Nucleic Acids Research最新文献

Inducible systems are crucial to metabolic engineering and synthetic biology, enabling organisms that function as biosensors and produce valuable compounds. However, almost all inducible systems are strain-specific, limiting comparative analyses and applications across strains rapidly. This study designed and presented a robust workflow for developing the cross-species inducible system. By applying this approach, two reconstructed inducible systems (a 2,4-diacetylphloroglucinol-inducible system PphlF3R1 and an anhydrotetracycline-inducible system Ptet2R2*) were successfully developed and demonstrated to function in three model microorganisms, including Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum. To enhance their practicality, both inducible systems were subsequently placed on the plasmid and genome for detailed characterization to determine the optimal expression conditions. Furthermore, the more efficient inducible system Ptet2R2* was employed to express various reporter proteins and gene clusters in these three strains. Moreover, the aTc-inducible system Ptet2R2*, combined with T7 RNA polymerase and dCas12a, was utilized to develop a single-input genetic circuit that enables the simultaneous activation and repression of gene expression. Overall, the cross-species inducible system serves as a stringent, controllable and effective tool for protein expression and metabolic pathway control in different bacteria.

edgeR is an R/Bioconductor software package for differential analyses of sequencing data in the form of read counts for genes or genomic features. Over the past 15 years, edgeR has been a popular choice for statistical analysis of data from sequencing technologies such as RNA-seq or ChIP-seq. edgeR pioneered the use of the negative binomial distribution to model read count data with replicates and the use of generalized linear models to analyze complex experimental designs. edgeR implements empirical Bayes moderation methods to allow reliable inference when the number of replicates is small. This article announces edgeR version 4, which includes new developments across a range of application areas. Infrastructure improvements include support for fractional counts, implementation of model fitting in C and a new statistical treatment of the quasi-likelihood pipeline that improves accuracy for small counts. The revised package has new functionality for differential methylation analysis, differential transcript expression, differential transcript and exon usage, testing relative to a fold-change threshold and pathway analysis. This article reviews the statistical framework and computational implementation of edgeR, briefly summarizing all the existing features and functionalities but with special attention to new features and those that have not been described previously.

Transcription factors bind to sequence motifs and act as activators or repressors. Transcription factors interface with a constellation of accessory cofactors to regulate distinct mechanistic steps to regulate transcription. We rapidly degraded the essential and pervasively expressed transcription factor ZNF143 to determine its function in the transcription cycle. ZNF143 facilitates RNA polymerase initiation and activates gene expression. ZNF143 binds the promoter of nearly all its activated target genes. ZNF143 also binds near the site of genic transcription initiation to directly repress a subset of genes. Although ZNF143 stimulates initiation at ZNF143-repressed genes (i.e. those that increase transcription upon ZNF143 depletion), the molecular context of binding leads to cis repression. ZNF143 competes with other more efficient activators for promoter access, physically occludes transcription initiation sites and promoter-proximal sequence elements, and acts as a molecular roadblock to RNA polymerases during early elongation. The term context specific is often invoked to describe transcription factors that have both activation and repression functions. We define the context and molecular mechanisms of ZNF143-mediated cis activation and repression.

The proteins SFPQ (splicing Factor Proline/Glutamine rich) and NONO (non-POU domain-containing octamer-binding protein) are mammalian members of the Drosophila Behaviour/Human Splicing (DBHS) protein family, which share 76% sequence identity in their conserved 320 amino acid DBHS domain. SFPQ and NONO are involved in all steps of post-transcriptional regulation and are primarily located in mammalian paraspeckles: liquid phase-separated, ribonucleoprotein sub-nuclear bodies templated by NEAT1 long non-coding RNA. A combination of structured and low-complexity regions provide polyvalent interaction interfaces that facilitate homo- and heterodimerisation, polymerisation, interactions with oligonucleotides, mRNA, long non-coding RNA, and liquid phase-separation, all of which have been implicated in cellular homeostasis and neurological diseases including neuroblastoma. The strength and competition of these interaction modes define the ability of DBHS proteins to dissociate from paraspeckles to fulfil functional roles throughout the nucleus or the cytoplasm. In this study, we define and dissect the coiled-coil interactions which promote the polymerisation of DBHS proteins, using a crystal structure of an SFPQ/NONO heterodimer which reveals a flexible coiled-coil interaction interface which differs from previous studies. We support this through extensive solution small-angle X-ray scattering experiments using a panel of SFPQ/NONO heterodimer variants which are capable of tetramerisation to varying extents. The QM mutant displayed a negligible amount of tetramerisation (quadruple loss of function coiled-coil mutant L535A/L539A/L546A/M549A), the Charged Single Alpha Helix (ΔCSAH) variant displayed a dimer-tetramer equilibrium interaction, and the disulfide-forming variant displayed constitutive tetramerisation (R542C which mimics the pathological Drosophila nonAdiss allele). We demonstrate that newly characterised coiled-coil interfaces play a role in the polymerisation of DBHS proteins in addition to the previously described canonical coiled-coil interface. The detail of these interactions provides insight into a process critical for the assembly of paraspeckles as well as the behaviour of SFPQ as a transcription factor, and general multipurpose auxiliary protein with functions essential to mammalian life. Our understanding of the coiled coil behaviour of SFPQ also enhances the explanatory power of mutations (often disease-associated) observed in the DBHS family, potentially allowing for the development of future medical options such as targeted gene therapy.