The Role of mRNA Alternative Splicing in Macrophages Infected with Mycobacterium tuberculosis: A Field Needing to Be Discovered.

Mycobacterium tuberculosis (Mtb) is one of the major causes of human death. In its battle with humans, Mtb has fully adapted to its host and developed ways to evade the immune system. At the same time, the human immune system has developed ways to respond to Mtb. The immune system responds to viral and bacterial infections through a variety of mechanisms, one of which is alternative splicing. In this study, we summarized the overall changes in alternative splicing of the transcriptome after macrophages were infected with Mtb. We found that after infection with Mtb, cells undergo changes, including (1) directly reducing the expression of splicing factors, which affects the regulation of gene expression, (2) altering the original function of proteins through splicing, which can involve gene truncation or changes in protein domains, and (3) expressing unique isoforms that may contribute to the identification and development of tuberculosis biomarkers. Moreover, alternative splicing regulation of immune-related genes, such as IL-4, IL-7, IL-7R, and IL-12R, may be an important factor affecting the activation or dormancy state of Mtb. These will help to fully understand the immune response to Mtb infection, which is crucial for the development of tuberculosis biomarkers and new drug targets.

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.

Competing RNA pairings in complex alternative splicing of a 3' variable region.

Alternative pre-mRNA splicing remarkably expands protein diversity in eukaryotes. Drosophila PGRP-LC can 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-LC pre-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 CG42235 and Pip Our findings will facilitate a better understanding of the regulatory mechanisms of highly complex alternative splicing as well as highly variable 3' processing.

A chelicerate-specific burst of nonclassical Dscam diversity.

BACKGROUND: The immunoglobulin (Ig) superfamily receptor Down syndrome cell adhesion molecule (Dscam) gene can generate tens of thousands of isoforms via alternative splicing, which is essential for both nervous and immune systems in insects. However, further information is required to develop a comprehensive view of Dscam diversification across the broad spectrum of Chelicerata clades, a basal branch of arthropods and the second largest group of terrestrial animals. RESULTS: In this study, a genome-wide comprehensive analysis of Dscam genes across Chelicerata species revealed a burst of nonclassical Dscams, categorised into four types-mDscam, sDscamα, sDscamß, and sDscamγ-based on their size and structure. Although the mDscam gene class includes the highest number of Dscam genes, the sDscam genes utilise alternative promoters to expand protein diversity. Furthermore, we indicated that the 5' cassette duplicate is inversely correlated with the sDscam gene duplicate. We showed differential and sDscam- biased expression of nonclassical Dscam isoforms. Thus, the Dscam isoform repertoire across Chelicerata is entirely dominated by the number and expression levels of nonclassical Dscams. Taken together, these data show that Chelicerata evolved a large conserved and lineage-specific repertoire of nonclassical Dscams. CONCLUSIONS: This study showed that arthropods have a large diversified Chelicerata-specific repertoire of nonclassical Dscam isoforms, which are structurally and mechanistically distinct from those of insects. These findings provide a global framework for the evolution of Dscam diversity in arthropods and offer mechanistic insights into the diversification of the clade-specific Ig superfamily repertoire.

Long-range RNA pairings contribute to mutually exclusive splicing.

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 Dscam exon cluster. Here, we reveal a molecular model for the regulation of the mutually exclusive splicing of the serpent pre-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 Dscam gene. 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.

Role and convergent evolution of competing RNA secondary structures in mutually exclusive splicing.

Exon 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 MRP1 genes 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 MRP exon 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.

Progress of FtsZ inhibitors as novel antibiotics leads.

Bacterial cell division is an attractive target for new antibiotics. FtsZ is a major cytoskeletal protein widespread among archaea and bacteria. FtsZ has a filament-forming GTPase and a structural homologue of eukaryotic tubulin. FtsZ has been validated as a target for antibiotics. This review summarizes the chemical features, binding sites, mechanisms of action, and minimum inhibitory concentration of FtsZ inhibitors.

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.

A large family of Dscam genes with tandemly arrayed 5' cassettes in Chelicerata.

Drosophila Dscam1 (Down Syndrome Cell Adhesion Molecules) and vertebrate clustered protocadherins (Pcdhs) are two classic examples of the extraordinary isoform diversity from a single genomic locus. Dscam1 encodes 38,016 distinct isoforms via mutually exclusive splicing in D. melanogaster, while the vertebrate clustered Pcdhs utilize alternative promoters to generate isoform diversity. Here we reveal a shortened Dscam gene family with tandemly arrayed 5' cassettes in Chelicerata. These cassette repeats generally comprise two or four exons, corresponding to variable Immunoglobulin 7 (Ig7) or Ig7-8 domains of Drosophila Dscam1. Furthermore, extraordinary isoform diversity has been generated through a combination of alternating promoter and alternative splicing. These sDscams have a high sequence similarity with Drosophila Dscam1, and share striking organizational resemblance to the 5' variable regions of vertebrate clustered Pcdhs. Hence, our findings have important implications for understanding the functional similarities between Drosophila Dscam1 and vertebrate Pcdhs, and may provide further mechanistic insights into the regulation of isoform diversity.

Antibiotic drugs targeting bacterial RNAs.

RNAs have diverse structures that include bulges and internal loops able to form tertiary contacts or serve as ligand binding sites. The recent increase in structural and functional information related to RNAs has put them in the limelight as a drug target for small molecule therapy. In addition, the recognition of the marked difference between prokaryotic and eukaryotic rRNA has led to the development of antibiotics that specifically target bacterial rRNA, reduce protein translation and thereby inhibit bacterial growth. To facilitate the development of new antibiotics targeting RNA, we here review the literature concerning such antibiotics, mRNA, riboswitch and tRNA and the key methodologies used for their screening.

Molecular basis underlying Mycobacterium tuberculosis D-cycloserine resistance. Is there a role for ubiquinone and menaquinone metabolic pathways?

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. tuberculosis is 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 coli and 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.

The structure, function, and regulation of Mycobacterium FtsZ.

FtsZ is a widely distributed major cytoskeletal protein involved in the archaea and bacteria cell division. It is the most critical component in the division machinery and similar to tubulin in structure and function. Four major roles of FtsZ have been characterized: cell elongation, GTPase, cell division, and bacterial cytoskeleton. FtsZ subunits can be assembled into protofilaments. Mycobacteria consist of a large family of medical and environmental important bacteria, such as M. leprae, M. tuberculosis, the pathogen of leprosy, and tuberculosis. Structure, function, and regulation of mycobacteria FtsZ are summarized here, together with the implication of FtsZ as potential novel drug target for anti-tuberculosis therapeutics.

Autophagy during Mycobacterium tuberculosis infection and implications for future tuberculosis medications.

Autophagy is a cellular homeostasis mechanism to eliminate unwanted or excessive organelles, or for the turnover of long-life cytosolic macromolecules. During Mycobacterium tuberculosis infection, autophagy represents not only an antimicrobial mechanism for the clearance of the intracellular pathogen, but also prevents excessive inflammation, avoiding the adverse effects on host. Here we focus on the anti-tuberculosis autophagy and signal pathways involved, and attempt to depict an integrative map of the interaction between autophagy and cytokine, ROS production, vitamin D, and inflammatory response. Novel autophagy-based therapy is also summarized. This integrative insight might add some novel thoughts for better tuberculosis medications.