The humoral immune system of Drosophila melanogaster, which is the best studied of all eukaryotes, is activated by the canonical IMD and Toll signalling pathways. Recently, long non-coding RNAs (lncRNAs) and genes encoding short polypeptides have been identified as potential regulators of the innate immune response. S2 cells are a macrophage-like cell line. They are used as a model system to study the molecular mechanisms of immune response gene activation. We used this cell line to study the effect of Escherichia coli and Micrococcus luteus bacteria on the transcription of the lncRNA-CR30055 and the CG45045 and CG44404 genes, encoding short polypeptides. We found that pathogens activate only CG45045, while the transcription levels of CR30055 and CG44404 remain unchanged. No activation of Cecropin C and some Bomanin family genes was observed, suggesting differing patterns of immune response gene activation in S2 cells and adult flies. The highest activation of CG45045 was observed between 6 and 12 hours of cell incubation with pathogens. The activation patterns of CG45045 after exposure to E. coli and M. luteus were similar, suggesting common mechanisms of transcriptional activation of this gene. Thus, CG45045 may be a novel gene involved in the humoral immune response of Drosophila.

In Drosophila, the 20-hydroxyecdysone (20E) hormone regulates numerous essential biological processes. Here, we studied the contribution of 20E to the activity of immune signaling pathways and antimicrobial activity using the model Drosophila S2 cells. We found that while 20E alone has no essential effect on this system, pretreating S2 cells with 20E followed by incubation with Escherichia coli or Micrococcus luteus stimulates the induction of a limited number of antimicrobial peptide (AMP) genes, such as Diptericin (Dpt) and Drosomycin (Drs). Contrary to this, cells pretreatment with 20E simulates the activity of numerous Bacillus subtilis-induced AMP genes. Interestingly, it also significantly promotes the expression of components of both the Toll (Dif, Dorsal, etc.) and the IMD pathways (Relish, IMD, etc.) in the presence of Bacillus subtilis. Unexpectedly, simultaneous treatment of S2 cells by 20E and all three bacteria shows another pattern of activity and leads to a suppression of Drosocin (Dro) induction, in particular. Our study reveals that the contribution of 20E to immune genes activity varies for different genes and depends on the mode of 20E interplay with the pathogen and the nature of the pathogen itself

The IMD and Toll signaling pathways in Drosophila melanogaster mediate the innate immune responses to Gram-negative and Gram-positive bacteria and fungi, respectively. Here we studied the involvement of the NF-κB transcription factor Relish, which is a mediator of the IMD pathway, in the humoral immune response to the Gram-positive bacteria Micrococcus luteus and Bacillus subtilis and the entomopathogenic fungus Metarhizium anisopliae, using D. melanogaster S2 cells as a model. Activation of Relish proteolysis was observed after S2 cell treatment with the control Gram-negative bacterium Escherichia coli. We found that M. luteus had also a noticeable effect on Relish activation, while B. subtilis and M. anisopliae effects were modest. Activation patterns of the genes encoding predominantly the IMD-pathway-dependent antimicrobial peptides (AMPs) and peptidoglycan recognition proteins (PGRPs), as well as the levels of Relish recruitment to the promoters of the genes, were found to be very similar in S2 cells treated with E. coli or M. luteus but were lower and differed in the case of B. subtilis and M. anisopliae. A Relish knockdown (KD) decreased the induction levels observed for all AMP and some PGRP genes in response to M. luteus treatment and the induction levels observed for several AMP genes after M. anisopliae and B. subtilis exposures. Therefore, our findings suggest that Relish plays a critical role in inducing the humoral immune response in Drosophila S2 cells, contributing primarily to the response against M. luteus and, to a lesser extent, to the responses against B. subtilis and M. anisopliae. ### Competing Interest Statement The authors have declared no competing interest.

Intracellular trafficking plays a critical role in the functioning of highly polarized cells, such as neurons. Transport of mRNAs, proteins, and other molecules to synaptic terminals maintains contact between neurons and ensures the transmission of nerve impulses. Cytoplasmic polyadenylation element binding (CPEB) proteins play an essential role in long-term memory (LTM) formation by regulating local translation in synapses. Here, we show that the 3′UTR of the Drosophila CPEB gene orb2 is required for targeting the orb2 mRNA and protein to synapses and that this localization is important for LTM formation. When the orb2 3′UTR is deleted, the orb2 mRNAs and proteins fail to localize in synaptic fractions, and pronounced LTM deficits arise. We found that the phenotypic effects of the orb2 3′UTR deletion were rescued by introducing the 3′UTR from the orb, another Drosophila CPEB gene. In contrast, the phenotypic effects of the 3′UTR deletion were not rescued by the 3′UTR from one of the Drosophila α-tubulin genes. Our results show that the orb2 mRNAs must be targeted to the correct locations in neurons and that proper targeting depends upon sequences in the 3′UTR.

