Oksana Maksimenko

The Drosophila protein male-specific lethal 1 (MSL1) is part of the five-subunit male-specific lethal (MSL) complex that binds only to the X chromosome in males. MSL1 contains a 93-amino-acid N-terminal unstructured region followed by a coiled-coil domain that forms a homodimer. The MSL1 dimer and male-specific lethal 2 (MSL2), which interacts with its coiled-coil domain, form a core that is primarily responsible for the specific recruitment of the MSL complex to X-chromosome high-affinity sites (HASs). In a yeast two-hybrid analysis, we found that the N-terminal region of MSL1, primarily amino acids 66–85 and the coiled-coil domain, interacts with many DNA-binding transcription factors. The coiled-coil domain of Drosophila virilis MSL1 (vCC) retains the ability to interact with Drosophila melanogaster MSL2 but not with zinc finger proteins. Expression of a double mutant of MSL1 (MSL1^Δ20vCC), in which amino acids 66–85 (Δ20) have been deleted and the coiled-coil domain has been replaced with the vCC domain, significantly reduced dosage compensation in males due to decreased binding of the MSL complex to the X chromosome. Substitution of MSL1 with MSL1^vCC in females ectopically expressing MSL2 also significantly reduced recruitment of the MSL complex to the X chromosome. Altogether, these results indicate a key role of the N-terminal region of MSL1 not only in the assembly of the MSL complex but also in its recruitment to the X chromosome in males.

In contrast to canonical cis-splicing, trans-splicing combines exons from two distinct transcripts, yielding chimeric mRNAs. One striking example is the ubiquitously expressed modifier of the mdg4 (mod(mdg4)) locus in Drosophila, where all mRNAs spanning over 30 isoforms are generated exclusively by trans-splicing. This process combines common constitutive exons with one of many alternative 3′ variable exons transcribed from independent promoters. Here, we demonstrate that trans-splicing is determined by a 600-bp region located in the proximal part of the last common intron. Unlike other known examples of trans-splicing, complementary sequences between donor and acceptor pre-mRNAs do not trigger trans-splicing of the mod(mdg4) gene. Deletion analysis revealed that the 600-bp intronic region contains multiple redundant motifs for RNA-binding proteins (RBPs) that collectively determine the level of trans-splicing. We identified specific binding of a large group of spliceosome-associated RBPs by affinity purification of the in vitro transcribed proximal part of the intronic RNA. A multifold reduction in mRNA expression caused by the partial removal of intronic sequences is partially compensated for by an increase in the translation efficiency of Mod(mdg4) isoforms. The results were consistent with a model in which multiple RBP motifs stabilize pre-mRNA binding to the spliceosome, thereby inducing trans-splicing.

Effective approaches for selecting nuclei in which a gene of interest is in an active state are necessary for studying spatial organization and gene expression regulation. In this work, a novel two-component genetic system was created for the parasegment-specific labeling of nuclei in which the regulatory domain iab-5, which stimulates the homeotic gene Abd-B, is active. The system is based on the integration of a transgene expressing the yeast GAL4 activator under the control of the minimal hsp70 gene promoter into the iab-5 domain, near the early embryonic enhancer. As a result, the iab-5 enhancer induces specific expression of GAL4, which strongly amplifies the expression of the mScarlet fluorescent protein, allowing for the efficient selection of labeled nuclei in which the iab-5 domain is activated. This approach can be used for the selection of target nuclei in which any regulatory element or gene of interest is in an active or repressed state.

C2H2 proteins, characterized by DNA-binding C2H2-type zinc finger domains, constitute the largest group of transcription factors. In addition to binding DNA, C2H2 domains can mediate protein–protein interactions, facilitating the oligomerization of C2H2 proteins. In this study, we identified eight C2H2 proteins in the Drosophila genome that feature a unique single C2H2 domain containing a conserved “CGxΦ” motif. Yeast two-hybrid assays, size-exclusion chromatography coupled with multi-angle light scattering, and chemical cross-linking experiments revealed a strong propensity of these domains to form dimers. Using NMR spectroscopy, we determined the solution structure of the dimeric C2H2 domain from the IMZF (Immune-mediated Zinc Finger) protein, providing structural evidence for the dimerization of C2H2 domains. Dimerization is mediated by the interface between the core C2H2 fold and the adjacent β-strand containing the CGxΦ motif, which was further validated by structure-guided mutagenesis. A bioinformatic survey showed that “CGxΦ”-type C2H2 domains are specific to Diptera. Finally, our predictions demonstrate that dimerizing C2H2 domains containing additional structural elements could be widespread among eukaryotic taxa, with the highest prevalence in insects. These findings establish that single C2H2 domains can mediate self-association and identify the CGxΦ-type C2H2 domains as a distinct structural subclass specific to dipteran insects.

