Biology subjects

Krasny, L.

Publications and source records attributed to Krasny, L..

12 recordsLinked to original sources

bioRxiv2024

Expanding the CarD interaction network: CrsL is a novel transcription factor in Mycobacterium smegmatis

Bacterial transcription regulation is critical for adaptation and survival. CarD is an essential transcription factor in mycobacteria involved in regulation of gene expression. We searched for CarD interaction partners in the model organism Mycobacterium smegmatis and identified two proteins: ApeB (MSMEG_5828) and an uncharacterized protein, which we named CrsL (MSMEG_5890). While ApeB interacted with CarD only when CarD was overexpressed, CrsL associated with CarD at its physiological levels. CrsL is a 5.7 kDa protein shown by NMR to be intrinsically disordered. CrsL homologs are present in actinobacteria including pathogenic species such as Mycobacterium tuberculosis. CrsL directly interacts with CarD and binds RNAP. ChIP-seq showed that CrsL associates with promoters of actively transcribed genes and [~]75 % of these regions are also associated with CarD. RNA-seq showed [~]50% and [~]66% overlap in differentially expressed genes between CrsL and CarD knockdowns during exponential and stationary phases, respectively. CrsL represses expression of DesA desaturase (MSMEG_5773) and DEAD/DEAH-box RNA helicase MSMEG_1930, which are important for adaptation to cold stress. Furthermore, CrsL promotes the growth of M. smegmatis at elevated temperature. In summary, this study identifies CrsL as a novel actinobacterial transcription factor and provides a basis for its further investigation.

microbiology↗

bioRxiv2023

The alternative sigma factor SigN of Bacillus subtilis is intrinsically toxic

Sigma factors bind and direct the RNA polymerase core to specific promoter sequences and alternative sigma factors direct transcription of different regulons of genes. Here, we study the pBS32 plasmid-encoded sigma factor SigN of Bacillus subtilis to determine how it contributes to DNA damage-induced cell death. We find that SigN causes cell death when expressed at high level and does so in the absence of its regulon suggesting it is intrinsically toxic. One way toxicity was relieved was by curing the pBS32 plasmid, which eliminated a positive feedback loop that lead to SigN hyper-accumulation. Another way toxicity was relieved was through mutating the chromosomally-encoded transcriptional repressor protein AbrB and derepressing a potent antisense transcript that antagonized SigN expression. We note that SigN exhibits a relatively high affinity for the RNA polymerase core, efficiently competing with the vegetative sigma factor SigA, suggesting that toxicity was due to the competitive inhibition of one or more essential transcripts. Why B. subtilis encodes a potentially toxic sigma factor is unclear but SigN may be related to phage-like genes also encoded on pBS32. SIGNIFICANCEAlternative sigma factors activate entire regulons of genes to improve viability in response to environmental stimuli. The pBS32 plasmid-encoded SigN of Bacillus subtilis is activated by the DNA damage response and leads to cellular demise. Here we find that SigN impairs viability by hyper-accumulating and outcompeting the vegetative sigma factor for the RNA polymerase core. Why B. subtilis retains a plasmid with a deleterious alternative sigma factor is unknown.

microbiology↗

bioRxiv2022

Proteomic profiling identifies co-regulated expression of splicing factors as a characteristic feature of intravenous leiomyomatosis

Intravenous leiomyomatosis (IVLM) is a rare benign smooth muscle tumour that is characterised by intravenous growth in the uterine and pelvic veins. Previous DNA copy number and transcriptomic studies have shown that IVLM harbours unique genomic and transcriptomic alterations when compared to uterine leiomyoma (uLM), which may account for their distinct clinical behaviour. Here we undertake the first comparative proteomic analysis of IVLM and other smooth muscle tumours (comprising uLM, soft tissue leiomyoma and benign metastasising leiomyoma) utilising data-independent acquisition mass spectrometry. We show that, at the protein level, IVLM is defined by the unique co-regulated expression of splicing factors. In particular, IVLM is enriched in two clusters composed of co-regulated proteins from the hnRNP, LSm, SR and Sm classes of the spliceosome complex. One of these clusters (Cluster 3) is associated with key biological processes including nascent protein translocation and cell signalling by small GTPases. Taken together, our study provides evidence of co-regulated expression of splicing factors in IVLM compared to other smooth muscle tumours which suggests a possible role for alternative splicing in the pathogenesis of IVLM.

