Publications by authors named "Hannah Mischo"

13 Publications

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Resilient SARS-CoV-2 diagnostics workflows including viral heat inactivation.

medRxiv 2021 Apr 10. Epub 2021 Apr 10.

There is a worldwide need for reagents to perform SARS-CoV-2 detection. Some laboratories have implemented kit-free protocols, but many others do not have the capacity to develop these and/or perform manual processing. We provide multiple workflows for SARS-CoV-2 nucleic acid detection in clinical samples by comparing several commercially available RNA extraction methods: QIAamp Viral RNA Mini Kit (QIAgen), RNAdvance Blood/Viral (Beckman) and Mag-Bind Viral DNA/RNA 96 Kit (Omega Bio-tek). We also compared One-step RT-qPCR reagents: TaqMan Fast Virus 1-Step Master Mix (FastVirus, ThermoFisher Scientific), qPCRBIO Probe 1-Step Go Lo-ROX (PCR Biosystems) and Luna Universal Probe One-Step RT-qPCR Kit (Luna, NEB). We used primer-probes that detect viral N (EUA CDC) and RdRP (PHE guidelines). All RNA extraction methods provided similar results. FastVirus and Luna proved most sensitive. N detection was more reliable than that of RdRP, particularly in samples with low viral titres. Importantly, we demonstrate that treatment of nasopharyngeal swabs with 70 degrees for 10 or 30 min, or 90 degrees for 10 or 30 min (both original variant and B 1.1.7) inactivates SARS-CoV-2 employing plaque assays, and that it has minimal impact on the sensitivity of the qPCR in clinical samples. These findings make SARS-CoV-2 testing portable to settings that do not have CL-3 facilities. In summary, we provide several testing pipelines that can be easily implemented in other laboratories and have made all our protocols and SOPs freely available at https://osf.io/uebvj/ .
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http://dx.doi.org/10.1101/2020.04.22.20074351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8043481PMC
April 2021

p38γ is essential for cell cycle progression and liver tumorigenesis.

Nature 2019 04 10;568(7753):557-560. Epub 2019 Apr 10.

Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain.

The cell cycle is a tightly regulated process that is controlled by the conserved cyclin-dependent kinase (CDK)-cyclin protein complex. However, control of the G0-to-G1 transition is not completely understood. Here we demonstrate that p38 MAPK gamma (p38γ) acts as a CDK-like kinase and thus cooperates with CDKs, regulating entry into the cell cycle. p38γ shares high sequence homology, inhibition sensitivity and substrate specificity with CDK family members. In mouse hepatocytes, p38γ induces proliferation after partial hepatectomy by promoting the phosphorylation of retinoblastoma tumour suppressor protein at known CDK target residues. Lack of p38γ or treatment with the p38γ inhibitor pirfenidone protects against the chemically induced formation of liver tumours. Furthermore, biopsies of human hepatocellular carcinoma show high expression of p38γ, suggesting that p38γ could be a therapeutic target in the treatment of this disease.
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http://dx.doi.org/10.1038/s41586-019-1112-8DOI Listing
April 2019

Nuclear fate of yeast snoRNA is determined by co-transcriptional Rnt1 cleavage.

Nat Commun 2018 05 3;9(1):1783. Epub 2018 May 3.

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK.

Small nucleolar RNA (snoRNA) are conserved and essential non-coding RNA that are transcribed by RNA Polymerase II (Pol II). Two snoRNA classes, formerly distinguished by their structure and ribonucleoprotein composition, act as guide RNA to target RNA such as ribosomal RNA, and thereby introduce specific modifications. We have studied the 5'end processing of individually transcribed snoRNA in S. cerevisiae to define their role in snoRNA biogenesis and functionality. Here we show that pre-snoRNA processing by the endonuclease Rnt1 occurs co-transcriptionally with removal of the mG cap facilitating the formation of box C/D snoRNA. Failure of this process causes aberrant 3'end processing and mislocalization of snoRNA to the cytoplasm. Consequently, Rnt1-dependent 5'end processing of box C/D snoRNA is critical for snoRNA-dependent methylation of ribosomal RNA. Our results reveal that the 5'end processing of box C/D snoRNA defines their distinct pathway of maturation.
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http://dx.doi.org/10.1038/s41467-018-04094-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5934358PMC
May 2018

Cell-Cycle Modulation of Transcription Termination Factor Sen1.

