Yu. V. Shidlovskii

Molecular components of JAK/STAT signaling pathway and its interaction with transcription machinery

JAK/STAT is a conserved signaling pathway in higher eukaryotes that plays a critical role in different processes of ontogenesis. This article reviews the pathway structure at the molecular level, mainly in Drosophila as a model organism. The review is focused on the data concerning the relationship between this signaling pathway and transcription machinery of higher eukaryotes.

Novel complex formed by the SAYP transcriptional coactivator

A multisubunit complex that contains the novel evolutionarily conserved transcription factor SAYP was isolated, and its protein composition was determined. SAYP was found to unite two complexes with different functions in transcription activation, namely, the chromatin-remodeling complex PBAP (SWI/SNF) and the general transcription factor TFIID, which is the main component of the RNA polymerase II preinitiation complex. The new supercomplex contained the total set of PBAP and TFIID subunits. All components of the supercomplex (SAYP, TFIID, and PBAP) are essential for its effective interaction with the promoters of SAYPdependent genes.

RNA immunoprecipitation technique for Drosophila melanogaster S2 cells

RNA-binding proteins play an important role in RNA metabolism, especially in mRNA biogenesis and subsequent expression patterns regulation. RNA immunoprecipitation (RIP) is a powerful tool for detecting protein–RNA associations. In this paper, we briefly cover the history of this method for analyzing RNA–protein interactions and reviewing a number of modifications of the RIP technique. We also present an adjusted RIP protocol that was modified for Drosophila S2 cell culture. The use of this protocol allows one to perform the efficient precipitation of RNA–protein complexes and harvest RNA in amounts that are sufficient for its downstream analysis.

Functioning of macromolecular complexes at successive stages of gene expression as self-coordinated molecular machines

The results of systematic investigations devoted to the role of Drosophila melanogaster proteins SAYP and ENY2 in the regulation of different stages of gene expression are presented. Gene expression is a system of multistage processes involving preparation of the chromatin matrix, initiation of transcription, mRNA synthesis, as well as formation of mRNP particles, their export, and translation in the cytoplasm. Each of these stages involves a great number of factors presented usually by protein complexes consisting of different subunits. Analysis of the available evidence indicates that the transcription coactivators SAYP and ENY2 act in gene expression as cooperative nanoregulators, thereby determining the molecular mechanisms of coordination of the spatial and temporal characteristics of the processes implementing the genetic information.

A novel conserved domain of SAYP coactivator mediates the interaction of TFIID and brahma transcription complexes

Using the S2 cell system of Drosophila melanogaster, it was shown that the key protein domain mediating the interaction of TFIID and Brahma transcriptional complexes within the BTFly supercomplex is the evolutionary conserved SAY domain of SAYP. TFIID and Brahma coactivators participated in the reporter gene activation induced by the SAY domain in cellular nuclei. The components of TFIID and Brahma directly interacting with SAY were identified.

Study of the Drosophila melanogaster trf2 gene and its protein product

The Drosophila melanogaster TRF2 protein regulates transcription of several genes. The trf2 gene structure was studied. The gene proved to code for two protein isoforms, a known 75-kDa isoform and a newly identified 175-kDa isoform. The new isoform combines the known isoform sequence with an extended N end containing a coiled-coil motif. The long TRF2 isoform was found to act as a component of a multiprotein complex, including ISWI ATPase as well.

Molecular machinery of the transcription initiation by RNA polymerase II

Transcription is the first stage of the realization of genetic information. In recent years, substantial progress in understanding of this process has been achieved. Eukaryotic cell nuclei were shown to possess extremely complex transcription machinery consisting of more that a hundred different factors. A series of our reviews is devoted to the first step of the transcription process, which is of key significance for the control of gene expression: transcription initiation on the genes transcribed by RNA polymerase II. This paper gives a short description of the main factors involved in this process.

The e(y)3 Gene Codes for SAYP, an Evolutionary Conserved Protein That Is Essential for Ontogeny

Enhancers of yellow (e(y)) is a group of genetically and functionally related genes. The proteins encoded by these genes are involved in transcription regulation. The e(y)3 gene under study codes for an ubiquitous nuclear protein, termed SAYP, which has homologs in various multicellular organisms. SAYP contains an AT-hook domain, two PHD domains, and a new evolutionarily conserved domain. SAYP mutants die during embryonic development. The weak e(y)ul mutation causes multiple malformations. The potent e(y)EMSl mutation causes embryonic death, implicating SAYP in early development.

Current technics for visualizing RNA in a cell

The study of RNA functions in a cell is based on the methods of labeling and detection of these molecules. In recent years, considerable progress has been made in this area, making it possible to detect RNA in amounts to one molecule with high specificity and to track the dynamics of spatial RNA distribution in the living cell. The review presents the latest developments in the field of in situ hybridization and the use of RNA aptamers and RNA-binding proteins. The prospects of using the CRISPR-Cas system for RNA visualization are discussed in detail.

Preparation and analysis of nuclear protein extract from Drosophila melanogaster embryos for studying transcription factors

Nuclear protein extract is an important substance for studying different molecular systems of the cell, particularly transcription machinery. The paper briefly describes extraction methods and provides a protocol for the preparation of extract from nuclei of Drosophila embryos using salt extraction. The extraction efficiency of transcription factors and chromatin proteins has been checked under different salt concentrations. The paper also provides data about chromatographic properties of some protein factors regulating gene activity.