A potential link between aromatics-induced oviposition repellency behaviors and specific odorant receptor of Aedes albopictus.

BACKGROUND: The Asian tiger mosquito, Aedes albopictus, is a competent vector for the spread of several viral arboviruses including dengue, chikungunya, and Zika. Several vital mosquito behaviors linked to survival and reproduction are primarily dependent on a sophisticated olfactory system for semiochemical perception. However, a limited number of studies has hampered our understanding of the relationship between the A. albopictus acute olfactory system and the complex chemical world. RESULTS: Here, we performed a qRT-PCR assay on antennae from A. albopictus of differing sex, age and physiological states, and found that AalbOr10 was enriched in blood-fed female mosquitoes. We then undertook single sensillum recording to de-orphan AalbOr10 using a panel of physiologically and behaviorally relevant odorants in a Drosophila 'empty neuron' system. The results indicated that AalbOr10 was activated by seven aromatic compounds, all of which hampered egg-laying in blood-fed mosquitoes. Furthermore, using a post-RNA interference oviposition assay, we found that reducing the transcript level of AalbOr10 affected repellent activity mediated by 2-ethylphenol at low concentrations (10-4 vol/vol). Computational modeling and molecular docking studies suggested that hydrogen bonds to Y68 and Y150 mediated the interaction of 2-ethylphenol with AalbOr10. CONCLUSION: We reveal a potential link between aromatics-induced oviposition repellency behaviors and a specific odorant receptor in A. albopictus. Our findings provide a foundation for identifying active semiochemicals for the monitoring or controlling of mosquito populations. © 2024 Society of Chemical Industry.

Confluence and convergence of Dscam and Pcdh cell-recognition codes.

The ability of neurites of the same neuron to avoid each other (self-avoidance) is a conserved feature in both invertebrates and vertebrates. The key to self-avoidance is the generation of a unique subset of cell-surface proteins in individual neurons engaging in isoform-specific homophilic interactions that drive neurite repulsion rather than adhesion. Among these cell-surface proteins are fly Dscam1 and vertebrate clustered protocadherins (cPcdhs), as well as the recently characterized shortened Dscam (sDscam) in the Chelicerata. Herein, we review recent advances in our understanding of how cPcdh, Dscam, and sDscam cell-surface recognition codes are expressed and translated into cellular functions essential for neural wiring.

A systematic CRISPR screen reveals redundant and specific roles for Dscam1 isoform diversity in neuronal wiring.

Drosophila melanogaster Down syndrome cell adhesion molecule 1 (Dscam1) encodes 19,008 diverse ectodomain isoforms via the alternative splicing of exon 4, 6, and 9 clusters. However, whether individual isoforms or exon clusters have specific significance is unclear. Here, using phenotype-diversity correlation analysis, we reveal the redundant and specific roles of Dscam1 diversity in neuronal wiring. A series of deletion mutations were performed from the endogenous locus harboring exon 4, 6, or 9 clusters, reducing to 396 to 18,612 potential ectodomain isoforms. Of the 3 types of neurons assessed, dendrite self/non-self discrimination required a minimum number of isoforms (approximately 2,000), independent of exon clusters or isoforms. In contrast, normal axon patterning in the mushroom body and mechanosensory neurons requires many more isoforms that tend to associate with specific exon clusters or isoforms. We conclude that the role of the Dscam1 diversity in dendrite self/non-self discrimination is nonspecifically mediated by its isoform diversity. In contrast, a separate role requires variable domain- or isoform-related functions and is essential for other neurodevelopmental contexts, such as axonal growth and branching. Our findings shed new light on a general principle for the role of Dscam1 diversity in neuronal wiring.

Emerging Progress of RNA-Based Antitumor Therapeutics.