Mol Cell 2018 04 12;70(2):312-326.e7. Epub 2018 Apr 12.

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Many non-coding transcripts (ncRNA) generated by RNA polymerase II in S. cerevisiae are terminated by the Nrd1-Nab3-Sen1 complex. However, Sen1 helicase levels are surprisingly low compared with Nrd1 and Nab3, raising questions regarding how ncRNA can be terminated in an efficient and timely manner. We show that Sen1 levels increase during the S and G2 phases of the cell cycle, leading to increased termination activity of NNS. Overexpression of Sen1 or failure to modulate its abundance by ubiquitin-proteasome-mediated degradation greatly decreases cell fitness. Sen1 toxicity is suppressed by mutations in other termination factors, and NET-seq analysis shows that its overexpression leads to a decrease in ncRNA production and altered mRNA termination. We conclude that Sen1 levels are carefully regulated to prevent aberrant termination. We suggest that ncRNA levels and coding gene transcription termination are modulated by Sen1 to fulfill critical cell cycle-specific functions.
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http://dx.doi.org/10.1016/j.molcel.2018.03.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5919780PMC
April 2018

Disengaging polymerase: terminating RNA polymerase II transcription in budding yeast.

Biochim Biophys Acta 2013 Jan 17;1829(1):174-85. Epub 2012 Oct 17.

Cancer Research UK London Research Institute, Blanche Lane South Mimms, Herts, UK.

Termination of transcription by RNA polymerase II requires two distinct processes: The formation of a defined 3' end of the transcribed RNA, as well as the disengagement of RNA polymerase from its DNA template. Both processes are intimately connected and equally pivotal in the process of functional messenger RNA production. However, research in recent years has elaborated how both processes can additionally be employed to control gene expression in qualitative and quantitative ways. This review embraces these new findings and attempts to paint a broader picture of how this final step in the transcription cycle is of critical importance to many aspects of gene regulation. This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation.
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http://dx.doi.org/10.1016/j.bbagrm.2012.10.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3793857PMC
January 2013

Gene loops enhance transcriptional directionality.

Science 2012 Nov 27;338(6107):671-5. Epub 2012 Sep 27.

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.

Eukaryotic genomes are extensively transcribed, forming both messenger RNAs (mRNAs) and noncoding RNAs (ncRNAs). ncRNAs made by RNA polymerase II often initiate from bidirectional promoters (nucleosome-depleted chromatin) that synthesize mRNA and ncRNA in opposite directions. We demonstrate that, by adopting a gene-loop conformation, actively transcribed mRNA encoding genes restrict divergent transcription of ncRNAs. Because gene-loop formation depends on a protein factor (Ssu72) that coassociates with both the promoter and the terminator, the inactivation of Ssu72 leads to increased synthesis of promoter-associated divergent ncRNAs, referred to as Ssu72-restricted transcripts (SRTs). Similarly, inactivation of individual gene loops by gene mutation enhances SRT synthesis. We demonstrate that gene-loop conformation enforces transcriptional directionality on otherwise bidirectional promoters.
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http://dx.doi.org/10.1126/science.1224350DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3563069PMC
November 2012

Yeast Sen1 helicase protects the genome from transcription-associated instability.

Mol Cell 2011 Jan;41(1):21-32

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.

