[Novel complexes of gene expression and their role in the appearance and evolution of the genus Homo].

Using genetic (yeast two-hybrid system) and biochemical (co-precipitation of proteins from cellular lysates) approaches, we have performed a whole-genome wide search for interacting partners of the previously described by us variants of hRPB11 subunit of human RNA polymerase II - hRPB1 1balpha, hRPB11calpha and hRPB1 1bbeta, hRPB 11cbeta - in fetal brain and Jurkat cell line libraries. In consequence, the main spectrum of the protein partners of these human specific isoforms of the RNA polymerase II subunit hRPB 11 (POLR2J) has been established. Functional characteristics of the uncovered protein partners of hRPB 11balpha and hRPB 11calpha isoforms clearly indicate that these isoforms, similarly to the main (major) subunit hRPB11a, are components of the distinct transcription complexes participating not only in the transcription of the specific DNA matrices, but involving also in the later stages of mRNA biogenesis. The RNA polymerase I-III common subunit hRPB6 (POLR2F) and basal component of the exon-exon junction complex Y14 (RBM8A) have been found among the protein partners of the isoforms hRPB 11bbeta and hRPB 11cbeta together with a number of proteins involved in the biogenesis of microRNAs, including a novel, not previously described variant of the microRNA processing nuclease DROSHA, which indicates the existence of a special coordination between processes of transcription and RNA interference in the nuclei of human cells.

A minor isoform of the human RNA polymerase II subunit hRPB11 (POLR2J) interacts with several components of the translation initiation factor eIF3.

Using the yeast two-hybrid (YTH) system we have uncovered interaction of the hRPB11cα minor isoform of Homo sapiens RNA polymerase II hRPB11 (POLR2J) subunit with three different subunits of the human translation initiation factor eIF3 (hEIF3): eIF3a, eIF3i, and eIF3m. One variant of eIF3m identified in the study is the product of translation of alternatively spliced mRNA. We have named a novel isoform of this subunit eIF3mß. By means of the YTH system we also have shown that the new eIF3mß isoform interacts with the eIF3a subunit. Whereas previously described subunit eIF3mα (GA17) has clear cytoplasmic localization, the novel eIF3mß isoform is detected predominantly in the cell nucleus. The discovered interactions of the hRPB11cα isoform with several hEIF3 subunits demonstrate a new type coordination between transcription and the following (downstream) stages of gene expression (such as mRNA transport from nucleus to the active ribosomes in cytoplasm) in Homo sapiens and point out the possibility of existence of nuclear hEIF3 subcomplexes.

[PSM2 and POLR2J gene families as molecular markers of the higher primate evolution].

We have studied the molecular evolution of two gene families specific for primates: POLR2J of the transcription system and PMS2 of the MMR repair system. The appearance and improvement of the genetic structure in each of the families was shown to strongly correlate with the main stages of the higher primates biological evolution. Our results indicate that the PSM2 and POLR2J genes can serve as helpful and reliable molecular markers of anthropogenesis.

[C-terminal domain (CTD) of the subunit Rpb1 of nuclear RNA polymerase II and its role in the transcription cycle].

The review deals with analysis of the structure, functions and molecular evolution of the CTD (C-terminal domain) of the largest subunit Rpb1 of nuclear RNA polymerase II. All the discovered ways of CTD modifications are summarized. Its interactions with various partners are described in the context of the CTD central role in coupling of the major transcription steps (initiation, elongation and termination) with the main stages of pre-mRNA processing (capping, splicing and polyadenylation) in eukaryotes during different stages of the cell cycle.

[New genes on human chromosome 7: bioinformatic analysis of a gene cluster from the POLR2J family]. / Novye geny na khromosome 7 cheloveka: bioinformatsionnyi analiz klastera genov iz semeistva POLR2J.

Four independent genes encoding various variants of the hRPB11 subunit of Homo sapiens RNA polymerase II were revealed in human chromosome 7. Three genes (POLR2J1, POLR2J2, and POLR2J3) form a cluster of total length 214530 bp in the genetic locus 7q22.1 on the long arm of chromosome 7 (contig NT_007933). The fourth gene (POLR2J4, 31040 bp) was localized in the cytogenetic locus 7p13 of the short arm of chromosome 7 (contig NT_007819). An analysis enabled us to refine dissimilar experimental data on the mapping of the hRPB11 subunit gene on chromosome 7. In particular, the presence of three sites of its localization according to data on hybridization with fluorescent-labeled probes (the FISH method) was explained. It was established that, upon the expression of the four human POLR2J genes, at least 14 types of mature mRNAs encoding somewhat differing hRPB11 isoforms can be synthesized. Eleven of these mRNAs were revealed (as full-length copies or clearly identifiable fragments) in the available databases of expressed sequence tags and cDNAs. The most probable scheme of origination of the multiple genes of the POLR2J family, as a result of three consecutive segmented duplications increasing in size, was proposed and substantiated. On the basis of the scheme, some assumptions on the pathways of evolution of separate human genes and the mechanisms of generation of protein diversity in higher eukaryotes were made. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 6; see also http://www.maik.ru.

[Structure and function of eukaryotic nuclear DNA-dependent RNA polymerase I]. / Structura i funktsii iadernoi DNK-zavisimoi RNK-polimerazy I éukariot.