cancer biology↗

bioRxiv2021

NAD+ capping of RNA in Archaea and Mycobacteria

Chemical modifications of RNA affect essential properties of transcripts, such as their translation, localization and stability. 5-end RNA capping with the ubiquitous redox cofactor nicotinamide adenine dinucleotide (NAD+) has been discovered in organisms ranging from bacteria to mammals. However, the hypothesis that NAD+ capping might be universal in all domains of life has not been proven yet, as information on this RNA modification is missing for Archaea. Likewise, this RNA modification has not been studied in the clinically important Mycobacterium genus. Here, we demonstrate that NAD+ capping occurs in the archaeal and mycobacterial model organisms Methanosarcina barkeri and Mycobacterium smegmatis. Moreover, we identify the NAD+-capped transcripts in M. smegmatis, showing that this modification is more prevalent in stationary phase, and revealing that mycobacterial NAD+-capped transcripts include non-coding small RNAs, such as Ms1. Furthermore, we show that mycobacterial RNA polymerase incorporates NAD+ into RNA, and that the genes of NAD+-capped transcripts are preceded by promoter elements compatible with {sigma}A/{sigma}F dependent expression. Taken together, our findings demonstrate that NAD+ capping exists in the archaeal domain of life, suggesting that it is universal to all living organisms, and define the NAD+-capped RNA landscape in mycobacteria, providing a basis for its future exploration.

molecular biology↗

bioRxiv2020

Mycobacterial HelD is a nucleic acids-clearing factor for RNA polymerase

RNA synthesis is central to life, and RNA polymerase depends on accessory factors for recovery from stalled states and adaption to environmental changes. Here we investigated the mechanism by which a helicase-like factor HelD recycles RNA polymerase. We report a cryo-EM structure of an unprecedented complex between the Mycobacterium smegmatis RNA polymerase and HelD. The crescent-shaped HelD simultaneously penetrates deep into two RNA polymerase channels that are responsible for DNA binding and substrate delivery to the active site, thereby locking RNA polymerase in an inactive state. We show that HelD prevents non-specific interactions between RNA polymerase and DNA and dissociates transcription elongation complexes, but does not inhibit RNA polymerase binding to the initiation {sigma} factor. The liberated RNA polymerase can either stay dormant, sequestered by HelD, or upon HelD release, restart transcription. Our results provide insights into the architecture and regulation of the highly medically-relevant mycobacterial transcription machinery and define HelD as a clearing factor that removes undesirable nucleic acids from RNA polymerase.

molecular biology↗

bioRxiv2020

Quasi-essentiality of RNase Y in Bacillus subtilis is caused by its critical role in the control of mRNA homeostasis

RNA turnover is essential in all domains of life. The endonuclease RNase Y (rny) is one of the key components involved in RNA metabolism of the model organism Bacillus subtilis. Essentiality of RNase Y has been a matter of discussion, since deletion of the rny gene is possible, but leads to severe phenotypic effects. In this work, we demonstrate that the rny mutant strain rapidly evolves suppressor mutations to at least partially alleviate these defects. All suppressor mutants had acquired a duplication of an about 60 kb long genomic region encompassing genes for all three core subunits of the RNA polymerase - , {beta}, {beta}'. When the duplication of the RNA polymerase genes was prevented by relocation of the rpoA gene in the B. subtilis genome, all suppressor mutants carried distinct single point mutations in evolutionary conserved regions of genes coding either for the {beta} or {beta} subunits of the RNA polymerase that were not tolerated by wild type bacteria. In vitro transcription assays with the mutated polymerase variants showed massive decreases in transcription efficiency. Altogether, our results suggest a tight cooperation between RNase Y and the RNA polymerase to establish an optimal RNA homeostasis in B. subtilis cells.

