Biology subjects

Gelsinger, D. R.

Publications and source records attributed to Gelsinger, D. R..

5 recordsLinked to original sources

bioRxiv2023

Antagonistic conflict between transposon-encoded introns and guide RNAs

TnpB nucleases represent the evolutionary precursors to CRISPR-Cas12 and are widespread in all domains of life, presumably due to the critical roles they play in transposon proliferation. IS605-family TnpB homologs function in bacteria as programmable homing endonucleases by exploiting transposon-encoded guide RNAs to cleave vacant genomic sites, thereby driving transposon maintenance through DSB-stimulated homologous recombination. Whether this pathway is conserved in other genetic contexts, and in association with other transposases, is unknown. Here we uncover molecular mechanisms of transposition and RNA-guided DNA cleavage by IS607-family elements that, remarkably, also encode catalytic, self-splicing group I introns. After reconstituting and systematically investigating each of these biochemical activities for a candidate IStron derived from Clostridium botulinum, we discovered sequence and structural features of the transposon-encoded RNA that satisfy molecular requirements of a group I intron and TnpB guide RNA, while still retaining the ability to be faithfully mobilized at the DNA level by the TnpA transposase. Strikingly, intron splicing was strongly repressed not only by TnpB, but also by the secondary structure of {omega}RNA alone, allowing the element to carefully control the relative levels of spliced products versus functional guide RNAs. Our results suggest that IStron transcripts have evolved a sensitive equilibrium to balance competing and mutually exclusive activities that promote transposon maintenance while limiting adverse fitness costs on the host. Collectively, this work explains how diverse enzymatic activities emerged during the selfish spread of IS607-family elements and highlights molecular innovation in the multi-functional utility of transposon-encoded noncoding RNAs.

molecular biology↗

bioRxiv2023

Bacterial genome engineering using CRISPR RNA-guided transposases

CRISPR-associated transposons (CASTs) have the potential to transform the technology landscape for kilobase-scale genome engineering, by virtue of their ability to integrate large genetic payloads with high accuracy, easy programmability, and no requirement for homologous recombination machinery. These transposons encode efficient, CRISPR RNA-guided transposases that execute genomic insertions in E. coli at efficiencies approaching [~]100%, generate multiplexed edits when programmed with multiple guides, and function robustly in diverse Gram-negative bacterial species. Here we present a detailed protocol for engineering bacterial genomes using CAST systems, including guidelines on the available homologs and vectors, customization of guide RNAs and DNA payloads, selection of common delivery methods, and genotypic analysis of integration events. We further describe a computational crRNA design algorithm to avoid potential off-targets and CRISPR array cloning pipeline for DNA insertion multiplexing. Starting from available plasmid constructs, the isolation of clonal strains containing a novel genomic integration event-of-interest can be achieved in 1 week using standard molecular biology techniques.

bioengineering↗

bioRxiv2020

Post-transcriptional regulation of redox homeostasis by the small RNA SHOxi in haloarchaea

Haloarchaea are highly resistant to oxidative stress, however, a comprehensive understanding of the processes regulating this remarkable response is lacking. Oxidative stress-responsive small non-coding RNAs (sRNAs) have been reported in the model archaeon, Haloferax volcanii, but targets and mechanisms have not been elucidated. Using a combination of high throughput and reverse molecular genetic approaches, we elucidated the functional role of the most up-regulated intergenic sRNA during oxidative stress in H. volcanii, named Small RNA in Haloferax Oxidative Stress (SHOxi). SHOxi was predicted to form a stable secondary structure with a conserved stem-loop region as the potential binding site for trans-targets. NAD-dependent malic enzyme mRNA, identified as a putative target of SHOxi, interacted directly with a putative "seed" region within the predicted stem loop of SHOxi. Malic enzyme is an enzyme of the tricarboxylic acid cycle that catalyzes the oxidative decarboxylation of malate into pyruvate using NAD+ as a cofactor. The destabilization of malic enzyme mRNA, and the decrease in the NAD+/NADH ratio, resulting from the direct RNA-RNA interaction between SHOxi and its trans-target was essential for the survival of H. volcanii to oxidative stress. These findings indicate that SHOxi likely regulates redox homeostasis during oxidative stress by the post-transcriptional destabilization of malic enzyme mRNA. SHOxi-mediated regulation provides evidence that the fine-tuning of metabolic cofactors could be a core strategy to mitigate damage from oxidative stress and confer resistance. This study is the first to establish the regulatory effects of sRNAs on mRNAs during the oxidative stress response in Archaea.

