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Alternative splicing is crucial for cancer progression and can be targeted pharmacologically, yet identifying driver exons genome-wide remains challenging. We propose identifying such exons by associating statistically gene-level cancer dependencies from knockdown viability screens with splicing profiles and gene expression. Our models predict the effects of splicing perturbations on cell proliferation from transcriptomic data, enabling in silico RNA screening and prioritizing targets for splicing-based therapies. We identified 1,073 exons impacting cell proliferation, many from genes not previously linked to cancer. Experimental validation confirms their influence on proliferation, especially in highly proliferative cancer cell lines. Integrating pharmacological screens with splicing dependencies highlights the potential driver exons affecting drug sensitivity. Our models also allow predicting treatment outcomes from tumor transcriptomes, suggesting applications in precision oncology. This study presents an approach to identifying cancer driver exon and their therapeutic potential, emphasizing alternative splicing as a cancer target., (© 2024. The Author(s).)

A non-pathogenic Mycoplasma pneumoniae-based chassis is leading the development of live biotherapeutic products (LBPs) for respiratory diseases. However, reports connecting Guillain-Barré syndrome (GBS) cases to prior M. pneumoniae infections represent a concern for exploiting such a chassis. Galactolipids, especially galactocerebroside (GalCer), are considered the most likely M. pneumoniae antigens triggering autoimmune responses associated with GBS development. In this work, we generated different strains lacking genes involved in galactolipids biosynthesis. Glycolipid profiling of the strains demonstrated that some mutants show a complete lack of galactolipids. Cross-reactivity assays with sera from GBS patients with prior M. pneumoniae infection showed that certain engineered strains exhibit reduced antibody recognition. However, correlation analyses of these results with the glycolipid profile of the engineered strains suggest that other factors different from GalCer contribute to sera recognition, including total ceramide levels, dihexosylceramide (DHCer), and diglycosyldiacylglycerol (DGDAG). Finally, we discuss the best candidate strains as potential GBS-free Mycoplasma chassis., Competing Interests: Declaration of competing interest The results published in this article are covered by patents PCT/EP2021/057122 and US2023/0310564 A1(licensed to Pulmobiotics S.L). L.S. and M.L.-S. are shareholders of Pulmobiotics S.L.. C.P.-L., R.M., and M.L.-S. are employees and have stock options of Pulmobiotics S.L. The remaining authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The regulation of exon inclusion through alternative splicing tunes the cell's behavior by increasing the functional diversity of the transcriptome and the proteome. Splicing factors work in concert to generate gene isoform pools that contribute to cell phenotypes yet their activity is controlled by multiple regulatory and signaling layers. This hinders identification of functional, phenotype-specific splicing factors using traditional single-omic measurements, such as their mutational state or expression. To address this challenge, we propose repurposing the virtual inference of protein activity by enriched regulon analysis (VIPER) to measure splicing factor activity solely from their downstream exon transcriptomic inclusion signatures. This approach is effective in assessing the effect of co-occurring splicing factor perturbations, as well as their post-translational regulation. As proof of concept, we dissect recurrent splicing factor programs underlying tumorigenesis including aberrantly activated factors acting as oncogenes and inactivated ones acting as tumor suppressors, which are undetectable by more conventional methodologies. Activation and inactivation of these cancer splicing programs effectively stratifies overall survival, as well as cancer hallmarks such as proliferation and immune evasion. Altogether, repurposing network-based inference of protein activity for splicing factor networks distills common, functionally relevant splicing programs in otherwise heterogeneous molecular contexts., Competing Interests: CONFLICTS OF INTEREST Dr. Califano is founder, equity holder, and consultant of DarwinHealth Inc., a company that has licensed some of the algorithms used in this manuscript from Columbia University. Columbia University is also an equity holder in DarwinHealth Inc. The rest of authors declare no conflicts of interest.

Identifying open reading frames (ORFs) being translated is not a trivial task. ProTInSeq is a technique designed to characterize proteomes by sequencing transposon insertions engineered to express a selection marker when they occur in-frame within a protein-coding gene. In the bacterium Mycoplasma pneumoniae, ProTInSeq identifies 83% of its annotated proteins, along with 5 proteins and 153 small ORF-encoded proteins (SEPs; ≤100 aa) that were not previously annotated. Moreover, ProTInSeq can be utilized for detecting translational noise, as well as for relative quantification and transmembrane topology estimation of fitness and non-essential proteins. By integrating various identification approaches, the number of initially annotated SEPs in this bacterium increases from 27 to 329, with a quarter of them predicted to possess antimicrobial potential. Herein, we describe a methodology complementary to Ribo-Seq and mass spectroscopy that can identify SEPs while providing other insights in a proteome with a flexible and cost-effective DNA ultra-deep sequencing approach., (© 2024. The Author(s).)

