A. N. Krasnov

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.

Tyrosine hydroxylase expression and activity in nigrostriatal dopaminergic neurons of MPTP-treated mice at the presymptomatic and symptomatic stages of parkinsonism

Progressive degeneration of nigrostriatal dopaminergic (DA-ergic) neurons is a key component in the pathogenesis of Parkinsons disease, which develops for a long time at the preclinical stage with no motor dysfunctions due to the initiation of compensatory processes. The goal of this study was to evaluate the changes in surviving nigrostriatal DA-ergic neurons with focus on tyrosine hydroxylase (TH) in MPTP-treated mice at the presymptomatic and early symptomatic stages of parkinsonism. According to our data, a partial degeneration of DA-ergic neurons at the presymptomatic stage was accompanied by: (i) no change in TH mRNA content in the substantia nigra (SN) suggesting a compensatory increase of TH gene expression in individual neurons; (ii) a decrease of TH protein content in the nigrostriatal system and no change in individual neurons, suggesting a slowdown of TH translation. When comparing DA-ergic neurons at the early symptomatic stage and presymptomatic stage, it becomes evident: (i) a decrease of TH mRNA content in the SN and hence gene expression in individual neurons; (ii) a decrease of TH content in the striatum and its increase in the SN and individual neurons suggesting an acceleration of TH translation. TH activity, an index of the rate of DA synthesis, was unchanged in the SN and decreased in the striatum to the same degree at both stages of parkinsonism. In the meantime, TH activity in individual neurons appeared to be compensatory increased, but to a higher degree at the symptomatic stage than at the presymptomatic one. These data first show that DA depletion, which provokes motor dysfunction, is not a result of the decrease of TH activity and the rate of DA synthesis but is rather related to either a decrease of DA release or an increase of DA uptake in striatal DA-ergic axons.

SAYP and Brahma are important for ‘repressive’ and ‘transient’ Pol II pausing

Drosophila SAYP, a homologue of human PHF10/BAF45a, is a metazoan coactivator associated with Brahma and essential for its recruitment on the promoter. The role of SAYP in DHR3 activator-driven transcription of the ftz-f1 gene, a member of the ecdysone cascade was studied. In the repressed state of ftz-f1 in the presence of DHR3, the Pol II complex is pre-recruited on the promoter; Pol II starts transcription but is paused 1.5 kb downstream of the promoter, with SAYP and Brahma forming a ‘nucleosomal barrier’ (a region of high nucleosome density) ahead of paused Pol II. SAYP depletion leads to the removal of Brahma, thereby eliminating the nucleosomal barrier. During active transcription, Pol II pausing at the same point correlates with Pol II CTD Ser2 phosphorylation. SAYP is essential for Ser2 phosphorylation and transcription elongation. Thus, SAYP as part of the Brahma complex participates in both ‘repressive’ and ‘transient’ Pol II pausing.

Heavy–light chain interrelations of MS-associated immunoglobulins probed by deep sequencing and rational variation ☆

The mechanisms triggering most of autoimmune diseases are still obscure. Autoreactive B cells play a crucial role in the development of such pathologies and, in particular, production of autoantibodies of different specificities. The combination of deep-sequencing technology with functional studies of antibodies selected from highly representative immunoglobulin combinatorial libraries may provide unique information on specific features in the repertoires of autoreactive B cells. Here, we have analyzed cross-combinations of the variable regions of human immunoglobulins against the myelin basic protein (MBP) previously selected from a multiple sclerosis (MS)-related scFv phage-display library. On the other hand, we have performed deep sequencing of the sublibraries of scFvs against MBP, Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1), and myelin oligodendrocyte glycoprotein (MOG). Bioinformatics analysis of sequencing data and surface plasmon resonance (SPR) studies have shown that it is the variable fragments of antibody heavy chains that mainly determine both the affinity of antibodies to the parent autoantigen and their cross-reactivity. It is suggested that LMP1-cross-reactive anti-myelin autoantibodies contain heavy chains encoded by certain germline gene segments, which may be a hallmark of the EBV-specific B cell subpopulation involved in MS triggering.

The DUBm subunit Sgf11 is required for mRNA export and interacts with Cbp80 in Drosophila

SAGA/TFTC is a histone acetyltransferase complex that has a second enzymatic activity because of the presence of a deubiquitination module (DUBm). Drosophila DUBm consists of Sgf11, ENY2 and Nonstop proteins. We show that Sgf11 has other DUBm-independent functions. It associates with Cbp80 component of the cap-binding complex and is thereby recruited onto growing messenger ribonucleic acid (mRNA); it also interacts with the AMEX mRNA export complex and is essential for hsp70 mRNA export, as well as for general mRNA export from the nucleus. Thus, Sgf11 functions as a component of both SAGA DUBm and the mRNA biogenesis machinery.

SAYP interacts with DHR3 nuclear receptor and participates in ecdysone-dependent transcription regulation

The role of metazoan coactivator SAYP in nuclear receptor-driven gene activation in the ecdysone cascade of Drosophila is considered. SAYP interacts with DHR3 nuclear receptor and activates the corresponding genes by recruiting the BTFly (Brahma and TFIID) coactivator supercomplex. The knockdown of SAYP leads to a decrease in the level of DHR3-activated transcription. DHR3 and SAYP interact during development and have multiple common targets across the genome.

ENY2: couple, triple...more?

ENY2/Sus1, a protein involved in the coupling of transcription with mRNA export, is a component of SAGA/TFTC and TREX-2/AMEX complexes. Recently, we have described the association of ENY2 with the third protein complex, THO. Moreover, our data indicate that ENY2 is also associated with other factors, both in the nucleus and cytoplasm. Thus, being a shared components of several protein complexes, ENY2 appears to function as an adapter molecule involved in integration of cellular processes, in particular, subsequent stages of gene expression.

Nuclear receptors: Structure and mechanisms of action

Nuclear receptors are a superfamily of conserved transcription factors with a unique domain structure. Nuclear receptors play an important role in the regulation of ontogeny, sexual maturation, and cell differentiation, as well as in various metabolic processes. Owing to the specifics of hormonal signaling, Drosophila melanogaster provides a promising model subject for studying the function and regulation of nuclear receptors. The review considers modern data on the molecular structure of nuclear receptors and the mechanisms of their interactions with ligands and transcription cofactors. The known functions of nuclear receptors in regulating embryo development and metamorphosis in D. melanogaster are described in detail.

A study of recombinant human lactoferrin secreted in milk of transgenic mice.

336 In this study, the properties of recombinant human lactoferrin isolated from milk of transgenic mice and natural human lactoferrin obtained isolated from breast milk were compared. Identity of both proteins was confirmed using by electrophoretic, immunological, and chromatographic methods. The lactoferrin from milk of transgenic mice exhibited the properties characteristic of human lactoferrin: it effectively chelated iron ions and binds to DNA, heparin, and bacterial lipopolysaccharide. Both proteins displayed identical bactericidal and fungicidal activities. The data obtained in this study allow the created genetic constructs to be used for obtaining producers of human lactoferrin on the basis of farm animals.

A review of the internet site practical molecular biology

In 2000, in the middle of September, the Russian Web site Practical Molecular Biology (http://molbiol.edu.ru) was opened. The main tasks of the project are (i) distribution of experimental work experience among Russian and foreign laboratories and (ii) organization of an information database in Russian for researchers connected with molecular biology or medicine.