RNA-based therapeutics (e.g., mRNAs, siRNAs, microRNAs, ASOs, and saRNAs) have considerable potential for tumor treatment. The development and optimization of RNA modifications and delivery systems enable the stable and efficient delivery of RNA cargos in vivo to elicit an antitumor response. Targeted RNA-based therapeutics with multiple specificities and high efficacies are now available. In this review, we discuss progress in RNA-based antitumor therapeutics, including mRNAs, siRNAs, miRNAs, ASOs, saRNAs, RNA aptamers, and CRISPR-based gene editing. We focus on the immunogenicity, stability, translation efficiency, and delivery of RNA drugs, and summarize their optimization and the development of delivery systems. In addition, we describe the mechanisms by which RNA-based therapeutics induce antitumor responses. Furthermore, we review the merits and limitations of RNA cargos and their therapeutic potential for cancers.

Cis mutagenesis in vivo reveals extensive noncanonical functions of Dscam1 isoforms in neuronal wiring.

Drosophila Down syndrome cell adhesion molecule 1 (Dscam1) encodes tens of thousands of cell recognition molecules via alternative splicing, which are required for neural function. A canonical self-avoidance model seems to provide a central mechanistic basis for Dscam1 functions in neuronal wiring. Here, we reveal extensive noncanonical functions of Dscam1 isoforms in neuronal wiring. We generated a series of allelic cis mutations in Dscam1, encoding a normal number of isoforms, but with an altered isoform composition. Despite normal dendritic self-avoidance and self-/nonself-discrimination in dendritic arborization (da) neurons, which is consistent with the canonical self-avoidance model, these mutants exhibited strikingly distinct spectra of phenotypic defects in the three types of neurons: up to ∼60% defects in mushroom bodies, a significant increase in branching and growth in da neurons, and mild axonal branching defects in mechanosensory neurons. Remarkably, the altered isoform composition resulted in increased dendrite growth yet inhibited axon growth. Moreover, reducing Dscam1 dosage exacerbated axonal defects in mushroom bodies and mechanosensory neurons but reverted dendritic branching and growth defects in da neurons. This splicing-tuned regulation strategy suggests that axon and dendrite growth in diverse neurons cell-autonomously require Dscam1 isoform composition. These findings provide important insights into the functions of Dscam1 isoforms in neuronal wiring.

Structural basis for the self-recognition of sDSCAM in Chelicerata.

To create a functional neural circuit, neurons develop a molecular identity to discriminate self from non-self. The invertebrate Dscam family and vertebrate Pcdh family are implicated in determining synaptic specificity. Recently identified in Chelicerata, a shortened Dscam (sDscam) has been shown to resemble the isoform-generating characters of both Dscam and Pcdh and represent an evolutionary transition. Here we presented the molecular details of sDscam self-recognition via both trans and cis interactions using X-ray crystallographic data and functional assays. Based on our results, we proposed a molecular zipper model for the assemblies of sDscam to mediate cell-cell recognition. In this model, sDscam utilized FNIII domain to form side-by-side interactions with neighboring molecules in the same cell while established hand-in-hand interactions via Ig1 domain with molecules from another cell around. Together, our study provided a framework for understanding the assembly, recognition, and evolution of sDscam.

Trans-splicing facilitated by RNA pairing greatly expands sDscam isoform diversity but not homophilic binding specificity.

The Down syndrome cell adhesion molecule 1 (Dscam1) gene can generate tens of thousands of isoforms via alternative splicing, which is essential for nervous and immune functions. Chelicerates generate approximately 50 to 100 shortened Dscam (sDscam) isoforms by alternative promoters, similar to mammalian protocadherins. Here, we reveal that trans-splicing markedly increases the repository of sDscamß isoforms in Tetranychus urticae. Unexpectedly, every variable exon cassette engages in trans-splicing with constant exons from another cluster. Moreover, we provide evidence that competing RNA pairing not only governs alternative cis-splicing but also facilitates trans-splicing. Trans-spliced sDscam isoforms mediate cell adhesion ability but exhibit the same homophilic binding specificity as their cis-spliced counterparts. Thus, we reveal a single sDscam locus that generates diverse adhesion molecules through cis- and trans-splicing coupled with alternative promoters. These findings expand understanding of the mechanism underlying molecular diversity and have implications for the molecular control of neuronal and/or immune specificity.

