The role of secondary structures in the functioning of 3' untranslated regions of mRNA: A review of functions of 3' UTRs' secondary structures and hypothetical involvement of secondary structures in cytoplasmic polyadenylation in Drosophila.

3' untranslated regions (3' UTRs) of mRNAs have many functions, including mRNA processing and transport, translational regulation, and mRNA degradation and stability. These different functions require cis-elements in 3' UTRs that can be either sequence motifs or RNA structures. Here we review the role of secondary structures in the functioning of 3' UTRs and discuss some of the trans-acting factors that interact with these secondary structures in eukaryotic organisms. We propose potential participation of 3'-UTR secondary structures in cytoplasmic polyadenylation in the model organism Drosophila melanogaster. Because the secondary structures of 3' UTRs are essential for post-transcriptional regulation of gene expression, their disruption leads to a wide range of disorders, including cancer and cardiovascular diseases. Trans-acting factors, such as STAU1 and nucleolin, which interact with 3'-UTR secondary structures of target transcripts, influence the pathogenesis of neurodegenerative diseases and tumor metastasis, suggesting that they are possible therapeutic targets.

The 3'UTR of the Drosophila CPEB translation factor gene orb2 plays a crucial role in spermatogenesis.

CPEB proteins are conserved translation regulators involved in multiple biological processes. One of these proteins in Drosophila, Orb2, is a principal player in spermatogenesis. It is required for meiosis and spermatid differentiation. During the later process, orb2 mRNA and protein are localized within the developing spermatid. To evaluate the role of the orb2 mRNA 3'UTR in spermatogenesis, we used the CRISPR/Cas9 system to generate a deletion of the orb2 3'UTR, orb2R. This deletion disrupts the process of spermatid differentiation but has no apparent effect on meiosis. Differentiation abnormalities include defects in the initial polarization of the 64-cell spermatid cysts, mislocalization of mRNAs and proteins in the elongating spermatid tails, altered morphology of the elongating spermatid tails, and defects in the assembly of the individualization complex. These disruptions in differentiation appear to arise because orb2 mRNA and protein are not properly localized within the 64-cell spermatid cyst.

The Reversible Carnitine Palmitoyltransferase 1 Inhibitor (Teglicar) Ameliorates the Neurodegenerative Phenotype in a Drosophila Huntington's Disease Model by Acting on the Expression of Carnitine-Related Genes.

Huntington's disease (HD) is a dramatic neurodegenerative disorder caused by the abnormal expansion of a CAG triplet in the huntingtin gene, producing an abnormal protein. As it leads to the death of neurons in the cerebral cortex, the patients primarily present with neurological symptoms, but recently metabolic changes resulting from mitochondrial dysfunction have been identified as novel pathological features. The carnitine shuttle is a complex consisting of three enzymes whose function is to transport the long-chain fatty acids into the mitochondria. Here, its pharmacological modification was used to test the hypothesis that shifting metabolism to lipid oxidation exacerbates the HD symptoms. Behavioural and transcriptional analyses were carried out on HD Drosophila model, to evaluate the involvement of the carnitine cycle in this pathogenesis. Pharmacological inhibition of CPT1, the rate-limiting enzyme of the carnitine cycle, ameliorates the HD symptoms in Drosophila, likely acting on the expression of carnitine-related genes.

Subunits of the PBAP Chromatin Remodeler Are Capable of Mediating Enhancer-Driven Transcription in Drosophila.

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.

Transcriptional quiescence in primordial germ cells.

