Biology subjects

Randau, L.

Publications and source records attributed to Randau, L..

6 recordsLinked to original sources

bioRxiv2025

A Multi-Layered Transcriptomic Analysis Sheds Light on Antisense Transcription, RNA Processing, and SmAP Interactions in Sulfolobus acidocaldarius

The archaeal domain contains organisms that are well-adapted to extreme conditions and changes in their habitat. Post-transcriptional regulation plays a key role in environmental adaptation, including rapid molecular responses to stress conditions. To understand the importance of RNA-based post-transcriptional regulation for these processes, a comprehensive analysis of the presence and processing of regulatory RNAs, as well as their interactions with other RNAs and proteins, is indispensable. Here, we combine the analysis of several RNA sequencing approaches to reveal the presence of a set of novel non-coding RNAs (ncRNAs), their expression in various conditions, processing, and molecular interactions in the transcriptome of Sulfolobus acidocaldarius, a model organism for Archaea. We expand its annotation by 160 intergenic ncRNAs (sRNAs) and 990 antisense RNAs (asRNAs), add the location and motifs of over 6000 transcript processing sites, and determine the interaction of transcripts with Sm-like archaeal proteins (SmAPs). We determined the correlation between the expression patterns of asRNAs and their cognate mRNAs, suggesting transcript-based regulation patterns in gene expression, particularly in response to changing environmental conditions. Additionally, we observed differential binding preferences of SmAP1 and SmAP2 towards mRNA and ncRNAs, suggesting a distribution of regulating roles of these chaperones. Finally, we provide an overview of our post-transcriptional data analysis results, optimized for custom exploration, in the form of a web-based transcriptome atlas (https://vicentebr.github.io/Sulfolobus_atlas/). IMPORTANCEPost-transcriptional regulation is a key control layer in gene expression. Yet, resources integrating asRNAs, RNA processing sites, and RNA-protein interactions are scarce for archaeal organisms. Here we combine multiple RNA-seq strategies and RIP-seq to expand the Sulfolobus acidocaldarius transcriptome with 990 asRNAs, thousands of transcript processing sites, and the interactomes of the essential RNA chaperones SmAP1 and SmAP2. Integrating the novel generated data for the re-analysis of heat-shock transcriptomics and proteomics reveals locus-specific antisense mechanisms that might help to explain the weak correlation between cellular RNA and protein levels reported previously. Moreover, our publicly accessible web atlas provides a community platform to explore these datasets and assist in the formulation of new hypotheses about archaeal RNA regulation.

systems biology↗

bioRxiv2024

Visualization of Type IV-A1 CRISPR-mediated repression of gene expression and plasmid replication

Type IV CRISPR-Cas effector complexes are often encoded on plasmids and are proposed to prevent the replication of competing plasmids. The Type IV-A1 CRISPR-Cas system of Pseudomonas oleovorans additionally harbors a CRISPR RNA (crRNA) that tightly regulates the transcript levels of a chromosomal target and represents a natural CRISPR interference (CRISPRi) tool. This study investigates CRISPRi effects of this system using synthetic crRNAs against genome and plasmid sequences. Targeting of reporter genes revealed extended interference in P. oleovorans and Escherichia coli cells producing recombinant CRISPR ribonucleoprotein (crRNP) complexes. RNA-Seq analyses of Type IV-A1 CRISPRi-induced transcriptome alterations demonstrated highly effective long-range down-regulation of histidine operon expression, whereas CRISPRi effects of dCas9 remained limited to the vicinity of its binding site. Single-molecule microscopy uncovered the localization dynamics of crRNP complexes. The tracks of fluorescently labeled crRNPs co-localized with regions of increased plasmid replication, supporting efficient plasmid targeting. These results identify mechanistic principles that facilitate the application of Type IV-A1 CRISPRi for the regulation of gene expression and plasmid replication.

microbiology↗

bioRxiv2023

Type IV-A3 CRISPR-Cas systems drive inter-plasmid conflicts by acquiring spacers in trans

Type IV-A CRISPR-Cas systems are primarily encoded on plasmids and form multi-subunit ribonucleoprotein complexes with unknown biological functions. In contrast to other CRISPR-Cas types, they lack the archetypical CRISPR acquisition module and encode a DinG helicase instead of a nuclease component. Type IV-A3 systems are carried by large conjugative plasmids that often harbor multiple antibiotic-resistance genes. Although their CRISPR array contents suggest a role in inter-plasmid conflicts, this function and the underlying mechanisms have remained unexplored. Here, we demonstrate that a plasmid-encoded type IV-A3 CRISPR-Cas system co-opts the type I-E adaptation machinery from its clinical Klebsiella pneumoniae host to update its CRISPR array. Furthermore, we demonstrate that robust interference of conjugative plasmids and phages is elicited through CRISPR RNA-dependent transcriptional repression. By targeting plasmid core functions, type IV-A3 can prevent the uptake of incoming plasmids, limit their horizontal transfer, and destabilize co-residing plasmids, altogether supporting type IV-A3s involvement in plasmid competition. Collectively, our findings shed light on the molecular mechanisms and ecological function of type IV-A3 systems and have broad implications for understanding and countering the spread of antibiotic resistance in clinically relevant strains.

