TREX-2-ORC Complex of D. melanogaster Participates in Nuclear Export of Histone mRNA.

The TREX-2-ORC protein complex of D. melanogaster is necessary for the export of the bulk of synthesized poly(A)-containing mRNA molecules from the nucleus to the cytoplasm through the nuclear pores. However, the role of this complex in the export of other types of RNA remains unknown. We have shown that TREX-2-ORC participates in the nuclear export of histone mRNAs: it associates with histone mRNPs, binds to histone H3 mRNA at the 3'-terminal part of the coding region, and participates in the export of histone mRNAs from the nucleus to the cytoplasm.

The Xmas-2 Protein of Drosophila melanogaster Undergoes Cleavage into Two Fragments.

The TREX-2 complex integrates several stages of gene expression, such as transcriptional activation and mRNA export. In D. melanogaster, TREX-2 consists of four major proteins: Xmas-2, ENY2, PCID2, and Sem1p. The Xmas-2 protein is the core subunit of the complex, with which other TREX-2 subunits interact. Xmas-2 homologues were found in all higher eukaryotes. Previously, it was shown that the human Xmas-2 homologue, GANP protein, can undergo cleavage into two parts, probably during apoptosis. We showed that the Xmas-2 protein of D. melanogaster can also split into two fragments. The resulting fragments of the protein correspond to the two large Xmas-2 domains. Protein splitting is observed both in vivo and in vitro. However, Xmas-2 cleavage in D. melanogaster is observed under normal conditions and is probably a part of the mechanism of transcription and mRNA export regulation in D. melanogaster.

The Human TREX-2 Complex Interacts with Subunits of the ORC Complex.

The TREX-2 protein complex is the key complex involved in the export of mRNA from the nucleus to the cytoplasm through the nuclear pores. Previously, a joint protein complex of TREX-2 with ORC was isolated in D. melanogaster. It was shown that the interaction of TREX-2 with ORC is necessary for efficient mRNA export from the nucleus to the cytoplasm. In this work, we showed that the TREX-2-ORC joint complex is also formed in human cells.

PCID2, a subunit of the Drosophila TREX-2 nuclear export complex, is essential for both mRNA nuclear export and its subsequent cytoplasmic trafficking.

The TREX-2 complex is essential for the general nuclear mRNA export in eukaryotes. TREX-2 interacts with the nuclear pore and transcriptional apparatus and links transcription to the mRNA export. However, it remains poorly understood how the TREX-2-dependent nuclear export is connected to the subsequent stages of mRNA trafficking. Here, we show that the PCID2 subunit of Drosophila TREX-2 is present in the cytoplasm of the cell. The cytoplasmic PCID2 directly interacts with the NudC protein and this interaction maintains its stability in the cytoplasm. Moreover, PCID2 is associated with the cytoplasmic mRNA and microtubules. The PCID2 knockdown blocks nuclear export of mRNA and also affects the general mRNA transport into the cytoplasm. These data suggest that PCID2 could be the link between the nuclear TREX-2-dependent export and the subsequent cytoplasmic trafficking of mRNA.

MLE Helicase Is a New Participant in the Transcription Regulation of the ftz-f1 Gene Encoding Nuclear Receptor in Higher Eukaryotes.

MLE helicase is an evolutionarily conserved eukaryotic protein involved in a wide range of processes in the regulation of gene expression. Previously, we studied the properties of MLE on the Drosophila melanogaster model. In the present work we continue studying the functions of MLE and show that MLE interacts with the components of the SWI/SNF chromatin remodelling complex. To clarify the work of MLE, the profile of MLE binding to the regulatory elements of the SWI/SNF-dependent ftz-f1 gene was analyzed. The effect of MLE on the expression of this gene, the transcription of which occurs by the RNA polymerase II pausing mechanism, was investigated. The data obtained indicate the important role of MLE in ensuring timely activation and high level of expression of the ftz-f1 gene in vivo.

Study of the Interaction between Xmas-2, the Main Protein of TREX-2 mRNA Export Complex, and the Orc3 Protein, a Subunit of ORC Complex of D. melanogaster.

The TREX-2 protein complex is the key participant in the export of mRNA from the nucleus to the cytoplasm through the nuclear pores. Previously, a protein complex of D. melanogaster consisting of TREX-2 and ORC complexes was purified. It was shown that, in the TREX-2-ORC complex, the Xmas-2 protein, which is the platform for TREX-2 assembly, interacts with the Orc3 protein. The aim of this work was to investigate what regions of the Xmas-2 amino acid sequence are involved in the interaction with Orc3. It was shown that the interaction of  Xmas-2 with Orc3 requires a C-terminal region of  Xmas-2 located downstream of the CID domain.

Multifunctional ENY2 Protein Interacts with RNA Helicase MLE.

ENY2 protein of Drosophila melanogaster was previously discovered and characterized in our laboratory [1, 2]. It was found that ENY2 is a subunit of several multiprotein complexes and connects various stages of gene expression [3-5]. This work is devoted to studying the interaction of ENY2 with RNA helicase MLE. This interaction was confirmed by independent methods. Data indicating that this interaction is conserved in evolution and is important for the functioning of MLE in both sexes were obtained.

[Protein complexes coordinating mRNA export from the nucleus into the cytoplasm].

The molecular mechanisms that coordinate transcription, processing, mRNP assembly, and mRNA export from the nucleus through nuclear pores into the cytoplasm have been the focus of intense research in recent years. Data demonstrating a tight association between the processes involved in gene expression are considered. The main protein complexes that play a role in mRNA export are described. The complexes are recruited to mRNA at steps preceding the mRNA export. The functions that the complexes perform at particular steps of gene expression are analyzed, and protein complexes responsible for quality control of mRNP discussed.