The CRISPR–Cas9 system has significantly advanced genome editing but remains constrained by its requirement for specific protospacer adjacent motifs (PAMs). To overcome this limitation, PAM-relaxed nucleases, including the novel near-PAMless chimeric SpRYc, have been developed. Here, we evaluated SpRYc editing activity across multiple experimental systems, including human HEK293 and CEM-R5 cells, as well as Drosophila melanogaster S2 cells and embryos. In HEK293 cells, SpRYc exhibited broad PAM compatibility, enabling editing at non-canonical PAMs, albeit with reduced and variable efficiency at canonical NGG sites compared to SpCas9. This context dependency was more pronounced in CEM-R5 T cells, where SpRYc activity at endogenous CXCR4 and B2M loci was largely restricted to NGG PAMs. In contrast, unlike SpCas9, SpRYc displayed negligible genome-editing activity in Drosophila embryos in vivo. Notably, the transcriptional activator dSpRYc-VPR showed robust activity in Drosophila S2 cells at both canonical and non-canonical PAMs. Reduced chromatin occupancy of dSpRYc-VPR suggests a balance between expanded PAM recognition and DNA-binding stability, providing a mechanistic explanation for context-dependent performance of SpRYc. Overall, our results highlight that expanded targeting flexibility comes at the cost of variable efficiency, underscoring the need for extensive locus- and context-specific validation of PAM-relaxed genome-editing tools.

CTCF (CCCTC-binding factor) is the best-studied architectural protein that is highly conserved among animals, including Drosophila and mammals. In Drosophi

The zinc finger-associated domain (ZAD), found in numerous Drosophila architectural proteins, such as Pita, enables homodimerization. Despite its prevalenc

Aptamers are promising synthetic molecular receptors that bind to specific targets by adopting a unique tertiary structure. However, their selection using standard SELEX protocols often does not allow the achievement of high affinity to the targets. Due to the lack and difficulty of obtaining data on the 3D structure of aptamers and their complexes, the design of known aptamers based on simple rules and software is in demand. The presented work considers the comparative characterization and design of DNA aptamers specific to the antibiotic kanamycin based on complementary interactions and structural motifs (bulges, mismatches, loops) predicted by NUPACK, RNAfold, and UNAFold software. The design included the elimination of non-functional parts of the aptamers and the stabilization of the kanamycin-binding loop. Seven novel aptamers, chosen based on these predictions, were synthesized, and their affinities were measured using an isothermal titration calorimetry technique. The prediction of end stem and hairpin loop structures was confirmed by comparison with circular dichroism data. As a result of sequential design with truncation of unnecessary nucleotides, a novel optimal 42-base-long aptamer was designed and demonstrated a dissociation constant of 109 ± 15 nM, which is 4.7-fold lower than the initial preparation (470 ± 40 nM) and overcomes all known aptamers to kanamycin.

Gene duplication, a major source of new genes in evolution, often occurs via reverse transcription of mRNA, leading to the integration of a retrocopy into a new genomic locus. Here, we performed an in-depth analysis of the evolutionary history of the e(y)2 gene in Metazoa. The E(y)2 protein is a shared subunit of two highly conserved complexes involved in transcription regulation (the DUB module of the SAGA complex) and mRNA transport (TREX-2). In Deuterostomes, the e(y)2 gene has undergone multiple independent retropositions, often giving rise to functional retrogenes. In contrast, among Protostomes, duplications of e(y)2 were identified only in Drosophilinae and a member of the Lepidoptera family (Manduca sexta). In Drosophila, the retrocopy e(y)2 acquired an almost ubiquitous expression pattern and compensates for the function of the parental gene in all tissues except the testes. The parental gene, e(y)2b, evolved a testis-specific expression pattern, lost the ability to incorporate into the DUB module, but retained nuclear envelope localization and the capacity to assemble into the TREX-2 complex. Knockout of the D. melanogaster e(y)2b gene resulted in reduced male fertility. Overall, our study highlights distinct evolutionary trajectories of the e(y)2 gene in Deuterostomes and Protostomes.

C2H2 proteins, characterized by DNA-binding C2H2-type zinc finger domains, constitute the largest group of transcription factors. In addition to binding DNA, C2H2 domains can mediate protein–protein interactions, facilitating oligomerization of C2H2 proteins. In this study, we identified eight C2H2 proteins in the Drosophila genome that feature a unique single C2H2 domain containing a conserved’CGEx’ motif. Yeast two-hybrid assays, SEC-MALS, and chemical cross-linking experiments revealed a strong propensity of these domains to form dimers. Using NMR spectroscopy, we determined the solution structure of the dimeric C2H2 domain from IMZF (Immune-mediated Zinc Finger) protein, providing structural evidence towards the dimerization of C2H2 domains. The structure revealed that dimerization is mediated by the interface between the core C2H2 fold and the adjacent β-strand containing’CGEx’ motif, which was further validated by structure-guided mutagenesis. A bioinformatic survey showed that’CGEx’-type C2H2 domains are specific to Diptera . Finally, we demonstrate that putatively dimerizing C2H2 domains containing additional structural elements beyond the canonical fold are widespread among diverse eukaryotic taxa, with the highest prevalence observed in insects. These findings establish that single C2H2 domains can mediate self-association and identify the’CGEx’-type C2H2 domain as a distinct structural subclass specific to Dipteran insects.