ReadZS detects cell type-specific and developmentally regulated RNA processing programs in single-cell RNA-seq.
2022; 23 (1): 226
RNA processing, including splicing and alternative polyadenylation, is crucial to gene function and regulation, but methods to detect RNA processing from single-cell RNA sequencing data are limited by reliance on pre-existing annotations, peak calling heuristics, and collapsing measurements by cell type. We introduce ReadZS, an annotation-free statistical approach to identify regulated RNA processing in single cells. ReadZS discovers cell type-specific RNA processing in human lung and conserved, developmentally regulated RNA processing in mammalian spermatogenesis-including global 3' UTR shortening in human spermatogenesis. ReadZS also discovers global 3' UTR lengthening in Arabidopsis development, highlighting the usefulness of this method in under-annotated transcriptomes.
View details for DOI 10.1186/s13059-022-02795-8
View details for PubMedID 36284317
Large-scale chemical-genetics yields new M. tuberculosis inhibitor classes.
New antibiotics are needed to combat rising levels of resistance, with new Mycobacterium tuberculosis (Mtb) drugs having the highest priority. However, conventional whole-cell and biochemical antibiotic screens have failed. Here we develop a strategy termed PROSPECT (primary screening of strains to prioritize expanded chemistry and targets), in which we screen compounds against pools of strains depleted of essential bacterial targets. We engineered strains that target 474 essential Mtb genes and screened pools of 100-150 strains against activity-enriched and unbiased compound libraries, probing more than 8.5million chemical-genetic interactions. Primary screens identified over tenfold more hits than screening wild-type Mtb alone, with chemical-genetic interactions providing immediate, direct target insights. We identified over 40 compounds that target DNA gyrase, the cell wall, tryptophan, folate biosynthesis and RNA polymerase, as well as inhibitors that target EfpA. Chemical optimization yielded EfpA inhibitors with potent wild-type activity, thus demonstrating the ability of PROSPECT to yield inhibitors against targets that would have eluded conventional drug discovery.
View details for DOI 10.1038/s41586-019-1315-z
View details for PubMedID 31217586
Euler buckling and nonlinear kinking of double-stranded DNA
NUCLEIC ACIDS RESEARCH
2013; 41 (21): 9881-9890
The bending stiffness of double-stranded DNA (dsDNA) at high curvatures is fundamental to its biological activity, yet this regime has been difficult to probe experimentally, and literature results have not been consistent. We created a 'molecular vise' in which base-pairing interactions generated a compressive force on sub-persistence length segments of dsDNA. Short dsDNA strands (<41 base pairs) resisted this force and remained straight; longer strands became bent, a phenomenon called 'Euler buckling'. We monitored the buckling transition via Förster Resonance Energy Transfer (FRET) between appended fluorophores. For low-to-moderate concentrations of monovalent salt (up to ∼150 mM), our results are in quantitative agreement with the worm-like chain (WLC) model of DNA elasticity, without the need to invoke any 'kinked' states. Greater concentrations of monovalent salts or 1 mM Mg(2+) induced an apparent softening of the dsDNA, which was best accounted for by a kink in the region of highest curvature. We tested the effects of all single-nucleotide mismatches on the DNA bending. Remarkably, the propensity to kink correlated with the thermodynamic destabilization of the mismatched DNA relative the perfectly complementary strand, suggesting that the kinked state is locally melted. The molecular vise is exquisitely sensitive to the sequence-dependent linear and nonlinear elastic properties of dsDNA.
View details for DOI 10.1093/nar/gkt739
View details for Web of Science ID 000327541800030
View details for PubMedID 23956222
View details for PubMedCentralID PMC3834817