Recent advances in RNA structurome.

RNA structures are essential to support RNA functions and regulation in various biological processes. Recently, a range of novel technologies have been developed to decode genome-wide RNA structures and novel modes of functionality across a wide range of species. In this review, we summarize key strategies for probing the RNA structurome and discuss the pros and cons of representative technologies. In particular, these new technologies have been applied to dissect the structural landscape of the SARS-CoV-2 RNA genome. We also summarize the functionalities of RNA structures discovered in different regulatory layers-including RNA processing, transport, localization, and mRNA translation-across viruses, bacteria, animals, and plants. We review many versatile RNA structural elements in the context of different physiological and pathological processes (e.g., cell differentiation, stress response, and viral replication). Finally, we discuss future prospects for RNA structural studies to map the RNA structurome at higher resolution and at the single-molecule and single-cell level, and to decipher novel modes of RNA structures and functions for innovative applications.

Self-avoidance alone does not explain the function of Dscam1 in mushroom body axonal wiring.

Alternative splicing of Drosophila Dscam1 into 38,016 isoforms provides neurons with a unique molecular code for self-recognition and self-avoidance. A canonical model suggests that the homophilic binding of identical Dscam1 isoforms on the sister branches of mushroom body (MB) axons supports segregation with high fidelity, even when only a single isoform is expressed. Here, we generated a series of mutant flies with a single exon 4, 6, or 9 variant, encoding 1,584, 396, or 576 potential isoforms, respectively. Surprisingly, most of the mutants in the latter two groups exhibited obvious defects in the growth, branching, and segregation of MB axonal sister branches. This demonstrates that the repertoires of 396 and 576 Dscam1 isoforms were not sufficient for the normal patterning of axonal branches. Moreover, reducing Dscam1 levels largely reversed the defects caused by reduced isoform diversity, suggesting a functional link between Dscam1 expression levels and isoform diversity. Taken together, these results indicate that canonical self-avoidance alone does not explain the function of Dscam1 in MB axonal wiring.

Hidden RNA pairings counteract the "first-come, first-served" splicing principle to drive stochastic choice in Dscam1 splice variants.

Drosophila melanogaster Dscam1 encodes 38,016 isoforms via mutually exclusive splicing; however, the regulatory mechanism behind this is not fully understood. Here, we found a set of hidden RNA secondary structures that balance the stochastic choice of Dscam1 splice variants (designated balancer RNA secondary structures). In vivo mutational analyses revealed the dual function of these balancer interactions in driving the stochastic choice of splice variants, through enhancement of the inclusion of distal exon 6s by cooperating with docking site-selector pairing to form a stronger multidomain pre-mRNA structure and through simultaneous repression of the inclusion of proximal exon 6s by antagonizing their docking site-selector pairings. Thus, we provide an elegant molecular model based on competition and cooperation between two sets of docking site-selector and balancer pairings, which counteracts the "first-come, first-served" principle. Our findings provide conceptual and mechanistic insight into the dynamics and functions of long-range RNA secondary structures.

Intron-targeted mutagenesis reveals roles for Dscam1 RNA pairing architecture-driven splicing bias in neuronal wiring.

Drosophila melanogaster Down syndrome cell adhesion molecule (Dscam1) can generate 38,016 different isoforms through largely stochastic, yet highly biased, alternative splicing. These isoforms are required for nervous functions. However, the functional significance of splicing bias remains unknown. Here, we provide evidence that Dscam1 splicing bias is required for mushroom body (MB) axonal wiring. We generate mutant flies with normal overall protein levels and an identical number but global changes in exon 4 and 9 isoform bias (DscamΔ4D-/- and DscamΔ9D-/-), respectively. In contrast to DscamΔ4D-/-, DscamΔ9D-/- exhibits remarkable MB defects, suggesting a variable domain-specific requirement for isoform bias. Importantly, changes in isoform bias cause axonal defects but do not influence the self-avoidance of axonal branches. We conclude that, in contrast to the isoform number that provides the molecular basis for neurite self-avoidance, isoform bias may play a role in MB axonal wiring by influencing non-repulsive signaling.