microbiology↗

bioRxiv2020

Ribosome Profiling in Archaea Reveals Leaderless Translation, Novel Translational Initiation Sites, and Ribosome Pausing at Single Codon Resolution

High-throughput methods, such as ribosome profiling, have revealed the complexity of translation regulation in Bacteria and Eukarya with large-scale effects on cellular functions. In contrast, the translational landscape in Archaea remains mostly unexplored. Here, we developed ribosome profiling in a model archaeon, Haloferax volcanii, elucidating, for the first time, the translational landscape of a representative of the third domain of life. We determined the ribosome footprint of H. volcanii to be comparable in size to that of the Eukarya. We linked footprint lengths to initiating and elongating states of the ribosome on leadered transcripts, operons, and on leaderless transcripts, the latter representing 70% of H. volcanii transcriptome. We manipulated ribosome activity with translation inhibitors to reveal ribosome pausing at specific codons. Lastly, we found that the drug harringtonine arrested ribosomes at initiation sites in this archaeon. This drug treatment allowed us to confirm known translation initiation sites and also reveal putative novel initiation sites in intergenic regions and within genes. Ribosome profiling revealed an uncharacterized complexity of translation in this archaeon with bacteria-like, eukarya-like, and potentially novel translation mechanisms. These mechanisms are likely to be functionally essential and to contribute to an expanded proteome with regulatory roles in gene expression.

molecular biology↗

bioRxiv2017

Transcriptional landscape and regulatory roles of small non-coding RNAs in the oxidative stress response of the haloarchaeon Haloferax volcanii

Haloarchaea in their natural environment are exposed to hyper-salinity, intense solar radiation, and desiccation, all of which generate high levels of oxidative stress. Previous work has shown that Haloarchaea are an order of magnitude more resistant to oxidative stress than most mesophilic organisms. Despite this resistance, the pathways Haloarchaea use to respond to oxidative stress damage are similar to that of non-resistant organisms suggesting that regulatory processes might be key to their robustness. Recently, small non-coding RNAs (sRNAs) were discovered in Archaea under a variety of environmental conditions. We report here the transcriptional landscape and functional roles of sRNAs in the regulation of the oxidative stress response of the model haloarchaeon Haloferax volcanii. Thousands of sRNAs, both intergenic and antisense, were discovered using strand-specific sRNA-seq, comprising around 30% of the transcriptome during non-challenged and oxidative stress conditions. We identified hundreds of differentially expressed sRNAs in response to hydrogen peroxide induced oxidative stress in H. volcanii. Targets of antisense sRNAs decreased in expression when sRNAs were up-regulated indicating that sRNAs are likely playing a negative regulatory role on mRNA targets at the transcript level. Target enrichment of these antisense sRNAs included mRNAs involved in transposons mobility, chemotaxis signaling, peptidase activity, and transcription factors.\n\nIMPORTANCEWhile a substantial body of experimental work has been done to uncover functions of sRNAs in gene regulation in Bacteria and Eukarya, the functional roles of sRNAs in Archaea are still poorly understood. This study is the first to establish the regulatory effects of sRNAs on mRNAs during the oxidative stress response in the haloarchaeon Haloferax volcanii. Our work demonstrates that common principles for the response to a major cellular stress exist across the 3 domains of life while uncovering pathways that might be specific to the Archaea. This work also underscores the relevance of sRNAs in adaptation to extreme environmental conditions.

microbiology↗