In most animal species, newly formed primordial germ cells (PGCs) acquire the special characteristics that distinguish them from the surrounding somatic cells. Proper fate specification of the PGCs is coupled with transcriptional quiescence, whether they are segregated by determinative or inductive mechanisms. Inappropriate differentiation of PGCs into somatic cells is thought to be prevented due to repression of RNA polymerase (Pol) II-dependent transcription. In the case of a determinative mode of PGC formation (Drosophila, Caenorhabditis elegans, etc.), there is a broad downregulation of Pol II activity. By contrast, PGCs display only gene-specific repression in organisms that rely on inductive signaling-based mechanism (e.g., mice). In addition to the global block of Pol II activity in PGCs, gene expression can be suppressed in other ways, such as chromatin remodeling and Piwi-mediated RNAi. Here, we discuss the mechanisms responsible for the transcriptionally silent state of PGCs in common experimental animals, such as Drosophila, C. elegans, Danio rerio, Xenopus, and mouse. While a PGC-specific downregulation of transcription is a common feature among these organisms, the diverse nature of underlying mechanisms suggests that this functional trait likely evolved independently on several instances. We discuss the possible biological relevance of these silencing mechanisms vis-a-vis fate determination of PGCs.

Association of uncoupling protein (Ucp) gene polymorphisms with cardiometabolic diseases.

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.

Multifunctional factor ENY2 is associated with the THO complex and promotes its recruitment onto nascent mRNA.

Metazoan E(y)2/ENY2 is a multifunctional protein important for transcription activation and mRNA export, being a component of SAGA/TFTC and the mRNA export complex AMEX. Here, we show that ENY2 in Drosophila is also stably associated with THO, the complex involved in mRNP biogenesis. The ENY2-THO complex is required for normal Drosophila development, functioning independently on SAGA and AMEX. ENY2 and THO arrive on the transcribed region of the hsp70 gene after its activation, and ENY2 plays an important role in THO recruitment. ENY2 and THO show no direct association with elongating RNA polymerase II. Recruitment of ENY2 and THO occurs by their loading onto nascent mRNA, apparently immediately after its synthesis, while the AMEX component Xmas-2 is loaded onto mRNA at a later stage. Knockdown of either ENY2 or THO, but not SAGA or AMEX, affects the processing of the transcript's 3' end. Thus, ENY2, as a shared subunit of several protein complexes governing the sequential steps of gene expression, plays an important role in the coordination of these steps.

Transcription co-activator SAYP mediates the action of STAT activator.

Jak/STAT is an important signaling pathway mediating multiple events in development. We describe participation of metazoan co-activator SAYP/PHF10 in this pathway downstream of STAT. The latter, via its activation domain, interacts with the conserved core of SAYP. STAT is associated with the SAYP-containing co-activator complex BTFly and recruits BTFly onto genes. SAYP is necessary for stimulating STAT-driven transcription of numerous genes. Mutation of SAYP leads to maldevelopments similar to those observed in STAT mutants. Thus, SAYP is a novel co-activator mediating the action of STAT.

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.

SWI/SNF Complex Connects Signaling and Epigenetic State in Cells of Nervous System.

SWI/SNF protein complexes are evolutionarily conserved epigenetic regulators described in all eukaryotes. In metameric animals, the complexes are involved in all processes occurring in the nervous system, from neurogenesis to higher brain functions. On the one hand, the range of roles is wide because the SWI/SNF complexes act universally by mobilizing the nucleosomes in a chromatin template at multiple loci throughout the genome. On the other hand, the complexes mediate the action of multiple signaling pathways that control most aspects of neural tissue development and function. The issues are discussed to provide insight into the molecular basis of the multifaceted role of SWI/SNFs in cell cycle regulation, DNA repair, activation of immediate-early genes, neurogenesis, and brain and connectome formation. An overview is additionally provided for the molecular basis of nervous system pathologies associated with the SWI/SNF complexes and their contribution to neuroinflammation and neurodegeneration. Finally, we discuss the idea that SWI/SNFs act as an integration platform to connect multiple signaling and genetic programs.

Genotypes of the UCP1 gene polymorphisms and cardiometabolic diseases: A multifactorial study of association with disease probability.

