Ontology highlight
ABSTRACT:
PROVIDER: PRJNA954882 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR24157200.fastq.gz | Fastqsanger.gz | |||
| SRR24157201.fastq.gz | Fastqsanger.gz | |||
| SRR24157202.fastq.gz | Fastqsanger.gz | |||
| SRR24157203.fastq.gz | Fastqsanger.gz | |||
| SRR24157204.fastq.gz | Fastqsanger.gz |
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Project description:Transposon-encoded tnpB and iscB genes encode RNA-guided DNA nucleases that promote their own selfish spread through targeted DNA cleavage and homologous recombination. These widespread gene families were repeatedly domesticated over evolutionary timescales, leading to the emergence of diverse CRISPR-associated nucleases including Cas9 and Cas12. We set out to test the hypothesis that TnpB nucleases may have also been repurposed for novel, unexpected functions other than CRISPR-Cas. Here, using phylogenetics, structural predictions, comparative genomics, and functional assays, we uncover multiple instances of programmable transcription factors that we name TnpB-like nuclease-dead repressors (TldR). These proteins employ naturally occurring guide RNAs to specifically target conserved promoter regions of the genome, leading to potent gene repression in a mechanism akin to CRISPRi technologies invented by humans. Focusing on a TldR clade found broadly in Enterobacteriaceae, we discover that bacteriophages exploit the combined action of TldR and an adjacently encoded phage gene to alter the expression and composition of the host flagellar assembly, a transformation with the potential to impact motility, phage susceptibility, and host immunity. Collectively, this work showcases the diverse molecular innovations that were enabled through repeated exaptation of transposon-encoded genes, and reveals the evolutionary trajectory of diverse RNA-guided transcription factors.
2024-04-22 | GSE245749 | GEO
Project description:RNA-guided endonucleases form the crux of diverse biological processes and technologies, including adaptive immunity, transposition, and genome editing. Some of these enzymes are components of insertion sequences (IS) in the IS200/IS605 and IS607 transposon families. Both IS families encode a TnpA transposase and TnpB nuclease, an RNA-guided enzyme ancestral to CRISPR-Cas12. In eukaryotes and their viruses, TnpB homologs occur as two distinct types, Fanzor1 and Fanzor2. We analyzed the evolutionary relationships between prokaryotic TnpBs and eukaryotic Fanzors, revealing that a clade of IS607 TnpBs with unusual active site arrangement found primarily in cyanobacteria likely gave rise to both types of Fanzors. The widespread nature of Fanzors imply that the properties of this particular group of IS607 TnpBs were particularly suited to adaptation and evolution in eukaryotes and their viruses. Biochemical analysis of a prokaryotic IS607 TnpB and virally encoded Fanzor1s revealed features that may have fostered co-evolution between TnpBs/Fanzors and their cognate transposases. These results provide insight into the evolutionary origins of a ubiquitous family of RNA-guided proteins that shows remarkable conservation across the three domains of life.
2023-11-18 | GSE246134 | GEO
Project description:Insertion sequences (IS) are compact and pervasive transposable elements found in bacteria, which encode only the genes necessary for their mobilization and maintenance. IS200/IS605 elements undergo ‘peel-and-paste’ transposition catalyzed by a TnpA transposase, but intriguingly, they also encode diverse, TnpB-family genes that are evolutionarily related to the CRISPR-associated effectors Cas9 and Cas12. Recent studies demonstrated that TnpB-family enzymes function as RNA-guided DNA endonucleases, but the broader biological role of this activity has remained enigmatic. Here we show that IscB and TnpB are essential to prevent loss of the donor IS element and potential transposon extinction as a consequence of the TnpA transposition mechanism. We first performed phylogenetic analysis of IscB/TnpB proteins and selected a family of related IS elements from Geobacillus stearothermophilus that we predicted would be mobilized by a common TnpA homolog. After reconstituting transposition using a heterologous expression system in E. coli, we found that IS elements were readily lost from the donor site due to the activity of TnpA in rejoining the flanking sequences back together upon excision. However, these IS elements also encode non-coding RNAs that guide TnpB and IscB nucleases to precisely recognize and cleave these excision products, leading either to elimination of the excision product or re-installation of the transposon through recombination. Indeed, under experimental conditions in which TnpA and TnpB-RNA complexes were co-expressed together with a genomically integrated IS element, transposon retention was significantly increased relative to conditions expressing TnpA alone. Remarkably, both TnpA and TnpB recognize the same AT-rich transposon-adjacent motif (TAM) during transposon excision and RNA-guided DNA cleavage, respectively, revealing a striking convergence in the evolution of DNA sequence specificity between transposase and nuclease. Collectively, our study reveals that RNA-guided DNA cleavage is a primal biochemical activity that arose to bias the selfish inheritance of transposable elements, which was later co-opted during the evolution of CRISPR-Cas adaptive immunity for antiviral defense.