microbiology↗

bioRxiv2020

A mouse SWATH-MS reference spectral library enables deconvolution of species-specific proteomic alterations in human tumour xenografts

SWATH-mass spectrometry (MS) enables accurate and reproducible proteomic profiling in multiple model organisms including the mouse. Here we present a comprehensive mouse reference spectral library (MouseRefSWATH) that permits quantification of up to 10,597 proteins (62.2% of the mouse proteome) by SWATH-MS. We exploit MouseRefSWATH to develop an analytical pipeline for species-specific deconvolution of proteomic alterations in human tumour xenografts (XenoSWATH). This method overcomes the challenge of high sequence similarity between mouse and human proteins, facilitating the study of host microenvironment-tumour interactions from bulk tumour measurements. We apply the XenoSWATH pipeline to characterise an intraductal xenograft model of breast ductal carcinoma in-situ and uncover complex regulation of cell migration pathways that are not restricted to tumour cells but also operate in the mouse stroma upon progression to invasive disease. MouseRefSWATH and XenoSWATH opens new opportunities for in-depth and reproducible proteomic assessment to address wide-ranging biological questions involving this important model organism.

biochemistry↗

bioRxiv2019

The alarmone (p)ppGpp is part of the heat shock response of Bacillus subtilis

Here, B. subtilis was used as a model organism to investigate how cells respond and adapt to proteotoxic stress conditions. Our experiments suggested that the stringent response, caused by raised levels of the (p)ppGpp alarmone, plays a role during thermotolerance development and the heat shock response. Accordingly, our experiments revealed a rapid increase of cellular (p)ppGpp levels upon heat shock as well as salt- and oxidative stress. Strains lacking (p)ppGpp exhibited increased stress sensitivity, while raised (p)ppGpp levels conferred increased stress tolerance to heat- and oxidative stress. During thermotolerance development, stress response genes were highly up-regulated together with a concurrent transcriptional down-regulation of the rRNA, which was influenced by the second messenger (p)ppGpp and the transcription factor Spx. Remarkably, we observed that (p)ppGpp appeared to control the cellular translational capacity and that during heat stress the raised cellular levels of the alarmone were able to curb the rate of protein synthesis. Furthermore, (p)ppGpp controls the heat-induced expression of Hpf and thus the formation of translationally inactive 100S disomes. These results indicate that B. subtilis cells respond to heat-mediated protein unfolding and aggregation, not only by raising the cellular repair capacity, but also by decreasing translation involving (p)ppGpp mediated stringent response to concurrently reduce the protein load for the cellular protein quality control system.\n\nAuthor SummaryHere we demonstrate that the bacterial stringent response, which is known to slow down translation upon sensing nutrient starvation, is also intricately involved in the stress response of B. subtilis cells. The second messengers (p)ppGpp act as pleiotropic regulators during the adaptation to heat stress. (p)ppGpp slows down translation and is also involved in the transcriptional down-regulation of the translation machinery, together with the transcriptional stress regulator Spx. The stress-induced elevation of cellular (p)ppGpp levels confers increased stress tolerance and facilitates an improved protein homeostasis by reducing the load on the protein quality control system.

microbiology↗

bioRxiv2019

Analysis of Nucleotide Pools in Bacteria Using HPLC-MS in HILIC Mode

Nucleotides, nucleosides and their derivatives are present in cells at varying concentrations that rapidly change with the nutritional, and energetic status of the cell. Knowledge of the concentration dynamics of these molecules is instrumental for understanding their regulatory effects. Determination of these concentrations is challenging due to the inherent instability of these molecules and, despite many decades of research, the reported values differ widely. Here, we present a comprehensive and easy-to-use approach to determine intracellular concentrations of >25 target molecular species. The approach uses rapid filtration and cold acidic extraction followed by high performance liquid chromatography (HPLC) in the hydrophilic interaction liquid chromatography (HILIC) mode using zwitterionic columns coupled with UV and MS detectors. The method reliably detects and quantifies all the analytes expected to be observed in the bacterial cell and paves the way for future studies correlating their concentrations with biological effects.