microbiology↗

bioRxiv2022

Identification of NAD-RNAs and ADPR-RNA decapping in the archaeal model organisms Sulfolobus acidocaldarius and Haloferax volcanii

NAD is a coenzyme central to metabolism that was also found to serve as a 5-terminal cap of bacterial and eukaryotic RNA species. The presence and functionality of NAD-capped RNAs (NAD-RNAs) in the archaeal domain remain to be characterized in detail. Here, by combining LC-MS and NAD captureSeq methodology, we quantified the total levels of NAD-RNAs and determined the identity of NAD-RNAs in the two model archaea, Sulfolobus acidocaldarius and Haloferax volcanii. A complementary differential RNA-Seq (dRNA-Seq) analysis revealed that NAD transcription start sites (NAD-TSS) correlate with well-defined promoter regions and often overlap with primary transcription start sites (pTSS). The population of NAD-RNAs in the two archaeal organisms shows clear differences, with S. acidocaldarius possessing more capped small non-coding RNAs (sncRNAs) and leader sequences. The NAD-cap did not prevent 5[->]3 exonucleolytic activity by the RNase Saci-aCPSF2. To investigate enzymes that facilitate the removal of the NAD-cap, four Nudix proteins of S. acidocaldarius were screened. None of the recombinant proteins showed NAD decapping activity. Instead, the Nudix protein Saci_NudT5 showed activity after incubating NAD-RNAs at elevated temperatures. Hyperthermophilic environments promote the thermal degradation of NAD into the toxic product ADPR. Incorporating NAD into RNAs and the regulation of ADPR-RNA decapping by Saci_NudT5 is proposed to provide additional layers of maintaining stable NAD levels in archaeal cells. ImportanceThis study reports the first characterization of 5-terminally modified RNA molecules in Archaea and establishes that NAD-RNA modifications, previously only identified in the other two domains of life, are also prevalent in the archaeal model organisms Sulfolobus acidocaldarius and Haloferax volcanii. We screened for NUDIX hydrolases that could remove the NAD-RNA cap and showed that none of these enzymes removed NAD modifications, but we discovered an enzyme that hydrolyzes ADPR-RNA. We propose that these activities influence the stabilization of NAD and its thermal degradation to potentially toxic ADPR products at elevated growth temperatures.

molecular biology↗

bioRxiv2020

An anti-CRISPR protein induces strong non-specific DNA binding activity in a CRISPR-Cas complex

Phages and other mobile genetic elements express anti-CRISPR proteins (Acrs) to protect their genomes from destruction by CRISPR-Cas systems. Acrs usually block the ability of CRISPR-Cas systems to bind or cleave their nucleic acid substrates. Here, we investigate an unusual Acr, AcrIF9, that induces a gain-of-function to a type I-F CRISPR-Cas (Csy) complex, causing it to bind strongly to DNA that lacks both a PAM sequence and sequence complementarity. We show that specific and non-specific dsDNA compete for the same site on the Csy:AcrIF9 complex with rapid exchange, but specific ssDNA appears to still bind through complemetarity to the CRISPR RNA. We also demonstrate that induction of non-specific DNA-binding is a conserved property of diverse AcrIF9 homologues, implying that this activity contributes the biologically relevant function of this Acr family. AcrIF9 provides another example of the surprising variety of mechanisms by which Acrs inhibit CRISPR-Cas systems.

microbiology↗

bioRxiv2019

Type IV CRISPR-Cas systems are highly diverse and involved in competition between plasmids

CRISPR-Cas systems provide prokaryotes with adaptive immune functions against viruses and other genetic parasites by leveraging small non-coding RNAs for nuclease-dependent degradation of their nucleic acid targets. In contrast to all other types of CRISPR-Cas systems, the mechanisms and biological roles of type IV systems have remained largely overlooked. Here, we describe a previously uncharted diversity of type IV gene cassettes, distributed across diverse prokaryotic genome backgrounds, and propose their classification into subtypes and variants. Congruent with recent findings, type IV modules were primarily found on plasmid-like elements. Remarkably, via a comprehensive analysis of their CRISPR spacer content, these systems were found to exhibit a strong bias towards the targeting of other plasmids. Our data indicate that the functions of type IV systems have diverged from those of other host-related CRISPR-Cas immune systems to adopt a yet unrecognised role in mediating conflicts between plasmids that compete to monopolize their hosts. Furthermore, we find evidence for cross-talk between certain type IV and type I CRISPR-Cas systems that co-exist intracellularly, thus providing an answer to the enigmatic absence of adaptation modules in these systems. Collectively, our results lead to the expansion and reclassification of type IV systems and provide novel insights into the biological function and evolution of these elusive systems.

microbiology↗