Three Essential Ribonucleases—RNase Y, J1, and III—Control the Abundance of a Majority of Bacillus subtilis mRNAs

Bacillus subtilis possesses three essential enzymes thought to be involved in mRNA decay to varying degrees, namely RNase Y, RNase J1, and RNase III. Using recently developed high-resolution tiling arrays, we examined the effect of depletion of each of these enzymes on RNA abundance over the whole genome. The data are consistent with a model in which the degradation of a significant number of transcripts is dependent on endonucleolytic cleavage by RNase Y, followed by degradation of the downstream fragment by the 5′–3′ exoribonuclease RNase J1. However, many full-size transcripts also accumulate under conditions of RNase J1 insufficiency, compatible with a model whereby RNase J1 degrades transcripts either directly from the 5′ end or very close to it. Although the abundance of a large number of transcripts was altered by depletion of RNase III, this appears to result primarily from indirect transcriptional effects. Lastly, RNase depletion led to the stabilization of many low-abundance potential regulatory RNAs, both in intergenic regions and in the antisense orientation to known transcripts

A novel framework for characterizing genomic haplotype diversity in the human immunoglobulin heavy chain locus

An incomplete ascertainment of genetic variation within the highly polymorphic immunoglobulin heavy chain locus (IGH) has hindered our ability to define genetic factors that influence antibody-mediated processes. Due to locus complexity, standard high-throughput approaches have failed to accurately and comprehensively capture IGH polymorphism. As a result, the locus has only been fully characterized two times, severely limiting our knowledge of human IGH diversity. Here, we combine targeted long-read sequencing with a novel bioinformatics tool, IGenotyper, to fully characterize IGH variation in a haplotype-specific manner. We apply this approach to eight human samples, including a haploid cell line and two mother-father-child trios, and demonstrate the ability to generate high-quality assemblies (>98% complete and >99% accurate), genotypes, and gene annotations, identifying 2 novel structural variants and 15 novel IGH alleles. We show multiplexing allows for scaling of the approach without impacting data quality, and that our genotype call sets are more accurate than short-read (>35% increase in true positives and >97% decrease in false-positives) and array/imputation-based datasets. This framework establishes a desperately needed foundation for leveraging IG genomic data to study population-level variation in antibody-mediated immunity, critical for bettering our understanding of disease risk, and responses to vaccines and therapeutics

Epigenomic characterization of Clostridioides difficile finds a conserved DNA methyltransferase that mediates sporulation and pathogenesis

Clostridioides (formerly Clostridium) difficile is a leading cause of healthcare-associated infections. Although considerable progress has been made in the understanding of its genome, the epigenome of C. difficile and its functional impact has not been systematically explored. Here, we perform a comprehensive DNA methylome analysis of C. difficile using 36 human isolates and observe a high level of epigenomic diversity. We discovered an orphan DNA methyltransferase with a well-defined specificity, the corresponding gene of which is highly conserved across our dataset and in all of the approximately 300 global C. difficile genomes examined. Inactivation of the methyltransferase gene negatively impacts sporulation, a key step in C. difficile disease transmission, and these results are consistently supported by multiomics data, genetic experiments and a mouse colonization model. Further experimental and transcriptomic analyses suggest that epigenetic regulation is associated with cell length, biofilm formation and host colonization. These findings provide a unique epigenetic dimension to characterize medically relevant biological processes in this important pathogen. This study also provides a set of methods for comparative epigenomics and integrative analysis, which we expect to be broadly applicable to bacterial epigenomic studies

Expression, maturation and turnover of DrrS, an unusually stable, DosR regulated small RNA in Mycobacterium tuberculosis

Mycobacterium tuberculosis depends on the ability to adjust to stresses encountered in a range of host environments, adjustments that require significant changes in gene expression. Small RNAs (sRNAs) play an important role as post-transcriptional regulators of prokaryotic gene expression, where they are associated with stress responses and, in the case of pathogens, adaptation to the host environment. In spite of this, the understanding of M. tuberculosis RNA biology remains limited. Here we have used a DosR-associated sRNA as an example to investigate multiple aspects of mycobacterial RNA biology that are likely to apply to other M. tuberculosis sRNAs and mRNAs. We have found that accumulation of this particular sRNA is slow but robust as cells enter stationary phase. Using reporter gene assays, we find that the sRNA core promoter is activated by DosR, and we have renamed the sRNA DrrS for DosR Regulated sRNA. Moreover, we show that DrrS is transcribed as a longer precursor, DrrS+, which is rapidly processed to the mature and highly stable DrrS. We characterise, for the first time in mycobacteria, an RNA structural determinant involved in this extraordinary stability and we show how the addition of a few nucleotides can lead to acute destabilisation. Finally, we show how this RNA element can enhance expression of a heterologous gene. Thus, the element, as well as its destabilising derivatives may be employed to post-transcriptionally regulate gene expression in mycobacteria in combination with different promoter variants. Moreover, our findings will facilitate further investigations into the severely understudied topic of mycobacterial RNA biology and into the role that regulatory RNA plays in M. tuberculosis pathogenesis

Assembly and diploid architecture of an individual human genome via single-molecule technologies

We present the first comprehensive analysis of a diploid human genome that combines single-molecule sequencing with single-molecule genome maps. Our hybrid assembly markedly improves upon the contiguity observed from traditional shotgun sequencing approaches, with scaffold N50 values approaching 30 Mb, and we identified complex structural variants (SVs) missed by other high-throughput approaches. Furthermore, by combining Illumina short-read data with long reads, we phased both single-nucleotide variants and SVs, generating haplotypes with over 99% consistency with previous trio-based studies. Our work shows that it is now possible to integrate single-molecule and high-throughput sequence data to generate de novo assembled genomes that approach reference quality

Global analysis of mRNA decay intermediates in Bacillus subtilis wild type and polynucleotide phosphorylase-deletions strains

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Assay of Bacillus subtilis ribonucleases in vitro

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