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).

Class-1 translation termination factors: invariant GGQ minidomain is essential for release activity and ribosome binding but not for stop codon recognition.

Previously, we have shown that all class-1 polypeptide release factors (RFs) share a common glycine-glycine-glutamine (GGQ) motif, which is critical for RF activity. Here, we subjected to site-directed mutagenesis two invariant amino acids, Gln185 and Arg189, situated in the GGQ minidomain of human eRF1, followed by determination of RF activity and the ribosome binding capacity for mutant eRF1. We show that replacement of Gln185 with polar amino acid residues causes partial inactivation of RF activity; Gln185Ile, Arg189Ala and Arg189Gln mutants are completely inactive; all mutants that retain partial RF activity respond similarly to three stop codons. We suggest that loss of RF activity for Gln185 and Arg189 mutants is caused by distortion of the conformation of the GGQ minidomain but not by damage of the stop codon recognition site of eRF1. Our data are inconsistent with the model postulating direct involvement of Gln185 side chain in orientation of water molecule toward peptidyl-tRNA ester bond at the ribosomal peptidyl transferase centre. Most of the Gln185 mutants exhibit reduced ability to bind to the ribosome, probably, to rRNA and/or (peptidyl)-tRNA(s). The data suggest that the GGQ motif is implicated both in promoting peptidyl-tRNA hydrolysis and binding to the ribosome.

Stop codon recognition in ciliates: Euplotes release factor does not respond to reassigned UGA codon.

In eukaryotes, the polypeptide release factor 1 (eRF1) is involved in translation termination at all three stop codons. However, the mechanism for decoding stop codons remains unknown. A direct interaction of eRF1 with the stop codons has been postulated. Recent studies focus on eRF1 from ciliates in which some stop codons are reassigned to sense codons. Using an in vitro assay based on mammalian ribosomes, we show that eRF1 from the ciliate Euplotes aediculatus responds to UAA and UAG as stop codons and lacks the capacity to decipher the UGA codon, which encodes cysteine in this organism. This result strongly suggests that in ciliates with variant genetic codes eRF1 does not recognize the reassigned codons. Recent hypotheses describing stop codon discrimination by eRF1 are not fully consistent with the set of eRF1 sequences available so far and require direct experimental testing.

Mg2+-induced tRNA folding.

Mg(2+)-induced folding of yeast tRNA(Phe) was examined at low ionic strength in steady-state and kinetic experiments. By using fluorescent labels attached to tRNA, four conformational transitions were revealed when the Mg(2+) concentration was gradually increased. The last two transitions were not accompanied by changes in the number of base pairs. The observed transitions were attributed to Mg(2+) binding to four distinct types of sites. The first two types are strong sites with K(diss) of 4 and 16 microM. The sites of the third and fourth types are weak with a K(diss) of 2 and 20 mM. Accordingly, the Mg(2+)-binding sites previously classified as "strong" and "weak" can be further subdivided into two subtypes each. Fluorescent transition I is likely to correspond to Mg(2+) binding to a unique strong site selective for Mg(2+); binding to this site causes only minor A(260) change. The transition at 2 mM Mg(2+) is accompanied by substantial conformational changes revealed by probing with ribonucleases T1 and V1 and likely enhances stacking of the tRNA bases. Fast and slow kinetic phases of tRNA refolding were observed. Time-resolved monitoring of Mg(2+) binding to tRNA suggested that the slow kinetic phase was caused by a misfolded tRNA structure formed in the absence of Mg(2+). Our results suggest that, similarly to large RNAs, Mg(2+)-induced tRNA folding exhibits parallel folding pathways and the existence of kinetically trapped intermediates stabilized by Mg(2+). A multistep scheme for Mg(2+)-induced tRNA folding is discussed.

The polypeptide chain release factor eRF1 specifically contacts the s(4)UGA stop codon located in the A site of eukaryotic ribosomes.