[Methods to study the RNA-protein interactions].

RNA-binding proteins (RBPs) play an important role in regulating gene expression at the posttranscriptional level, including the steps of pre-mRNA splicing, polyadenylation, mRNA stabilization, mRNA export from the nucleus to the cytoplasm, mRNA localization, and translation. RBPs regulate these processes primarily by binding to specific sequence elements in newly synthesized or mature transcripts. While many RPBs are known to recognize certain nucleotide sequences in RNA, information is insufficient for others. In particular, RBPs often compete for RNA binding or interact with RNA cooperatively. Hence, it is of importance to study the RNA-protein interactions in vivo. Numerous methods have been developed to identify the target nucleotide sequences of RBPs. The methods include the electrophoretic mobility shift assay (EMSA), systematic evolution of ligands by exponential enrichment (SELEX), RNA pull-down assay, RNA footprinting, RNA immunoprecipitation (RIP), UV-induced crosslinking immunoprecipitation (CLIP) and its variants, and measurement of the level for newly synthesized transcripts. Each of the methods has its limitation, and several methods supplementing each other should be employed in order to detect the RNA sequence to which a protein binds.

[E(y)2, the novel component of SAGA/TFTC complex in eucaryotes participates in export of mRNP from nucleus and couples transcription with nuclear pore].

For many years transcription was studied independently from the following stages of gene expression. However the tight connection between different stages of gene expression became evident during the last years. This review discusses the new molecular mechanisms coordinating transcription and mRNA export from the nucleus and coupling of transcription and position of the gene in the nucleus. The new protein E(y)2 which plays an important role in these processes is described.

[Conservative E(y)2/Sus1 protein interacts with the Su(Hw)-dependent insulators in Drosophila].

Insulators are regulatory elements having two properties. First, they are able to disturb the interaction between promoters and enhancers/silencers. Second, they are able to block distribution of the heterochromatin. The best-studied are the Su(Hw)-dependent insulators of Drosophila melanogaster, activity of which is determined by the Su(Hw) protein. In this study it was demonstrated that novel, evolutionary conservative transcription factor E(y)2/Sus1 interacted with the Su(Hw) zinc-finger domain and was present in the protein complex, associated with the Su(Hw)-dependent insulators.

[Conservative E(y)2/Sus1 protein is the member of SAGA complex and new nuclear pore-associated complex in Drosophila].

The SGA/TFTC complex plays an important role in the regulation of transcription. We have examined the significance of the gene positioning in the nucleus for its transcription and subsequent export of nascent mRNA. It was demonstrated that E(y)2 protein was a subunit of the SAGA/TFTC histone acetyl transferase complex in Drosophila and that E(y)2 concentrated at the nuclear periphery. An interaction between E(y)2 and the nuclear pore complex (NPC) was demonstrated, as well as that SAGA/TFTC also contacted the NPC at nuclear periphery. In addition, it was shown that E(y)2 formed complex with the Xmas-2 protein (X-linked male sterile 2) both in normal conditions and after heat shock. Importantly, the E(y)2 and Xmas-2 knockdown decreased the contact between the heat-shock protein 70 (hsp70) gene loci and the nuclear envelope before and after activation, and interfered with the transcription. Thus, E(y)2 and Xmas-2 together with SAGA/TFTC functioned in the anchoring of a subset of transcription sites to the NPCs to achieve efficient transcription and mRNA export.

The role of SAGA coactivator complex in snRNA transcription.

The general snRNA gene transcription apparatus has been extensively studied. However, the role of coactivators in this process is far from being clearly understood. Here, we have demonstrated that the Drosophila SAGA complex interacts with the PBP complex, the key component of the snRNA gene transcription apparatus, and is present at the promoter regions of the snRNA genes transcribed by both the RNA polymerase II and RNA polymerase III (U6 snRNA). We show that SAGA interacts with the Brf1 transcription factor, which is a part of the RNA polymerase III transcription apparatus and is present at promoters of a number of Pol III-transcribed genes. Mutations inactivating several SAGA subunit genes resulted in reduced snRNA levels in adult flies, indicating that SAGA is indeed the transcriptional coactivator for the snRNA genes. The transcription of the Pol II and Pol III-transcribed U genes was reduced by mutations in all tested SAGA complex subunits. Therefore, the transcription of the Pol II and Pol III-transcribed U genes was reduced by the mutations in the deubiquitinase module, as well as in the acetyltransferase module of the SAGA, indicating that the whole complex is essential for their transcription. Therefore, the SAGA complex activates snRNA genes suggesting its wide involvement in the regulation of gene transcription, and consequently, in the maintenance of cellular homeostasis.

[Principles of functioning of the complex of transcription initiation by RNA polymerase II].

This final review describes the general principle and mechanisms that underlie functioning of the machinery of transcription initiation by polymerase II in eukaryotes. The main models of transcription initiation, the module principle underlying the structure of the transcription complex, the regulation of transcription initiation, and the association between the nuclear architecture and transcription are discussed.

[Study of the novel tissue-specific RNA polymerase II transcription factor].

It has been established that retrogenes lose introns and regulatory regions. Most of them become pseudogenes, but some acquire tissue-specific functions. In this study, a contrary situation is described, when a retrogene from Drosophila melanogaster performs the main functions and is expressed in all tissues, while the initial gene is active only in a small part of the male germ cells. It is suggested that this phenomenon resulted from retroposition of the initial precursor gene in the transcription-suitable region of the genome.