2023-06-01 | GSE223127 | GEO
Project description:TnpB nucleases represent the evolutionary precursors to CRISPR-Cas12 and are widespread in all domains of life. IS605-family TnpB homologs function in bacteria as programmable RNA-guided homing endonucleases driving transposon maintenance through DSB-stimulated homologous recombination. Here we uncover molecular mechanisms of transposition lifecycle of IS607-family elements that, remarkably, also encode group I introns. We discover molecular features for a candidate ‘IStron’ from Clostridium botulinum that allow 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 highlights molecular innovation in the multi-functional utility of transposon-encoded noncoding RNAs.
2024-05-06 | GSE261343 | GEO
Project description:TnpB nucleases represent the evolutionary precursors to CRISPR-Cas12 and are widespread in all domains of life. IS605-family TnpB homologs function in bacteria as programmable RNA-guided homing endonucleases driving transposon maintenance through DSB-stimulated homologous recombination. Here we uncover molecular mechanisms of transposition lifecycle of IS607-family elements that, remarkably, also encode group I introns. We discover molecular features for a candidate ‘IStron’ from Clostridium botulinum that allow 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 highlights molecular innovation in the multi-functional utility of transposon-encoded noncoding RNAs.
2024-05-06 | GSE261342 | GEO
Project description:In mice, the PIWI-piRNA pathway is essential to re-establish transposon silencing during male germline reprogramming. The cytoplasmic PIWI protein MILI mediates piRNA-guided transposon RNA cleavage as well as piRNA amplification. MIWI2-bound piRNAs and its nuclear localization are proposed to be dependent upon MILI function. Here, we demonstrate the existence of a piRNA biogenesis pathway that in the absence of MILI that sustains partial MIWI2 function and reprogramming activity.
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Project description:As the ancestor of CRISPR-Cas12 nucleases, TnpB represents the most compact gene editing tool currently available. Recent studies have identified multiple TnpB systems with gene editing activity in mammalian cells, and the potential of TnpB in treating diseases has been demonstrated in animal models. However, the editing characteristics of various TnpB systems, comparable to CRISPR tools, require more extensive investigation. Using a standardized evaluation framework, we conducted a thorough analysis of the editing properties of four TnpB variants alongside representative Cas12 and Cas9 tools. Overall, TnpBs exhibit intermediate editing activity and safety profiles among all tested systems, with ISYmu1 TnpB demonstrating a good performance in both editing activity and specificity. Considering its compact size, potent editing efficiency and high specificity, ISYmu1 TnpB represents a promising candidate for in vivo gene therapy applications.
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Project description:Mutation effects prediction is a fundamental challenge in biotechnology and biomedicine. State-of-the-art computational methods have demonstrated the benefits of including semantically rich representations learned from protein sequences, but leave structural constraints out of reach. Here we developed Protein Mutational Effect Predictor (ProMEP), a general and multimodal deep representation learning method that simultaneously learns sequence context and structural constraints from proteins at the scale of evolution. ProMEP markedly outperforms current leading methods and enables accurate zero-shot mutational effects prediction across a variety of deep mutational scanning experiments. The application of ProMEP in the transposon-associated TnpB enzyme engineering task further demonstrates its ability for high-throughput protein space exploration. Without prior knowledge of TnpB, ProMEP accurately identifies multiple mutations that significantly improve the editing efficiency from millions of variants.
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