It has been shown previously [Brown, C.M. & Tate, W.P. (1994) J. Biol. Chem. 269, 33164-33170.] that the polypeptide chain release factor RF2 involved in translation termination in prokaryotes was able to photocrossreact with mini-messenger RNAs containing stop signals in which U was replaced by 4-thiouridine (s4U). Here, using the same strategy we have monitored photocrosslinking to eukaryotic ribosomal components of 14-mer mRNA in the presence of tRNA(f)(Met), and 42-mer mRNA in the presence of tRNA(Asp) (tRNA(Asp) gene transcript). We show that: (a) both 14-mer and 42-mer mRNAs crossreact with ribosomal RNA and ribosomal proteins. The patterns of the crosslinked ribosomal proteins are similar with both mRNAs and sensitive to ionic conditions; (b) the crosslinking patterns obtained with 42-mer mRNAs show characteristic modification upon addition of tRNA(Asp) providing evidence for appropriate mRNA phasing onto the ribosome. Similar changes are not detected with the 14-mer mRNA.tRNA(f)(Met) pairs; (c) when eukaryotic polypeptide chain release factor 1 (eRF1) is added to the ribosome.tRNA(Asp) complex it crossreacts with the 42-mer mRNA containing the s(4)UGA stop codon located in the A site, but not with the s(4)UCA sense codon; this crosslink involves the N-terminal and middle domains of eRF1 but not the C domain which interacts with eukaryotic polypeptide chain release factor 3 (eRF3); (d) addition of eRF3 has no effect on the yield of eRF1-42-mer mRNA crosslinking and eRF3 does not crossreact with 42-mer mRNA. These experiments delineate the in vitro conditions allowing optimal phasing of mRNA on the eukaryotic ribosome and demonstrate a direct and specific contact of 'core' eRF1 and s(4)UGA stop codon within the ribosomal A site.

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 factor aRF1 from the archaeon Methanococcus jannaschii is active with eukaryotic ribosomes.

Class-1 translation termination factors (release factors (RFs)) from Eukarya (eRF1) and Archaea (aRF1) exhibit a high degree of amino acid sequence homology and share many common motifs. In contrast to eRF1, function(s) of aRF1 have not yet been studied in vitro. Here, we describe for the first time the cloning and expression in Escherichia coli of the gene encoding the peptide chain RF from the hyperthermophilic archaeon Methanococcus jannaschii (MjaRF1). In an in vitro assay with mammalian ribosomes, MjaRF1, which was overproduced in E. coli, was active as a RF with all three termination codon-containing tetraplets, demonstrating the functional resemblance of aRF1 and eRF1. This observation confirms the earlier prediction that eRF1 and aRF1 form a common structural-functional eRF1/aRF1 protein family, originating from a common ancient ancestor.

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/.

Ap4A induces apoptosis in human cultured cells.

Diadenosine oligophosphates (Ap(n)A) have been proposed as intracellular and extracellular signaling molecules in animal cells. The ratio of diadenosine 5',5'''-P1,P3-triphosphate to diadenosine 5',5'''-P1,P4-tetraphosphate (Ap3A/Ap4A) is sensitive to the cellular status and alters when cultured cells undergo differentiation or are treated with interferons. In cells undergoing apoptosis induced by DNA topoisomerase II inhibitor VP16, the concentration of Ap3A decreases significantly while that of Ap4A increases. Here, we have examined the effects of exogenously added Ap3A and Ap4A on apoptosis and morphological differentiation. Penetration of Ap(n)A into cells was achieved by cold shock. Ap4A at 10 microM induced programmed cell death in human HL60, U937 and Jurkat cells and mouse VMRO cells and this effect appeared to require Ap4A breakdown as hydrolysis-resistant analogues of Ap4A were inactive. On its own, Ap3A induced neither apoptosis nor cell differentiation but did display strong synergism with the protein kinase C activators 12-deoxyphorbol-13-O-phenylacetate and 12-deoxyphorbol-13-O-phenylacetate-20-acetate in inducing differentiation of HL60 cells. We propose that Ap4A and Ap3A are physiological antagonists in determination of the cellular status: Ap4A induces apoptosis whereas Ap3A is a co-inductor of differentiation. In both cases, the mechanism of signal transduction remains unknown.

Comparative allelotyping of the short arm of human chromosome 3 in epithelial tumors of four different types.

Comparative allelotyping of the short arm of human chromosome 3 (3p) in four types of epithelial carcinomas was performed using an identical set of polymorphic markers. In total, 117 samples of non-papillary renal cell carcinoma (RCC), non-small cell lung carcinoma (NSCLC), carcinoma of uterine cervix (CC), and breast carcinoma (BC) were screened for loss of heterozygosity (LOH) with 10 di-, tri- and tetrameric markers covering nine bands of 3p. High LOH frequencies were detected in at least one locus: RCC (36/43, 84%), BC (20/26, 77%), NSCLC (16/24, 67%), and CC (15/24, 62%). Small interstitial deletions prevailed in BC and CC whereas large continuous and discontinuous deletions were mainly found in RCC and NSCLC. Different epithelial tumors displayed unique LOH profiles with partial overlaps in 3p26.1, 3p21.31, and 3p13. The overlap around D3S2409 (3p21.31) appeared common for RCC, BC and CC.

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.