In the eukaryotic cell, normal protein biosynthesis is sustained by several million ribosomes, which contain rRNA as an essential component. The high-molecular-weight precursor of large and 5.8S rRNAs is synthesized by DNA-dependent RNA polymerase I (Pol I) in the nucleolus. Data on DNA regulatory elements, protein factors involved in rDNA transcription by Pol I, subunit composition of Pol I, and on the interactions and possible functions of individual subunits are summarized.

Rpc19 and Rpc40, two alpha-like subunits shared by nuclear RNA polymerases I and III, are interchangeable between the fission and budding yeasts.

The cDNAs and genes encoding the common subunits Rpc19 and Rpc40 of nuclear RNA polymerases I and III of Schizosaccharomyces pombe were isolated from cDNA and genomic libraries of the fission yeast and tested for their ability to substitute for the homologous genes in Saccharomyces cerevisiae by heterospecific complementation of corresponding null alleles and temperature-sensitive mutations. The results obtained indicate that both Sz. pombe genes (rpc19(+) and rpc40(+)) are able to replace their S. cerevisiae counterparts in vivo. The primary structure and general organization of both genes were established: rpc40(+) is an intronless gene, while rpc19(+) contains three introns (73, 48 and 77 bp long); rpc19(+) is situated on the long arm of chromosome I and rpc40(+) on the long arm of chromosome II.

[Molecular cloning of some components of the translation apparatus of fission yeast Schizosaccharomyces pombe and a list of its cytoplasm ic proteins genes]. / Molekuliarnoe klonirovanie nekotorykh komponentov apparata transliatsii deliashchikhsia drozhzhei Schizosaccharomyces pombe i pervichnyi unifitsirovannyi spisok genov tsitoplasmaticheskikh ribosomnykh belkov etogo mikroorganizma.

Full-length cDNAs of four new genes encoding cytoplasmic ribosomal proteins L14 and L20 (large ribosomal subunit) and S1 and S27 (small ribosomal subunit) were isolated and sequenced during the analysis of the fission yeast Schizosaccharomyces pombe genome. One of the Sz. pombe genes encoding translation elongation factor EF-2 was also cloned and its precise position on chromosome I established. A unified nomenclature was proposed, and the list of all known genetic determinants encoding cytoplasmic ribosomal proteins of Sz. pombe was compiled. By now, 76 genes/cDNAs encoding different ribosomal proteins have been identified in the fission yeast genome. Among them, 35 genes are duplicated and three homologous genes are identified for each of the ribosomal proteins L2, L16, P1, and P2.

[Characteristics of the cDNA of the Schizosaccharomyces pombe rpa43+ gene: structural similarity of the Rpa43 subunit of RNA-polymerase I with the Rpc25 subunit of RNA-polymerase III]. / Kharakteristika kDNK gena rpa43+ Schizosaccharomyces pombe: strukturnoeskhodstvo sub''edinitsy Rpa43 RNK-polimerazy I s sub''edinitsei Rpc25 RNK-polimerazy III.

We isolated and characterized full-length cDNA of the rpa43+ gene encoding one of subunits of the nuclear RNA polymerase I of Schizosaccharomyces pombe. The gene contains two introns and is located on chromosome II. Comparison of the primary structure of the subunit Rpa43 of Sz. pombe (173 aa; M 19,385 Da; pl 5.36), deduced from the cDNA obtained, with the amino acid sequences of subunits A43 from Saccharomyces cerevisiae and Drosophila melanogaster demonstrates a high divergence of this protein in evolution. A comparison of the Rpa43 with other proteins from the SwissProt database revealed a similarity of this subunit to subunit Rpc25 of RNA polymerase III, which, as was shown previously, is structurally similar to subunit Rpb7 of RNA polymerase II. Thus, including the Rpa43<-->Rpc25<-->Rpb7 family, nuclear RNA polymerases I-III contain at least 11 identical and/or similar subunits. This fact illustrates a pronounced resemblance of the organization of all three enzymes of the eukaryotic transcription apparatus. Moreover, at least ten out of these eleven families of eukaryotic RNA polymerase subunits have homologues in the 13-subunit archaeal RNA polymerase.

[Molecular cloning and characteristics of rpc19+ and rpc40+ Schizosaccharomyces pombe genes, coding for common subunits of nuclear RNA polymerase I and III]. / Molekuliarnoe klonirovanie i kharakteristika genov rpc19+ i rpc40+ Schizosaccharomyces pombe, kodiruiushchikh obshchie sub''edinitsy iadernykh RNK-polimeraz I i III.

Full-length copies of cDNAs of the rpc19+ and rpc40+ genes encoding the common subunits of nuclear RNA polymerases I and III and the corresponding fragments of chromosomes were isolated from genomic and cDNA libraries of Schizosaccharomyces pombe and characterized. It was established that the cloned genes are located on chromosomes III and II of the fission yeast, respectively. The rpc40+ gene lacks introns, and the rpc19+ gene contains two intervening sequences. The comparison of subunits Rpc19 (125 aa; M 13 722 Da; pI 4.51) and Rpc40 (348 aa; M 39 141 Da; pI 5.40) of Sz. pombe, whose characteristics were deduced from the sequences of their cDNAs, with the orthologous components of other eukaryotes allowed the most conserved structure-functional domains of these proteins to be identified.