Complex RNA Secondary Structures Mediate Mutually Exclusive Splicing of Coleoptera Dscam1.

Mutually exclusive splicing is an important mechanism for expanding protein diversity. An extreme example is the Down syndrome cell adhesion molecular (Dscam1) gene of insects, containing four clusters of variable exons (exons 4, 6, 9, and 17), which potentially generates tens of thousands of protein isoforms through mutually exclusive splicing, of which regulatory mechanisms are still elusive. Here, we systematically analyzed the variable exon 4, 6, and 9 clusters of Dscam1 in Coleoptera species. Through comparative genomics and RNA secondary structure prediction, we found apparent evidence that the evolutionarily conserved RNA base pairing mediates mutually exclusive splicing in the Dscam1 exon 4 cluster. In contrast to the fly exon 6, most exon 6 selector sequences in Coleoptera species are partially located in the variable exon region. Besides, bidirectional RNA-RNA interactions are predicted to regulate the mutually exclusive splicing of variable exon 9 of Dscam1. Although the docking sites in exon 4 and 9 clusters are clade specific, the docking sites-selector base pairing is conserved in secondary structure level. In short, our result provided a mechanistic framework for the application of long-range RNA base pairings in regulating the mutually exclusive splicing of Coleoptera Dscam1.

RNA structures in alternative splicing and back-splicing.

Alternative splicing greatly expands the transcriptomic and proteomic diversities related to physiological and developmental processes in higher eukaryotes. Splicing of long noncoding RNAs, and back- and trans- splicing further expanded the regulatory repertoire of alternative splicing. RNA structures were shown to play an important role in regulating alternative splicing and back-splicing. Application of novel sequencing technologies made it possible to identify genome-wide RNA structures and interaction networks, which might provide new insights into RNA splicing regulation in vitro to in vivo. The emerging transcription-folding-splicing paradigm is changing our understanding of RNA alternative splicing regulation. Here, we review the insights into the roles and mechanisms of RNA structures in alternative splicing and back-splicing, as well as how disruption of these structures affects alternative splicing and then leads to human diseases. This article is categorized under: RNA Processing > Splicing Regulation/Alternative Splicing RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems.

Chelicerata sDscam isoforms combine homophilic specificities to define unique cell recognition.

Thousands of Down syndrome cell adhesion molecule (Dscam1) isoforms and ∼60 clustered protocadhrein (cPcdh) proteins are required for establishing neural circuits in insects and vertebrates, respectively. The strict homophilic specificity exhibited by these proteins has been extensively studied and is thought to be critical for their function in neuronal self-avoidance. In contrast, significantly less is known about the Dscam1-related family of ∼100 shortened Dscam (sDscam) proteins in Chelicerata. We report that Chelicerata sDscamα and some sDscamß protein trans interactions are strictly homophilic, and that the trans interaction is meditated via the first Ig domain through an antiparallel interface. Additionally, different sDscam isoforms interact promiscuously in cis via membrane proximate fibronectin-type III domains. We report that cell-cell interactions depend on the combined identity of all sDscam isoforms expressed. A single mismatched sDscam isoform can interfere with the interactions of cells that otherwise express an identical set of isoforms. Thus, our data support a model by which sDscam association in cis and trans generates a vast repertoire of combinatorial homophilic recognition specificities. We propose that in Chelicerata, sDscam combinatorial specificity is sufficient to provide each neuron with a unique identity for self-nonself discrimination. Surprisingly, while sDscams are related to Drosophila Dscam1, our results mirror the findings reported for the structurally unrelated vertebrate cPcdh. Thus, our findings suggest a remarkable example of convergent evolution for the process of neuronal self-avoidance and provide insight into the basic principles and evolution of metazoan self-avoidance and self-nonself discrimination.

