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The Drosophila melanogastergene Dscam1potentially 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 Dscam1in the two midge species (Belgica antarcticaand Clunio marinus). Surprisingly, midge Dscam1encodes 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 Dscam1alternative splicing.

Alternative pre-mRNA splicing remarkably expands protein diversity in eukaryotes. Drosophila PGRP-LCcan generate three major 3′ splice isoforms that exhibit distinct innate immune recognition and defenses against various microbial infections. However, the regulatory mechanisms underlying the uniquely biased splicing pattern at the 3′ variable region remain unclear. Here we show that competing RNA pairings control the unique splicing of the 3′ variable region of Drosophila PGRP-LCpre-mRNA. We reveal three roles by which these RNA pairings jointly regulate the 3′ variant selection through activating the proximal 3′ splice site and concurrently masking the intron-proximal 5′ splice site, in combination with physical competition of RNA pairing. We also reveal that competing RNA pairings regulate alternative splicing of the highly complex 3′ variable regions of Drosophila CG42235and Pip. Our findings will facilitate a better understanding of the regulatory mechanisms of highly complex alternative splicing as well as highly variable 3′ processing.

ABSTRACTExon or cassette duplication is an important means of expanding protein and functional diversity through mutually exclusive splicing. However, the mechanistic basis of this process in non-arthropod species remains poorly understood. Here, we demonstrate that MRP1genes underwent tandem exon duplication in Nematoda, Platyhelminthes, Annelida, Mollusca, Arthropoda, Echinodermata, and early-diverging Chordata but not in late-diverging vertebrates. Interestingly, these events were of independent origin in different phyla, suggesting convergent evolution of alternative splicing. Furthermore, we showed that multiple sets of clade-conserved RNA pairings evolved to guide species-specific mutually exclusive splicing in Arthropoda. Importantly, we also identified a similar structural code in MRPexon clusters of the annelid, Capitella teleta, and chordate, Branchiostoma belcheri, suggesting an evolutionarily conserved competing pairing-guided mechanism in bilaterians. Taken together, these data reveal the molecular determinants and RNA pairing-guided evolution of species-specific mutually exclusive splicing spanning more than 600 million years of bilaterian evolution. These findings have a significant impact on our understanding of the evolution of and mechanism underpinning isoform diversity and complex gene structure.

Mutually exclusive splicing is an important means of increasing the protein repertoire, by which the Down's syndrome cell adhesion molecule (Dscam) gene potentially generates 38,016 different isoforms in Drosophila melanogaster. However, the regulatory mechanisms remain obscure due to the complexity of the Dscamexon cluster. Here, we reveal a molecular model for the regulation of the mutually exclusive splicing of the serpentpre-mRNA based on competition between upstream and downstream RNA pairings. Such dual RNA pairings confer fine tuning of the inclusion of alternative exons. Moreover, we demonstrate that the splicing outcome of alternative exons is mediated in relative pairing strength-correlated mode. Combined comparative genomics analysis and experimental evidence revealed similar bidirectional structural architectures in exon clusters 4 and 9 of the Dscamgene. Our findings provide a novel mechanistic framework for the regulation of mutually exclusive splicing and may offer potentially applicable insights into long-range RNA–RNA interactions in gene regulatory networks.

Introduction:Tuberculosis remains a formidable threat to global public health. Multidrug-resistant tuberculosis presents increasing burden on the control strategy. D-Cycloserine (DCS) is an effective second-line drug against Mycobacterium tuberculosis(M. tuberculosis), the causative agent of tuberculosis. Though less potent than isoniazid (INH) and streptomycin, DCS is crucial for antibiotic-resistant tuberculosis. One advantage of DCS is that less drug-resistant M. tuberculosisis reported in comparison with first-line antituberculosis drugs such as INH and rifampin.Areas covered:In this review, we summarise our current knowledge of DCS, and review the drug target and low-level resistance of DCS in M. tuberculosis.We summarise the metabolism of D-alanine (D-Ala) and peptidoglycan biosynthesis in bacteria. We first compared the amino acid similarity of Mycobacterium alanine racemase and D-Ala:D-alanine ligase and quite unexpectedly found that the two enzymes are highly conserved among Mycobacterium.Expert opinion:We summarise the drug targets of DCS and possible mechanisms underlying its low-level resistance for the first time. One significant finding is that ubiquinone and menaquinone metabolism-related genes are novel genes underlying DCS resistance in Escherichia coliand with homologues in M. tuberculosis.Further understanding of DCS targets and basis for its low-level resistance might inspire us to improve the use of DCS or find better drug targets.

Drosophila melanogasterDown 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 Dscam1splicing 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.