M. M. Kurshakova

SAGA and a novel Drosophila export complex anchor efficient transcription and mRNA export to NPC

SAGA/TFTC‐type multiprotein complexes play important roles in the regulation of transcription. We have investigated the importance of the nuclear positioning of a gene, its transcription and the consequent export of the nascent mRNA. We show that E(y)2 is a subunit of the SAGA/TFTC‐type histone acetyl transferase complex in Drosophila and that E(y)2 concentrates at the nuclear periphery. We demonstrate an interaction between E(y)2 and the nuclear pore complex (NPC) and show that SAGA/TFTC also contacts the NPC at the nuclear periphery. E(y)2 forms also a complex with X‐linked male sterile 2 (Xmas‐2) to regulate mRNA transport both in normal conditions and after heat shock. Importantly, E(y)2 and Xmas‐2 knockdown decreases the contact between the heat‐shock protein 70 (hsp70) gene loci and the nuclear envelope before and after activation and interferes with transcription. Thus, E(y)2 and Xmas‐2 together with SAGA/TFTC function in the anchoring of a subset of transcription sites to the NPCs to achieve efficient transcription and mRNA export.

Two Isoforms of Drosophila TRF2 Are Involved in Embryonic Development, Premeiotic Chromatin Condensation, and Proper Differentiation of Germ Cells of Both Sexes

ABSTRACT The Drosophila TATA box-binding protein (TBP)-related factor 2 (TRF2 or TLF) was shown to control a subset of genes different from that controlled by TBP. Here, we have investigated the structure and functions of the trf2 gene. We demonstrate that it encodes two protein isoforms: the previously described 75-kDa TRF2 and a newly identified 175-kDa version in which the same sequence is preceded by a long N-terminal domain with coiled-coil motifs. Chromatography of Drosophila embryo extracts revealed that the long TRF2 is part of a multiprotein complex also containing ISWI. Both TRF2 forms are detected at the same sites on polytene chromosomes and have the same expression patterns, suggesting that they fulfill similar functions. A study of the manifestations of the trf2 mutation suggests an essential role of TRF2 during embryonic Drosophila development. The trf2 gene is strongly expressed in germ line cells of adult flies. High levels of TRF2 are found in nuclei of primary spermatocytes and trophocytes with intense transcription. In ovaries, TRF2 is present both in actively transcribing nurse cells and in the transcriptionally inactive oocyte nuclei. Moreover, TRF2 is essential for premeiotic chromatin condensation and proper differentiation of germ cells of both sexes.

A retrocopy of a gene can functionally displace the source gene in evolution.

The e(y)2 gene of Drosophila melanogaster encodes the ubiquitous evolutionarily conserved co-activator of RNA polymerase II that is involved in transcription regulation of a high number of genes. The Drosophila e(y)2b gene, paralogue of the e(y)2 has been found. The analysis of structure of the e(y)2, e(y)2b and its orthologues from other species reveals that the e(y)2 gene derived as a result of retroposition of the e(y)2b during Drosophila evolution. The mRNA-derived retrogenes lack introns or regulatory regions; most of them become pseudogenes whereas some acquire tissue-specific functions. Here we describe the different situation: the e(y)2 retrogene performs the general function and is ubiquitously expressed, while the source gene is functional only in a small group of male germ cells. This must have resulted from retroposition into a transcriptionally favorable region of the genome.

Integrity of the Mod(mdg4)-67.2 BTB Domain Is Critical to Insulator Function in Drosophila melanogaster

ABSTRACT The Drosophila gypsy insulator contains binding sites for the Suppressor of Hairy-wing [Su(Hw)] protein. Enhancer and silencer blocking require Su(Hw) recruitment of Mod(mdg4)-67.2, a BTB/POZ domain protein that interacts with Su(Hw) through a carboxyl-terminal acidic domain. Here we conducted mutational analyses of the Mod(mdg4)-67.2 BTB domain. We demonstrate that this domain is essential for insulator function, in part through direction of protein dimerization. Our studies revealed the presence of a second domain (DD) that contributes to Mod(mdg4)-67.2 dimerization when the function of the BTB domain is compromised. Additionally, we demonstrate that mutations in amino acids of the charged pocket in the BTB domain that retain dimerization of the mutated protein cause a loss of insulator function. In these cases, the mutant proteins failed to localize to chromosomes, suggesting a role for the BTB domain in chromosome association. Interestingly, replacement of the Mod(mdg4)-67.2 BTB domain with the GAF BTB domain produced a nonfunctional protein. Taken together, these data suggest that the Mod(mdg4)-67.2 BTB domain confers novel activities to gypsy insulator function.

ORC interacts with THSC/TREX-2 and its subunits promote Nxf1 association with mRNP and mRNA export in Drosophila

Abstract The origin recognition complex (ORC) of eukaryotes associates with the replication origins and initiates the pre-replication complex assembly. In the literature, there are several reports of interaction of ORC with different RNAs. Here, we demonstrate for the first time a direct interaction of ORC with the THSC/TREX-2 mRNA nuclear export complex. The THSC/TREX-2 was purified from the Drosophila embryonic extract and found to bind with a fraction of the ORC. This interaction occurred via several subunits and was essential for Drosophila viability. Also, ORC was associated with mRNP, which was facilitated by TREX-2. ORC subunits interacted with the Nxf1 receptor mediating the bulk mRNA export. The knockdown of Orc5 led to a drop in the Nxf1 association with mRNP, while Orc3 knockdown increased the level of mRNP-bound Nxf1. The knockdown of Orc5, Orc3 and several other ORC subunits led to an accumulation of mRNA in the nucleus, suggesting that ORC participates in the regulation of the mRNP export.

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.

Involvement of general transcriptional factors in the regulation of transcription of the hsp70 gene in vivo.

475 Gene transcription by RNA polymerase II is activated with the involvement of general transcriptional factors (GTFs)—TFIIB, TFIID, TFIIE, TFIIF, and TFIIH [1]. The main factor involved in promoter recognition is the multiprotein complex TFIID consisting of the TATA-box-binding protein (TBP) and 13 to 15 TAF proteins associated with TBP [2, 3]. Some TAF proteins are also present in the TFTC complex, which is also involved in transcription activation bur contains no TBP. It also contains histone acetyltransferase GCN5, TRRAP, and Spt proteins [4–6]. The Mediator complex ensures interaction between specific transcription activators and GTF [7, 8]. The hsp70 gene of Drosophila melanogaster is a convenient model for studying transcription activation by heat shock in vivo [9]. In this work, we studied the involvement of transcriptional factors in transcription activation in vivo. It was shown that, before heat induction, nearly all general transcriptional factors are present on the hsp70 gene promoter. However, after heat exposure, the amount of TBP, TAF, TFIIB, and TFIIF decreases, whereas the amount of RNA polymerase II, TFIIH, GCN5, TRRAP, and Med13 increases.

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.

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.

The role of SAGA coactivator complex in snRNA transcription

ABSTRACT 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.