RNA secondary structures in Dscam1 mutually exclusive splicing: unique evolutionary signature from the midge.

The Drosophila melanogaster gene Dscam1 potentially generates 38,016 distinct isoforms via mutually exclusive splicing, which are required for both nervous and immune functions. However, the mechanism underlying splicing regulation remains obscure. Here we show apparent evolutionary signatures characteristic of competing RNA secondary structures in exon clusters 6 and 9 of Dscam1 in the two midge species (Belgica antarctica and Clunio marinus). Surprisingly, midge Dscam1 encodes only ∼6000 different isoforms through mutually exclusive splicing. Strikingly, the docking site of the exon 6 cluster is conserved in almost all insects and crustaceans but is specific in the midge; however, the docking site-selector base-pairings are conserved. Moreover, the docking site is complementary to all predicted selector sequences downstream from every variable exon 9 of the midge Dscam1, which is in accordance with the broad spectrum of their isoform expression. This suggests that these cis-elements mainly function through the formation of long-range base-pairings. This study provides a vital insight into the evolution and mechanism of Dscam1 alternative splicing.

Role of RNA secondary structures in regulating Dscam alternative splicing.

Alternative splicing of mRNA precursors is a versatile mechanism of expanding proteomic diversity. The most striking example of this is the Drosophila melanogaster Down syndrome cell adhesion molecule (Dscam1) gene, which potentially encodes 38,016 distinct isoforms by mutually exclusive splicing. The genomic organization of Dscam1 is largely conserved across the pancrustaceans, although the number of splice isoforms varies from 2240 in the clam shrimp (Eulimnadia texana) to 121,104 in the whiteleg shrimp (Litopenaeus vannamei). RNA secondary structure plays a pivotal role in mutually exclusive splicing of Dscam1. Here, we review recent progress in the identification, evolution, and regulatory roles of RNA secondary structure in alternative splicing of Dscam1.

Effects of a 15-amino-acid isoform of amyloid- ß expressed by silkworm pupae on B6C3-Tg Alzheimer's disease transgenic mice.

Silkworms are an economically important insect.Silkworm pupae are also a nutrient-rich food and can be used as a pharmaceutical intermediate.The N-terminus of Aß includes 1-15 amino acid residues with a B cell surface antigen that is necessary to produce antibody and prevent the adverse reactions observed in response to the full Aß42 peptide. In this study, we used silkworm pupae to develop a safer vaccine for Alzheimer's disease (AD) patients. Aß15 peptide was fused with the cholera toxin B subunit (CTB) and expressed in silkworm pupae. Then, we tested an oral vaccine with the peptide expressed by silkworm pupae in a transgenic mouse model of AD. The results show that anti-Aß antibodies were induced, Aß deposition in the brain decreased, the content of malondialdehyde was lower than in the other group, and memory and cognition of the mice improved. These results suggest that the high-nutrient CTB-Aß15 silkworm pupa vaccine has a potential clinical application for the prevention of AD.

Study of the whole genome, methylome and transcriptome of Cordyceps militaris.

The complete genome of Cordyceps militaris was sequenced using single-molecule real-time (SMRT) sequencing technology at a coverage over 300×. The genome size was 32.57 Mb, and 14 contigs ranging from 0.35 to 4.58 Mb with an N50 of 2.86 Mb were assembled, including 4 contigs with telomeric sequences on both ends and an additional 8 contigs with telomeric sequences on either the 5' or 3' end. A methylome database of the genome was constructed using SMRT and m4C and m6A methylated nucleotides, and many unknown modification types were identified. The major m6A methylation motif is GA and GGAG, and the major m4C methylation motif is GC or CG/GC. In the C. militaris genome DNA, there were four types of methylated nucleotides that we confirmed using high-resolution LCMS-IT-TOF. Using PacBio Iso-Seq, a total of 31,133 complete cDNA sequences were obtained in the fruiting body. The conserved domains of the nontranscribed regions of the genome include TATA boxes, which are the initial regions of genome replication. There were 406 structural variants between the HN and CM01 strains, and there were 1,114 structural variants between the HN and ATCC strains.