Contribution of UCP1 single nucleotide polymorphisms (SNPs) to susceptibility for cardiometabolic pathologies (CMP) and their involvement in specific risk factors for these conditions varies across populations. We tested whether UCP1 SNPs A-3826G, A-1766G, Ala64Thr and A-112C are associated with common CMP and their risk factors across Armenia, Greece, Poland, Russia and United Kingdom. This case-control study included genotyping of these SNPs, from 2,283 Caucasians. Results were extended via systematic review and meta-analysis. In Armenia, GA genotype and A allele of Ala64Thr displayed ~2-fold higher risk for CMP compared to GG genotype and G allele, respectively (p<0.05). In Greece, A allele of Ala64Thr decreased risk of CMP by 39%. Healthy individuals with A-3826G GG genotype and carriers of mutant allele of A-112C and Ala64Thr had higher body mass index compared to those carrying other alleles. In healthy Polish, higher waist-to-hip ratio (WHR) was observed in heterozygotes A-3826G compared to AA homozygotes. Heterozygosity of A-112C and Ala64Thr SNPs was related to lower WHR in CMP individuals compared to wild type homozygotes (p<0.05). Meta-analysis showed no statistically significant odds-ratios across our SNPs (p>0.05). Concluding, the studied SNPs could be associated with the most common CMP and their risk factors in some populations.

Components of the translation apparatus, including ribosomal proteins, have been found in cell nuclei in various organisms. Components of the translation apparatus are involved in various nuclear processes, particularly those associated with genome integrity control and the nuclear stages of gene expression, such as transcription, mRNA processing, and mRNA export. Components of the translation apparatus control intranuclear trafficking; the nuclear import and export of RNA and proteins; and regulate the activity, stability, and functional recruitment of nuclear proteins. The nuclear translocation of these components is often involved in the cell response to stimulation and stress, in addition to playing critical roles in oncogenesis and viral infection. Many components of the translation apparatus are moonlighting proteins, involved in integral cell stress response and coupling of gene expression subprocesses. Thus, this phenomenon represents a significant interest for both basic and applied molecular biology. Here, we provide an overview of the current data regarding the molecular functions of translation factors and ribosomal proteins in the cell nucleus.

The chromatin remodeler SWI/SNF is an important participant in gene activation, functioning predominantly by opening the chromatin structure on promoters and enhancers. Here, we describe its novel mode of action in which SWI/SNF factors mediate the targeted action of an enhancer. We studied the functions of two signature subunits of PBAP subfamily, BAP170 and SAYP, in Drosophila. These subunits were stably tethered to a transgene reporter carrying the hsp70 core promoter. The tethered subunits mediate transcription of the reporter in a pattern that is generated by enhancers close to the insertion site in multiple loci throughout the genome. Both tethered SAYP and BAP170 recruit the whole PBAP complex to the reporter promoter. However, we found that BAP170-dependent transcription is more resistant to the depletion of other PBAP subunits, suggesting that BAP170 may play a more critical role in establishing enhancer-dependent transcription.

The main control of the synthesis of the antimicrobial peptides in the cells of Drosophila melanogaster is carried out by two signalling pathways IMD and Toll, which include the classic transcription factors of the NF‐kB family (Dif, Dorsal and Relish). The Toll pathway is activated in response to gram‐positive bacteria and fungi, and the IMD pathway is activated in response to gram‐negative bacteria.

The hereditary aspect of obesity is a major focus of modern medical genetics. The genetic background is known to determine a higher-than-average prevalence of obesity in certain regions, like Oceania. There is evidence that dysfunction of brown adipose tissue (BAT) may be a risk factor for obesity and type 2 diabetes (T2D). A significant number of studies in the field focus on the UCP family. The Ucp genes code for electron transport carriers. UCP1 (thermogenin) is the most abundant protein of the UCP superfamily and is expressed in BAT, contributing to its capability of generating heat. Single nucleotide polymorphisms (SNPs) of Ucp1–Ucp3 were recently associated with risk of cardiometabolic diseases. This review covers the main Ucp SNPs A–3826G, A–1766G, A–112C, Met229Leu, Ala64Thr ( Ucp1 ), Ala55Val, G–866A ( Ucp2 ), and C–55 T ( Ucp3 ), which may be associated with the development of obesity, disturbance in lipid metabolism, T2D, and cardiovascular diseases.

Early stages of transcription from eukaryotic promoters include two principal events: the capping of newly synthesized mRNA and the transition of RNA polymerase II from the preinitiation complex to the productive elongation state. The capping checkpoint model implies that these events are tightly coupled, which is necessary for ensuring the proper capping of newly synthesized mRNA. Recent findings also show that the capping machinery has a wider effect on transcription and the entire gene expression process. The molecular basis of these phenomena is discussed.

The Paip2 protein is a factor regulating mRNA translation and stability in the cytoplasm. It has also been found in the nuclei of several cell types in Drosophila. Here, we aim to elucidate the functions of Paip2 in the cell nucleus. We find that nuclear Paip2 is a component of an ~300‐kDa protein complex. Paip2 interacts with mRNA capping factor and factors of RNA polymerase II (Pol II) transcription initiation and early elongation. Paip2 functionally cooperates with the Cbp80 subunit of the cap‐binding complex, with both proteins ensuring proper Pol II C‐terminal domain (CTD) Ser5 phosphorylation at the promoter. Thus, Paip2 is a novel player at the stage of mRNA capping and early Pol II elongation.

Paip2 (Poly(A)-binding protein - interacting protein 2) is a conserved metazoan-specific protein that has been implicated in regulating the translation and stability of mRNAs. However, we have found that Paip2 is not restricted to the cytoplasm but is also found in the nucleus in Drosophila embryos, salivary glands, testes, and tissue culture cells. Nuclear Paip2 is associated with chromatin, and in chromatin immunoprecipitation experiments it maps to the promoter regions of active genes. However, this chromatin association is indirect, as it is RNA-dependent. Thus, Paip2 is one more item in the growing list of translation factors that are recruited to mRNAs co-transcriptionally.

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.

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.