Biology subjects

Milek, M.

Publications and source records attributed to Milek, M..

5 recordsLinked to original sources

bioRxiv2024

Subcellular mRNA kinetic modeling reveals nuclear retention as rate-limiting

Eukaryotic mRNAs are transcribed, processed, translated, and degraded in different subcellular compartments. Here, we measured mRNA flow rates between subcellular compartments in mouse embryonic stem cells. By combining metabolic RNA labeling, biochemical fractionation, mRNA sequencing, and mathematical modeling, we determined the half-lives of nuclear pre-, nuclear mature, cytosolic, and membrane-associated mRNAs from over 9000 genes. In addition, we estimated transcript elongation rates. Many matured mRNAs have long nuclear half-lives, indicating nuclear retention as the rate-limiting step in the flow of mRNAs. In contrast, mRNA transcripts coding for transcription factors show fast kinetic rates, and in particular short nuclear half-lives. Differentially localized mRNAs have distinct rate constant combinations, implying modular regulation. Membrane stability is high for membrane-localized mRNA and cytosolic stability is high for cytosol-localized mRNA. mRNAs encoding target signals for membranes have low cytosolic and high membrane half-lives with minor differences between signals. Transcripts of nuclear-encoded mitochondrial proteins have long nuclear retention and cytoplasmic kinetics that do not reflect co-translational targeting. Our data and analyses provide a useful resource to study spatiotemporal gene expression regulation.

systems biology↗

bioRxiv2023

IL-33 controls IL-22-dependent antibacterial defense by modulating the microbiota

IL-22 plays a critical role in defending against mucosal infections, but how IL-22 production is regulated is incompletely understood. Here, we show that mice lacking IL-33 or its receptor ST2 (IL-1RL1) were more resistant to Streptococcus pneumoniae lung infection than wild-type animals, and that single nucleotide polymorphisms in IL33 and IL1RL1 were associated with pneumococcal pneumonia in humans. The effect of IL-33 on S. pneumoniae infection was mediated by negative regulation of IL-22 production in innate lymphoid cells (ILCs), but independent of ILC2s as well as IL-4 and IL-13 signaling. Moreover, IL-33s influence on antibacterial defense was dependent on housing conditions of the mice, and mediated by the modulatory effect of IL-33 on the microbiota. Collectively, we provide insight into the bidirectional crosstalk between the innate immune system and the microbiota. We identify a mechanism, dependent on both genetic and environmental factors, that impacts the efficacy of antibacterial immune defense and thus susceptibility to pneumonia. SIGNIFICANCE STATEMENTLower respiratory tract infections are the fifth leading cause of death. Here, we describe a mechanism influenced by genetic and environmental factors that affects the efficacy of pulmonary antibacterial immune responses. We show that IL-33 controls antibacterial defense by regulating the production of IL-22, a cytokine with known functions in antimicrobial immunity in lungs. The effect of IL-33 on IL-22-dependent defense was influenced by the hygienic status of the mice and mediated by IL-33s modulatory effect on the animal microbiota. In addition, genetic variation in genes involved in IL-33 signaling was associated with bacterial pneumonia in humans. Our findings may be important for our understanding of the factors influencing predisposition to lower respiratory tract infections.

immunology↗

bioRxiv2023

Robust detection of clinically relevant features in single-cell RNA profiles of patient-matched fresh and formalin-fixed paraffin-embedded (FFPE) lung cancer tissue

Single-cell transcriptional profiling reveals cell heterogeneity and clinically relevant traits in intra-operatively collected patient-derived tissue. However, the established approach to perform such analyses on freshly collected tissue constitutes an important limitation since it requires prospective collection and immediate processing. Therefore, the ability to perform single-cell RNA sequencing from archived tissues would be very beneficial in a clinical setting. Here, we benchmark single-cell gene expression profiles from patient-matched fresh, cryopreserved and FFPE cancer tissue. We find that fresh tissue and FFPE routine blocks can be employed for the robust detection of clinically relevant traits on the single-cell level. Specifically, single-cell maps of fresh patient tissues and corresponding FFPE tissue blocks could be integrated into common low-dimensional representations, and cell subtype clusters showed highly correlated transcriptional strengths of signaling pathways, Hallmark and clinically useful signatures, despite some variability in expression of individual genes due to technological differences. FFPE tissue blocks revealed higher cell diversity compared to fresh tissue. In contrast, single-cell profiling of cryopreserved tissue was prone to artifacts in the clinical setting. Our analysis suggests that single-cell RNA sequencing from FFPE tissues is comparable to and can replace analyses from fresh tissue. This highlights the potential of single-cell profiling in the analysis of retrospectively and prospectively collected archival pathology cohorts and dramatically increases the applicability in translational projects.

pathology↗

bioRxiv2020

CRNKL1 is a highly selective regulator of intron-retaining HIV-1 and cellular mRNAs

The HIV-1 Rev protein is a nuclear export factor for unspliced and incompletely-spliced HIV-1 RNAs. Without Rev, these intron-retaining RNAs are trapped in the nucleus. A genome-wide screen identified nine proteins of the spliceosome which all enhanced expression from the HIV-1 unspliced RNA after CRISPR/Cas knock-down. Depletion of DHX38, WDR70 and four proteins of the Prp19-associated complex (ISY1, BUD31, XAB2, CRNKL1) resulted in a more than 20-fold enhancement of unspliced HIV-1 RNA levels in the cytoplasm. Targeting of CRNKL1, DHX38, and BUD31 affected nuclear export efficiencies of the HIV-1 unspliced RNA to a much larger extent than splicing. Transcriptomic analyses further revealed that CRNKL1 also suppresses cytoplasmic levels of cellular mRNAs with selectively retained introns. Thus, CRNKL1 dependent nuclear retention seems to be a novel mechanism for the regulation of cytoplasmic levels of intron-retaining cellular mRNAs that is harnessed by HIV-1 to direct its complex splicing pattern.

molecular biology↗

bioRxiv2019

Loss of macp{Psi} ribosomal RNA modification is a major feature of cancer

The ribosome is an RNA-protein complex essential for translation in all domains of life. The structural and catalytic core of the ribosome is its ribosomal RNA (rRNA). While mutations in ribosomal protein (RP) genes are known drivers of oncogenesis, oncogenic rRNA variants have remained elusive. We discovered a cancer-specific single nucleotide variation in 18S rRNA at nucleotide 1248.U in up to 45.9% of colorectal carcinoma (CRC) patients and present across >22 cancer types. This is the site of a unique hyper-modified base, 1-methyl-3--amino--carboxyl-propyl pseudouridine (m1acp3{Psi}), a >1 billion years conserved RNA modification at the ribosomes peptidyl decoding-site. A sub-set of CRC tumors we term hypo-m1acp3{Psi}, show sub-stoichiometric m1acp3{Psi}-modification unlike normal control tissues. A m1acp3{Psi} knockout model and hypo-m1acp3{Psi} patient tumors share a translational signature, characterized by highly abundant ribosomal proteins. Thus, m1acp3{Psi}-deficient rRNA forms an uncharacterized class of onco-ribosome which may serve as a chemotherapeutic target for treating cancer patients.

cancer biology↗