Biology subjects

Sykora, M.

Publications and source records attributed to Sykora, M..

7 recordsLinked to original sources

bioRxiv2023

Cbp1-Cren7 chromatinization of CRISPR arrays favours transcription from leader- over cryptic promoters

CRISPR arrays form the physical memory of CRISPR adaptive immune systems by incorporating foreign DNA as spacers that are often AT-rich and derived from viruses. As promoter elements such as the TATA-box are AT-rich, CRISPR arrays are prone to harbouring cryptic promoters. Sulfolobales harbor extremely long CRISPR arrays spanning several kilobases, a feature that is accompanied by the CRISPR-specific transcription factor Cbp1. Aberrant Cbp1 expression modulates CRISPR array transcription, but the molecular mechanisms underlying this regulation are unknown. Here, we characterise the genome-wide Cbp1 binding at nucleotide resolution and characterise the binding motifs on distinct CRISPR arrays, as well as on unexpected non-canonical binding sites associated with transposasons. Cbp1 recruits Cren7 forming together chimeric chromatin-like structures at CRISPR arrays. We dissect Cbp1 function in vitro and in vivo and show that the third HTH domain is responsible for Cren7 recruitment, and that Cbp1-Cren7 chromatinization plays a dual role in the transcription of CRISPR arrays. It suppresses spurious transcription from cryptic promoters within CRISPR arrays but enhances CRISPR RNA transcription directed from their cognate promoters in their leader region. Our results show that Cbp1-Cren7 chromatinization drives the productive expression of long CRISPR arrays.

microbiology↗

bioRxiv2022

High-resolution single-cell atlas reveals diversity and plasticity of tissue-resident neutrophils in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) is characterized by molecular heterogeneity with diverse immune cell infiltration patterns, which has been linked to both, therapy sensitivity and resistance. However, full understanding of how immune cell phenotypes vary across different patient and tumor subgroups is lacking. Here, we dissect the NSCLC tumor microenvironment at high resolution by integrating 1,212,463 single-cells from 538 samples and 309 patients across 29 datasets, including our own dataset capturing cells with low mRNA content. Based on the cellular composition we stratified patients into immune deserted, B cell, T cell, and myeloid cell subtypes. Using bulk samples with genomic and clinical information, we identified specific cellular components associated with tumor histology and genotypes. Analysis of cells with low mRNA content uncovered distinct subpopulations of tissue-resident neutrophils (TRNs) that acquire new functional properties in the tissue microenvironment, providing evidence for the plasticity of TRNs. TRN-derived gene signature was associated with anti-PD-L1 treatment failure in a large NSCLC cohort. In briefSalcher, Sturm, Horvath et al. integrate single-cell datasets to generate the largest transcriptome atlas in NSCLC, refining patient stratification based on tumor immune phenotypes, and revealing associations of histological subtypes and genotypes with specific cellular composition patterns. Coverage of cells with low mRNA content by single-cell sequencing identifies distinct tissue-resident neutrophil subpopulations, which acquire new properties within the tumor microenvironment. Gene signature from tissue-resident neutrophils is associated with immune checkpoint inhibitor treatment failure. The integrated atlas is publicly available online (https://luca.icbi.at), allowing the dissection of tumor-immune cell interactions in NSCLC. HighlightsO_LIHigh-resolution single-cell atlas of the tumor microenvironment (TME) in NSCLC. C_LIO_LIHistological tumor subtypes and driver genes imprint specific cellular TME patterns. C_LIO_LIscRNA-seq of cells with low transcript count identifies distinct tissue-resident neutrophil (TRN) subpopulations and non-canonical functional properties in the TME niche. C_LIO_LITRN gene signature identifies patients who are refractory to treatment with PD-L1 inhibitors. C_LI O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/491204v1_ufig1.gif" ALT="Figure 1"> View larger version (63K): org.highwire.dtl.DTLVardef@1050b6aorg.highwire.dtl.DTLVardef@3088c8org.highwire.dtl.DTLVardef@6408b8org.highwire.dtl.DTLVardef@1788c22_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer 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

Transcripts of vaccinia virus postreplicative genes do not contain a 5' methylguanosine cap

Vaccinia virus (VACV) is a prototypical poxvirus originally used for eradication of smallpox. Investigation into VACV mRNAs carried out almost half a century ago substantially contributed to the fundamental discovery of the 5 mRNA cap, a hallmark of all eukaryotic and many viral mRNAs. VACV research also facilitated the identification and understanding of the general mechanism of 5 mRNA cap synthesis. We characterized the VACV transcripts at the individual mRNA molecule level and found that vaccinia postreplicative mRNAs, containing nontemplated 5 poly(A) leaders, surprisingly lack the 5 cap structure in vivo. We also show that the lengths of the nontemplated leaders and the presence or absence of cap structures at the 5 mRNA ends are controlled by the initiator sequence of the VACV postreplicative promoters. One Sentence SummaryThe promoter sequence determines the synthesis of the 5 cap and poly(A) leaders in vaccinia virus postreplicative mRNAs.

molecular biology↗

bioRxiv2019

Temporal Transcriptome and Promoter Architecture ofthe African Swine Fever Virus

The African Swine Fever Virus causes haemorrhagic fever in domestic pigs and presents the biggest global threat to animal farming in recorded history. Despite its importance, very little is known the mechanisms and temporal regulation of transcription in ASFV. Here we report the first detailed viral transcriptome analysis of ASFV during early and late infection of Vero cells. In addition to total RNA sequencing, we have characterised the transcription start sites and transcription termination sites at nucleotide-resolution, revealing the distinct DNA consensus motifs of early and late promoters, as well as the sequence determinants for transcription termination. ASFV can utilise alternative promoters to generate distinct proteins from the same transcription unit that differ with respect to the polypeptide N-terminus. Finally, our results reveal that the ASFV-RNAP undergoes transcript slippage at the 5 end of transcription units that in a promoter sequence-specific manner results in the addition of 5-AT and 5-ATAT tails to mRNAs.

genomics↗

bioRxiv2018

Messenger RNAs transcribed from yeast linear cytoplasmic plasmids possess unconventional 5’ and 3’ UTRs and suggest a novel mechanism of translation

Linear plasmids with almost identical compact genetic organization have been found in the cytoplasm of yeast species from nine genera. We employed pGKL1,2 plasmids from Kluyveromyces lactis as a model to investigate the previously unstudied transcriptome of yeast cytoplasmic linear plasmids. We performed 5 and 3 RACE analysis of all the pGKL1,2 mRNAs and found them not 3 polyadenylated and containing mostly uncapped 5 poly(A) leaders that are not complementary to the plasmid DNA. The degree of 5 capping and/or 5 polyadenylation is specific to each gene and is controlled by the corresponding promoter regions. We refined the description of the pGKL1,2 promoters and found new alternative promoters of several genes. We also provide evidence that K2ORF3 encodes an mRNA cap guanine-N7-methyltransferase and that 5 capped pGKL1,2 transcripts contain N7-methylated caps. Translation of pGKL1,2 transcripts is enhanced in Ism1{Delta} and pab1{Delta} strains and is independent of eIF4E and Pab1 translation factors. We suggested a model of a primitive regulation of pGKL1,2 plasmids gene expression where degree of 5 mRNA capping, degree of 5 non-template polyadenylation and presence of negative regulators as PAB1 and Lsm1 play an important role. Our data also suggest a close relationship between linear plasmids and poxviruses.

molecular biology↗

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↗