change in the aminoacylation rate at both Mg2+ concentrations; (iv) phage transcripts with double substitutions A20U+A35C and A20U+A36C were very poor substrates for FRS. Collectively, the results indicate the non-identical mode of tRNA(Phe) recognition by E. coli FRS at low and high Mg2+ concentrations. Probably, along with identity elements, the local tRNA conformation is essential for recognition by FRS.

Human chromosome 3: high-resolution fluorescence in situ hybridization mapping of 40 unique NotI linking clones homologous to genes and cDNAs.

Forty new NotI linking clones representing sequence tagged sites (STSs) were mapped by fluorescence in situ hybridization (FISH) to different regions of human chromosome 3 (HSA3). Clone NL1-245, containing human aminoacylase 1, was localized to 3p21.2-p21.1. Our previous localization of the CLC-2 chloride channel protein gene was refined to 3q27. Clone NL2-316 most likely contains a translocon-associated protein gamma-subunit gene and was mapped to 3q23-q24. To our knowledge, this is the first time this gene has been mapped. One NotI linking clone (NL1-229) probably contains a new protein phosphatase gene. This clone was mapped to 3p25. Five NotI linking clones probably contain human expressed sequence tags (ESTs), as they possess sequences with a high level of identity (> 90%) to cDNA clones. Other clones show 56-85% homology to known mammalian and human genes with various functions, including oncogenes and tumour-suppressor genes. These clones might represent new genes.