Cardiometabolic diseases (CMDs) are complex disorders with a heterogenous phenotype, which are caused by multiple factors including genetic factors. Single nucleotide polymorphisms (SNPs) rs45539933 (p.Ala64Thr), rs10011540 (c.-112A>C), rs3811791 (c.-1766A>G), and rs1800592 (c.-3826A>G) in the UCP1 gene have been analyzed for association with CMDs in many studies providing controversial results. However, previous studies only considered individual UCP1 SNPs and did not evaluate them in an integrated manner, which is a more powerful approach to uncover genetic component of complex diseases. This study aimed to investigate associations between UCP1 genotype combinations and CMDs or CMD risk factors in the context of non-genetic factors. We performed multiple logistic regression analysis and proposed new methodology of testing different combinations of SNP genotypes. We found that probability of CMDs increased in presence of the three-SNP combination of genotypes with minor alleles of c.-3826A>G and p.Ala64Thr and wild allele of c.-112A>C, with increasing age, body mass index (BMI), body fat percentage (BF%) and may differ between sexes and between countries. The combination of genotypes with c.-3826A>G minor allele and wild homozygotes of c.-112A>C and p.Ala64Thr was associated with increased probability of diabetes. While combination of genotypes with minor alleles of all three SNPs reduced the CMD probability. The present results suggest that age, BMI, sex, and UCP1 three-SNP combinations of genotypes significantly contribute to CMD probability. Varying of c.-112A>C alleles in the genotype combination with minor alleles of c.-3826A>G and p.Ala64Thr markedly changes CMD probability.

lncRNA read-through regulates the BX-C insulator Fub-1.

Though long non-coding RNAs (lncRNAs) represent a substantial fraction of the Pol II transcripts in multicellular animals, only a few have known functions. Here we report that the blocking activity of the Bithorax complex (BX-C) Fub-1 boundary is segmentally regulated by its own lncRNA. The Fub-1 boundary is located between the Ultrabithorax (Ubx) gene and the bxd/pbx regulatory domain, which is responsible for regulating Ubx expression in parasegment PS6/segment A1. Fub-1 consists of two hypersensitive sites, HS1 and HS2. HS1 is an insulator while HS2 functions primarily as an lncRNA promoter. To activate Ubx expression in PS6/A1, enhancers in the bxd/pbx domain must be able to bypass Fub-1 blocking activity. We show that the expression of the Fub-1 lncRNAs in PS6/A1 from the HS2 promoter inactivates Fub-1 insulating activity. Inactivation is due to read-through as the HS2 promoter must be directed toward HS1 to disrupt blocking.

Slik maintains tissue homeostasis by preventing JNK-mediated apoptosis.

BACKGROUND: The c-Jun N-terminal kinase (JNK) pathway is an evolutionarily conserved regulator of cell death, which is essential for coordinating tissue homeostasis. In this study, we have characterized the Drosophila Ste20-like kinase Slik as a novel modulator of JNK pathway-mediated apoptotic cell death. RESULTS: First, ectopic JNK signaling-triggered cell death is enhanced by slik depletion whereas suppressed by Slik overexpression. Second, loss of slik activates JNK signaling, which results in enhanced apoptosis and impaired tissue homeostasis. In addition, genetic epistasis analysis suggests that Slik acts upstream of or in parallel to Hep to regulate JNK-mediated apoptotic cell death. Moreover, Slik is necessary and sufficient for preventing physiologic JNK signaling-mediated cell death in development. Furthermore, introduction of STK10, the human ortholog of Slik, into Drosophila restores slik depletion-induced cell death and compromised tissue homeostasis. Lastly, knockdown of STK10 in human cancer cells also leads to JNK activation, which is cancelled by expression of Slik. CONCLUSIONS: This study has uncovered an evolutionarily conserved role of Slik/STK10 in blocking JNK signaling, which is required for cell death inhibition and tissue homeostasis maintenance in development.

Long-Term Memory Formation in Drosophila Depends on the 3'UTR of CPEB Gene orb2.