Sen1 of S. cerevisiae is a known component of the NRD complex implicated in transcription termination of nonpolyadenylated as well as some polyadenylated RNA polymerase II transcripts. We now show that Sen1 helicase possesses a wider function by restricting the occurrence of RNA:DNA hybrids that may naturally form during transcription, when nascent RNA hybridizes to DNA prior to its packaging into RNA protein complexes. These hybrids displace the nontranscribed strand and create R loop structures. Loss of Sen1 results in transient R loop accumulation and so elicits transcription-associated recombination. SEN1 genetically interacts with DNA repair genes, suggesting that R loop resolution requires proteins involved in homologous recombination. Based on these findings, we propose that R loop formation is a frequent event during transcription and a key function of Sen1 is to prevent their accumulation and associated genome instability.
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http://dx.doi.org/10.1016/j.molcel.2010.12.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3314950PMC
January 2011

Fail-safe transcriptional termination for protein-coding genes in S. cerevisiae.

Mol Cell 2009 Oct;36(1):88-98

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.

Transcription termination of RNA polymerase II (Pol II) on protein-coding genes in S. cerevisiae relies on pA site recognition by 3' end processing factors. Here we demonstrate the existence of two alternative termination mechanisms that rescue polymerases failing to disengage from the template at pA sites. One of these fail-safe mechanisms is mediated by the NRD complex, similar to termination of short noncoding genes. The other termination mechanism is mediated by Rnt1 cleavage of the nascent transcript. Both fail-safe termination mechanisms trigger degradation of readthrough transcripts by the exosome. However, Rnt1-mediated termination can also enhance the usage of weak pA signals and thereby generate functional mRNA. We propose that these alternative Pol II termination pathways serve the dual function of avoiding transcription interference and promoting rapid removal of aberrant transcripts.
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http://dx.doi.org/10.1016/j.molcel.2009.07.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2779338PMC
October 2009

Budding yeast RNA polymerases I and II employ parallel mechanisms of transcriptional termination.

Genes Dev 2008 Apr;22(8):1082-92

Sir William Dunn School of Pathology, Oxford OX1 3RE, United Kingdom.

Both RNA polymerase I and II (Pol I and Pol II) in budding yeast employ a functionally homologous "torpedo-like" mechanism to promote transcriptional termination. For two well-defined Pol II-transcribed genes, CYC1 and PMA1, we demonstrate that both Rat1p exonuclease and Sen1p helicase are required for efficient termination by promoting degradation of the nascent transcript associated with Pol II, following mRNA 3' end processing. Similarly, Pol I termination relies on prior Rnt1p cleavage at the 3' end of the pre-rRNA 35S transcript. This is followed by the combined actions of Rat1p and Sen1p to degrade the Pol I-associated nascent transcript that consequently promote termination in the downstream rDNA spacer sequence. Our data suggest that the previously defined in vitro Pol I termination mechanism involving the action of the Reb1p DNA-binding factor to "road-block" Pol I transcription close to the termination region may have overlooked more complex in vivo molecular processes.
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http://dx.doi.org/10.1101/gad.463408DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2335328PMC
April 2008

Small nuclear RNAs encoded by Herpesvirus saimiri upregulate the expression of genes linked to T cell activation in virally transformed T cells.

Curr Biol 2005 May;15(10):974-9

Howard Hughes Medical Institute, Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06536, USA.

Seven small nuclear RNAs of the Sm class are encoded by Herpesvirus saimiri (HVS), a gamma Herpesvirus that causes aggressive T cell leukemias and lymphomas in New World primates and efficiently transforms T cells in vitro. The Herpesvirus saimiri U RNAs (HSURs) are the most abundant viral transcripts in HVS-transformed, latently infected T cells but are not required for viral replication or transformation in vitro. We have compared marmoset T cells transformed with wild-type or a mutant HVS lacking the most highly conserved HSURs, HSURs 1 and 2. Microarray and Northern analyses reveal that HSUR 1 and 2 expression correlates with significant increases in a small number of host mRNAs, including the T cell-receptor beta and gamma chains, the T cell and natural killer (NK) cell-surface receptors CD52 and DAP10, and intracellular proteins--SKAP55, granulysin, and NKG7--linked to T cell and NK cell activation. Upregulation of three of these transcripts was rescued after transduction of deletion-mutant-HVS-transformed cells with a lentiviral vector carrying HSURs 1 and 2. These changes indicate an unexpected role for the HSURs in regulating a remarkably defined and physiologically relevant set of host targets involved in the activation of virally transformed T cells during latency.
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http://dx.doi.org/10.1016/j.cub.2005.04.034DOI Listing
May 2005

Actinomycin D induces histone gamma-H2AX foci and complex formation of gamma-H2AX with Ku70 and nuclear DNA helicase II.