The development of advanced genetic tools is boosting microbial engineering which can potentially tackle wide-ranging challenges currently faced by our society. Here we present SURE editing, a multi-recombinase engineering rationale combining oligonucleotide recombineering with the selective capacity of antibiotic resistance via transient insertion of selector plasmids. We test this method in Mycoplasma pneumoniae, a bacterium with a very inefficient native recombination machinery. Using SURE editing, we can seamlessly generate, in a single step, a wide variety of genome modifications at high efficiencies, including the largest possible deletion of this genome (30 Kb) and the targeted complementation of essential genes in the deletion of a region of interest. Additional steps can be taken to remove the selector plasmid from the edited area, to obtain markerless or even scarless edits. Of note, SURE editing is compatible with different site-specific recombinases for mediating transient plasmid integration. This battery of selector plasmids can be used to select different edits, regardless of the target sequence, which significantly reduces the cloning load associated to genome engineering projects. Given the proven functionality in several microorganisms of the machinery behind the SURE editing logic, this method is likely to represent a valuable advance for the synthetic biology field., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Independent Component Analysis (ICA) allows the dissection of omic datasets into modules that help to interpret global molecular signatures. The inherent randomness of this algorithm can be overcome by clustering many iterations of ICA together to obtain robust components. Existing algorithms for robust ICA are dependent on the choice of clustering method and on computing a potentially biased and large Pearson distance matrix., Results: We present robustica, a Python-based package to compute robust independent components with a fully customizable clustering algorithm and distance metric. Here, we exploited its customizability to revisit and optimize robust ICA systematically. Of the 6 popular clustering algorithms considered, DBSCAN performed the best at clustering independent components across ICA iterations. To enable using Euclidean distances, we created a subroutine that infers and corrects the components' signs across ICA iterations. Our subroutine increased the resolution, robustness, and computational efficiency of the algorithm. Finally, we show the applicability of robustica by dissecting over 500 tumor samples from low-grade glioma (LGG) patients, where we define two new gene expression modules with key modulators of tumor progression upon IDH1 and TP53 mutagenesis., Conclusion: robustica brings precise, efficient, and customizable robust ICA into the Python toolbox. Through its customizability, we explored how different clustering algorithms and distance metrics can further optimize robust ICA. Then, we showcased how robustica can be used to discover gene modules associated with combinations of features of biological interest. Taken together, given the broad applicability of ICA for omic data analysis, we envision robustica will facilitate the seamless computation and integration of robust independent components in large pipelines., (© 2022. The Author(s).)

Retinal photoreceptors have a distinct transcriptomic profile compared to other neuronal subtypes, likely reflecting their unique cellular morphology and function in the detection of light stimuli by way of the ciliary outer segment. We discovered a layer of this molecular specialization by revealing that the vertebrate retina expresses the largest number of tissue-enriched microexons of all tissue types. A subset of these microexons is included exclusively in photoreceptor transcripts, particularly in genes involved in cilia biogenesis and vesicle-mediated transport. This microexon program is regulated by Srrm3 , a paralog of the neural microexon regulator Srrm4 . Despite the fact that both proteins positively regulate retina microexons in vitro, only Srrm3 is highly expressed in mature photoreceptors. Its deletion in zebrafish results in widespread down-regulation of microexon inclusion from early developmental stages, followed by other transcriptomic alterations, severe photoreceptor defects, and blindness. These results shed light on the transcriptomic specialization and functionality of photoreceptors, uncovering unique cell type-specific roles for Srrm3 and microexons with implications for retinal diseases.

Mycoplasmas have exceptionally streamlined genomes and are strongly adapted to their many hosts, which provide them with essential nutrients. Owing to their relative genomic simplicity, Mycoplasmas have been used to develop chassis for biotechnological applications. However, the dearth of robust and precise toolkits for genomic manipulation and tight regulation has hindered any substantial advance. Herein we describe the construction of a robust genetic toolkit for M. pneumoniae, and its successful deployment to engineer synthetic gene switches that control and limit Mycoplasma growth, for biosafety containment applications. We found these synthetic gene circuits to be stable and robust in the long-term, in the context of a minimal cell. With this work, we lay a foundation to develop viable and robust biosafety systems to exploit a synthetic Mycoplasma chassis for live attenuated vectors for therapeutic applications., (© 2022. The Author(s).)

The removal of unwanted genetic material is a key aspect in many synthetic biology efforts and often requires preliminary knowledge of which genomic regions are dispensable. Typically, these efforts are guided by transposon mutagenesis studies, coupled to deepsequencing (TnSeq) to identify insertion points and gene essentiality. However, epistatic interactions can cause unforeseen changes in essentiality after the deletion of a gene, leading to the redundancy of these essentiality maps. Here, we present LoxTnSeq, a new methodology to generate and catalogue libraries of genome reduction mutants. LoxTnSeq combines random integration of lox sites by transposon mutagenesis, and the generation of mutants via Cre recombinase, catalogued via deep sequencing. When LoxTnSeq was applied to the naturally genome reduced bacterium Mycoplasma pneumoniae, we obtained a mutant pool containing 285 unique deletions. These deletions spanned from > 50 bp to 28 Kb, which represents 21% of the total genome. LoxTnSeq also highlighted large regions of non-essential genes that could be removed simultaneously, and other non-essential regions that could not, providing a guide for future genome reductions., (© 2020 The Authors. Microbial Biotechnology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)