Activation of local translation in neurites in response to stimulation is an important step in the formation of long-term memory (LTM). CPEB proteins are a family of translation factors involved in LTM formation. The Drosophila CPEB protein Orb2 plays an important role in the development and function of the nervous system. Mutations of the coding region of the orb2 gene have previously been shown to impair LTM formation. We found that a deletion of the 3'UTR of the orb2 gene similarly results in loss of LTM in Drosophila. As a result of the deletion, the content of the Orb2 protein remained the same in the neuron soma, but significantly decreased in synapses. Using RNA immunoprecipitation followed by high-throughput sequencing, we detected more than 6000 potential Orb2 mRNA targets expressed in the Drosophila brain. Importantly, deletion of the 3'UTR of orb2 mRNA also affected the localization of the Csp, Pyd, and Eya proteins, which are encoded by putative mRNA targets of Orb2. Therefore, the 3'UTR of the orb2 mRNA is important for the proper localization of Orb2 and other proteins in synapses of neurons and the brain as a whole, providing a molecular basis for LTM formation.

3'UTR of mRNA Encoding CPEB Protein Orb2 Plays an Essential Role in Intracellular Transport in Neurons.

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.

Transcription coactivator SAYP combines chromatin remodeler Brahma and transcription initiation factor TFIID into a single supercomplex.

Transcription activation by RNA polymerase II is a complicated process driven by combined, precisely coordinated action of a wide array of coactivator complexes, which carry out chromatin-directed activities and nucleate the assembly of the preinitiation complex on the promoter. Using various techniques, we have shown the existence of a stable coactivator supercomplex consisting of the chromatin-remodeling factor Brahma (SWI/SNF) and the transcription initiation factor TFIID, named BTFly (Brahma and TFIID in one assembly). The coupling of Brahma and TFIID is mediated by the SAYP factor, whose evolutionarily conserved activation domain SAY can directly bind to both BAP170 subunit of Brahma and TAF5 subunit of TFIID. The integrity of BTFly is crucial for its ability to activate transcription. BTFly is distributed genome-wide and appears to be a means of effective transcription activation.

Application of the 3C Method to Study the Developmental Genes in Drosophila Larvae.

A transition from one developmental stage to another is accompanied by activation of developmental programs and corresponding gene ensembles. Changes in the spatial conformation of the corresponding loci are associated with this activation and can be investigated with the help of the Chromosome Conformation Capture (3C) methodology. Application of 3C to specific developmental stages is a sophisticated task. Here, we describe the use of the 3C method to study the spatial organization of developmental loci in Drosophila larvae. We critically analyzed the existing protocols and offered our own solutions and the optimized protocol to overcome limitations. To demonstrate the efficiency of our procedure, we studied the spatial organization of the developmental locus Dad in 3rd instar Drosophila larvae. Differences in locus conformation were found between embryonic cells and living wild-type larvae. We also observed the establishment of novel regulatory interactions in the presence of an adjacent transgene upon activation of its expression in larvae. Our work fills the gap in the application of the 3C method to Drosophila larvae and provides a useful guide for establishing 3C on an animal model.

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.

The role of CPEB family proteins in the nervous system function in the norm and pathology.

Posttranscriptional gene regulation includes mRNA transport, localization, translation, and regulation of mRNA stability. CPEB (cytoplasmic polyadenylation element binding) family proteins bind to specific sites within the 3'-untranslated region and mediate poly- and deadenylation of transcripts, activating or repressing protein synthesis. As part of ribonucleoprotein complexes, the CPEB proteins participate in mRNA transport and localization to different sub-cellular compartments. The CPEB proteins are evolutionarily conserved and have similar functions in vertebrates and invertebrates. In the nervous system, the CPEB proteins are involved in cell division, neural development, learning, and memory. Here we consider the functional features of these proteins in the nervous system of phylogenetically distant organisms: Drosophila, a well-studied model, and mammals. Disruption of the CPEB proteins functioning is associated with various pathologies, such as autism spectrum disorder and brain cancer. At the same time, CPEB gene regulation can provide for a recovery of the brain function in patients with fragile X syndrome and Huntington's disease, making the CPEB genes promising targets for gene therapy.

Localization and Functional Roles of Components of the Translation Apparatus in the Eukaryotic Cell Nucleus.

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.