Long Noncoding RNA GMAN, Up-regulated in Gastric Cancer Tissues, Is Associated With Metastasis in Patients and Promotes Translation of Ephrin A1 by Competitively Binding GMAN-AS.

BACKGROUND & AIMS: We aimed to identify long noncoding RNAs (lncRNAs) that are up-regulated in gastric cancer tissues from patients and study their function in gastric tumor metastasis. METHODS: We collected gastric tumor and nontumor tissues from patients in China and analyzed levels of lncRNAs by microarray analysis, proteins by immunohistochemistry, and RNAs by quantitative reverse-transcription polymerase chain reaction; we compared these with survival times of patients and tumor progression. RNA levels were knocked down or knocked out in BGC-823, SGC-7901, and MKN45 cell lines using small interfering or short hairpin RNAs or clustered regularly interspaced short palindromic repeats (ie, CRISPR)/CRISPR associated protein 9 (ie, Cas9) vectors. Genes were overexpressed from transfected plasmids in HGC-27 cells. Cells were analyzed by Northern blot and immunoblot, polysome profiling assay, and cell invasion assay. Cells were injected into the tail veins or spleens of nude mice or SCID mice; lung and liver tissues were collected, and metastases were counted. lncRNAs were cloned by using rapid amplification of complementary DNA ends. Their interactions with other genes were determined by RNA pulldown and mapping assays. RESULTS: In microarray analyses, we identified 151 lncRNAs expressed at significantly higher levels in gastric tumor vs nontumor tissues. Levels of an lncRNA that we called gastric cancer metastasis associated long noncoding RNA (GMAN) were increased in gastric tumor tissues, compared with nontumor tissues; its up-regulation was associated with tumor metastasis and shorter survival times of patients. The GMAN gene overlaps with the ephrin A1 gene (EFNA1) and was highly expressed in BGC-823 and MKN45 cells. Knockdown of GMAN in these cells did not affect proliferation, colony formation, or adhesion but did reduce their invasive activity in Transwell assays. Ectopic expression of GMAN increased the invasive activity of HGC-27 cells. BGC-823 and MKN45 cells with knockdown of GMAN formed fewer metastases after injection into tail veins of nude mice. Knockdown or knockout of GMAN also reduced levels of ephrin A1 protein in cells. We found that GMAN promoted translation of ephrin A1 messenger RNA into protein by binding to the antisense GMAN RNA (GMAN-AS)-this antisense sequence is also complementary to that of ephrin A1 mRNA. Levels of ephrin A1 protein were also increased in gastric tumors from patients with metastases than in those without metastases. Knockout of ephrin A1 in BGC-823 cells reduced their invasive activity in Transwell assays and ability to form metastases after injection into SCID mice. Ectopic expression of ephrin A1 in BGC-823 cells with knockdown or knockout of GMAN restored their invasive activities and ability form metastases in nude or SCID mice. A CRISPR/Cas9-based strategy to disrupt the GMAN gene significantly reduced the numbers of metastases formed from SGC-7901 cells in mice. CONCLUSIONS: We identified an lncRNA, which we call GMAN, that is increased in gastric tumors from patients and associated with survival and formation of metastases. It regulates translation of ephrin A1 mRNA by binding competitively to GMAN-AS. Knockdown or knockout of GMAN or ephrin A1 in gastric cancer cell lines reduces their invasive activity and ability to form metastases after injection into mice. These genes might be targeted to prevent or reduce gastric cancer metastasis.