microbiology↗

bioRxiv2019

Conduit integrity is compromised during acute lymph node expansion

Lymph nodes (LNs) work as filtering organs, constantly sampling peripheral cues. This is facilitated by the conduit network, a parenchymal tubular-like structure formed of bundles of aligned extracellular matrix (ECM) fibrils ensheathed by fibroblastic reticular cells (FRCs). LNs undergo 5-fold expansion with every adaptive immune response and yet these ECM-rich structures are not permanently damaged. Whether conduit integrity and filtering functions are affected during cycles of LN expansion and resolution is not known. Here we show that the conduit structure is disrupted during acute LN expansion but FRC-FRC contacts remain intact. In homeostasis, polarised FRCs adhere to the underlying substrate to deposit ECM ba-solaterally. ECM production by FRCs is regulated by the C-type lectin CLEC-2, expressed by dendritic cells (DCs), at transcriptional and secretory levels. Inflamed LNs maintain conduit size-exclusion, but flow becomes leaky, which allows soluble antigens to reach more antigen-presenting cells. We show how dynamic communication between peripheral tissues and LNs changes during immune responses, and describe a mechanism that enables LNs to prevent inflammation-induced fibrosis. HighlightsO_LIFRCs use polarized microtubule networks to guide matrix deposition C_LIO_LICLEC-2/PDPN controls matrix production at transcriptional and post-transcriptional levels C_LIO_LIFRCs halt matrix production and decouple from conduits during acute LN expansion C_LIO_LIConduits leak soluble antigen during acute LN expansion C_LI

immunology↗

bioRxiv2018

Transcription apparatus of the yeast killer DNA plasmids: Architecture, function, and evolutionary origin

Transcription of extrachromosomal elements such as organelles, viruses, and plasmids is dependent on cellular RNA polymerase (RNAP) or intrinsic RNAP encoded by these elements. The yeast Kluyveromyces lactis contains killer DNA plasmids that bear putative non-canonical RNAP genes. Here, we describe the architecture and evolutionary origin of this transcription machinery. We show that the two RNAP subunits interact in vivo, and this complex interacts with another two plasmid-encoded proteins - the mRNA capping enzyme, and a putative helicase which interacts with plasmid-specific DNA. Further, we identify a promoter element that causes 5 polyadenylation of plasmid-specific transcripts via RNAP slippage during transcription initiation, and structural elements that precede the termination sites. As a result, we present a first model of the yeast killer plasmid transcription initiation and intrinsic termination. Finally, we demonstrate that plasmid RNAP and its promoters display high similarity to poxviral RNAP and promoters of early poxviral genes, respectively.

molecular biology↗

bioRxiv2017

“CapZyme-Seq” comprehensively defines promoter-sequence determinants for RNA 5’ capping with NAD+

Nucleoside-containing metabolites such as NAD+ can be incorporated as \"5' caps\" on RNA by serving as non-canonical initiating nucleotides (NCINs) for transcription initiation by RNA polymerase (RNAP). Here, we report \"CapZyme-Seq,\" a high-throughput-sequencing method that employs NCIN-decapping enzymes NudC and Rai1 to detect and quantify NCIN-capped RNA. By combining CapZyme-Seq with multiplexed transcriptomics, we determine efficiencies of NAD+ capping by Escherichia coli RNAP for ~16,000 promoter sequences. The results define preferred transcription start-site (TSS) positions for NAD+ capping and define a consensus promoter sequence for NAD+ capping: HRRASWW (TSS underlined). By applying CapZyme-Seq to E. coli total cellular RNA, we establish that sequence determinants for NCIN capping in vivo match the NAD+-capping consensus defined in vitro, and we identify and quantify NCIN-capped small RNAs. Our findings define the promoter-sequence determinants for NCIN capping with NAD+ and provide a general method for analysis of NCIN capping in vitro and in vivo.

biochemistry↗