J Biol Chem 2005 Mar 21;280(10):9586-94. Epub 2004 Dec 21.

Departments of Biochemistry and Molecular Biology, Institute of Molecular Biotechnology, Postfach 100 813, D-07708 Jena, Germany.

Formation of gamma-H2AX foci is a P. O.cellular response to genotoxic stress, such as DNA double strand breaks or stalled replication forks. Here we show that gamma-H2AX foci were also formed when cells were incubated with 0.5 microg/ml DNA intercalating agent actinomycin D. In untreated cells, gamma-H2AX co-immunoprecipitated with Ku70, a subunit of DNA-dependent protein kinase, as well as with nuclear DNA helicase II (NDH II), a DEXH family helicase also known as RNA helicase A or DHX9. This association was increased manifold after actinomycin D treatment. DNA degradation diminished the amount of Ku70 associated with gamma-H2AX but not that of NDH II. In vitro binding studies with recombinant NDH II and H2AX phosphorylated by DNA-dependent protein kinase confirmed a direct physical interaction between NDH II and gamma-H2AX. Thereby, the NDH II DEXH domain alone, i.e. its catalytic core, was able to support binding to gamma-H2AX. Congruently, after actinomycin D treatment, NDH II accumulated in RNA-containing nuclear bodies that predominantly co-localized with gamma-H2AX foci. Taken together, these results suggest that histone gamma-H2AX promotes binding of NDH II to transcriptionally stalled sites on chromosomal DNA.
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http://dx.doi.org/10.1074/jbc.M411444200DOI Listing
March 2005

The Herpesvirus saimiri small nuclear RNAs recruit AU-rich element-binding proteins but do not alter host AU-rich element-containing mRNA levels in virally transformed T cells.

Mol Cell Biol 2004 May;24(10):4522-33

Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University, New Haven, Connecticut 06536, USA.

Herpesvirus saimiri (HVS) encodes seven Sm-class small nuclear RNAs, called HSURs (for Herpesvirus saimiri U RNAs), that are abundantly expressed in HVS-transformed, latently infected marmoset T cells but are of unknown function. HSURs 1, 2, and 5 have highly conserved 5'-end sequences containing the AUUUA pentamer characteristic of AU-rich elements (AREs) that regulate the stability of many host mRNAs, including those encoding most proto-oncogenes and cytokines. To test whether the ARE-containing HSURs act to sequester host proteins that regulate the decay of these mRNAs, we demonstrate their in vivo interaction with the ARE-binding proteins hnRNP D and HuR in HVS-transformed T cells using a new cross-linking assay. Comprehensive Northern and microarray analyses revealed, however, that the levels of endogenous ARE-containing mRNAs are not altered in T cells latently infected with HVS mutants lacking HSURs 1 and 2. HSUR 1 binds the destabilizing ARE-binding protein tristetraprolin induced following activation of HVS-transformed T cells, but even in such stimulated cells, the levels of host ARE-containing mRNAs are not altered by deletion of HSURs 1 and 2. Instead, HSUR 1 itself is degraded by an ARE-dependent pathway in HVS-transformed T cells, suggesting that HVS may take advantage of the host ARE-mediated mRNA decay pathway to regulate HSUR expression. This is the first example of posttranscriptional regulation of the expression of an Sm small nuclear RNA.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC400482PMC
http://dx.doi.org/10.1128/mcb.24.10.4522-4533.2004DOI Listing
May 2004