Elena K. Shematorova

Members of the SAGA and Mediator complexes are partners of the transcription elongation factor TFIIS.

TFIIS, an elongation factor encoded by DST1 in Saccharomyces cerevisiae, stimulates transcript cleavage in arrested RNA polymerase II. Two components of the RNA polymerase II machinery, Med13 (Srb9) and Spt8, were isolated as two‐hybrid partners of the conserved TFIIS N‐terminal domain. They belong to the Cdk8 module of the Mediator and to a subform of the SAGA co‐activator, respectively. Co‐immunoprecipitation experiments showed that TFIIS can bind the Cdk8 module and SAGA in cell‐free extracts. spt8Δ and dst1Δ mutants were sensitive to nucleotide‐depleting drugs and epistatic to null mutants of the RNA polymerase II subunit Rpb9, suggesting that their elongation defects are mediated by Rpb9. rpb9Δ, spt8Δ and dst1Δ were lethal in cells lacking the Rpb4 subunit. The TFIIS N‐terminal domain is also strictly required for viability in rpb4Δ, although it is not needed for binding to RNA polymerase II or for transcript cleavage. It is proposed that TFIIS and the Spt8‐containing form of SAGA co‐operate to rescue RNA polymerase II from unproductive elongation complexes, and that the Cdk8 module temporarily blocks transcription during transcript cleavage.

Ancient origin, functional conservation and fast evolution of DNA-dependent RNA polymerase III

RNA polymerase III contains seventeen subunits in yeasts (Saccharomyces cerevisiae and Schizosaccharomyces pombe) and in human cells. Twelve of them are akin to the core RNA polymerase I or II. The five other are RNA polymerase III-specific and form the functionally distinct groups Rpc31-Rpc34-Rpc82 and Rpc37-Rpc53. Currently sequenced eukaryotic genomes revealed significant homology to these seventeen subunits in Fungi, Animals, Plants and Amoebozoans. Except for subunit Rpc31, this also extended to the much more distantly related genomes of Alveolates and Excavates, indicating that the complex subunit organization of RNA polymerase III emerged at a very early stage of eukaryotic evolution. The Sch.pombe subunits were expressed in S.cerevisiae null mutants and tested for growth. Ten core subunits showed heterospecific complementation, but the two largest catalytic subunits (Rpc1 and Rpc2) and all five RNA polymerase III-specific subunits (Rpc82, Rpc53, Rpc37, Rpc34 and Rpc31) were non-functional. Three highly conserved RNA polymerase III-specific domains were found in the twelve-subunit core structure. They correspond to the Rpc17-Rpc25 dimer, involved in transcription initiation, to an N-terminal domain of the largest subunit Rpc1 important to anchor Rpc31, Rpc34 and Rpc82, and to a C-terminal domain of Rpc1 that presumably holds Rpc37, Rpc53 and their Rpc11 partner.

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

Abstract 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.

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 eIF3mβ. By means of the YTH system we also have shown that the new eIF3mβ isoform interacts with the eIF3a subunit. Whereas previously described subunit eIF3mα (GA17) has clear cytoplasmic localization, the novel eIF3mβ 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.

PMS2 and POLR2J gene families as molecular markers of the higher primates evolution

We have studied the molecular evolution of two gene families specific for primates: POLR2J of the transcription system and PMS2 of the mismatch repair (MMR) system. The appearance of these families and upgrading of their genetic structures was shown to neatly correlate with the main stages of the biological evolution of higher primates. Our results indicate that the PMS2 and POLR2J genes can serve as helpful and reliable molecular markers of anthropogenesis.

New Genes on Human Chromosome 7: Bioinformation Analysis of a Cluster of Genes from the POLR2J Family

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 of 214 530 bp in the genetic locus 7q22.1 on the long arm of chromosome 7 (contig NT_007933). The fourth gene (POLR2J4, 31 040 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.

A key enzyme of animal steroidogenesis can function in plants enhancing their immunity and accelerating the processes of growth and development

BackgroundThe initial stage of the biosynthesis of steroid hormones in animals occurs in the mitochondria of steroidogenic tissues, where cytochrome P450SCC (CYP11A1) encoded by the CYP11A1 gene catalyzes the conversion of cholesterol into pregnenolone – the general precursor of all the steroid hormones, starting with progesterone. This stage is missing in plants where mitochondrial cytochromes P450 (the mito CYP clan) have not been found. Generating transgenic plants with a mitochondrial type P450 from animals would offer an interesting option to verify whether plant mitochondria could serve as another site of P450 monooxygenase reaction for the steroid hormones biosynthesis.ResultsFor a more detailed comparison of steroidogenic systems of Plantae and Animalia, we have created and studied transgenic tobacco and tomato plants efficiently expressing mammalian CYP11A1 cDNA. The detailed phenotypic characterization of plants obtained has shown that through four generations studied, the transgenic tobacco plants have reduced a period of vegetative development (early flowering and maturation of bolls), enlarged biomass and increased productivity (quantity and quality of seeds) as compared to the only empty-vector containing or wild type plants. Moreover, the CYP11A1 transgenic plants show resistance to such fungal pathogen as Botrytis cinerea. Similar valuable phenotypes (the accelerated course of ontogenesis and/or stress resistance) are also visible in two clearly distinct transgenic tomato lines expressing CYP11A1 cDNA: one line (No. 4) has an accelerated rate of vegetative development, while the other (No. 7) has enhanced immunity to abiotic and biotic stresses. The progesterone level in transgenic tobacco and tomato leaves is 3–5 times higher than in the control plants of the wild type.ConclusionsFor the first time, we could show the compatibility in vivo of even the most specific components of the systems of biosynthesis of steroid hormones in Plantae and Animalia. The hypothesis is proposed and substantiated that the formation of the above-noted special phenotypes of transgenic plants expressing mammalian CYP11A1 cDNA is due to the increased biosynthesis of progesterone that can be considered as a very ancient bioregulator of plant cells and the first real hormone common to plants and animals.

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 (coprecipitation of proteins from cellular lysates) approaches, we have performed a whole-genome wide search for interacting partners of variants of the hRPB11 subunit of human RNA polymerase II that we have previously described (hRPB11bα, hRPB11cα, hRPB11bβ, hRPB11cβ) in fetal brain and the Jurkat cell line libraries. In consequence, the main spectra of the protein partners of these human-specific isoforms of the RNA polymerase II subunit hRPB11 (POLR2J) were defined. Functional characteristics of the uncovered protein partners of the hRPB11bα and hRPB11cα isoforms clearly indicate that these isoforms, like the major subunit hRPB11a, are components of distinct transcription complexes that not only participate in transcription of specific DNA matrices, but are also involved in the later stages of mRNA biogenesis. The common subunit of RNA polymerases I?III hRPB6 (POLR2F) and basal component of the exon-exon junction complex Y14 (RBM8A) were found among the protein partners of the isoforms hRPB11bβ and hRPB11cβ, together with a number of proteins involved in biogenesis of microRNAs, including a novel, previously undescribed variant of the microRNA processing nuclease DROSHA. These data indicate the existence of a special coordination between processes of transcription and RNA interference in the nuclei of human cells.