Use of CRISPR/Cas9 with Homology-Directed Repair to Gene-Edit Topoisomerase IIβin Human Leukemia K562 Cells: Generation of a Resistance Phenotype

Journal of Pharmacology and Experimental Therapeutics, Journal Year: 2024, Volume and Issue: 389(2), P. 186 - 196

Published: March 20, 2024

DNA topoisomerase IIβ (TOP2β/180; 180 kDa) is a nuclear enzyme that regulates topology by generation of short-lived double-strand breaks primarily during transcription. TOP2β/180 can be target for damage-stabilizing anticancer drugs, whose efficacy often limited chemoresistance. Our laboratory previously demonstrated reduced levels (and the paralog TOP2α/170) in an acquired etoposide-resistant K562 clonal cell line, K/VP.5 part due to overexpression microRNA-9-3p/5p impacting post-transcriptional events. To evaluate effect on drug sensitivity upon reduction/elimination TOP2ß/180, premature stop codon was generated at gene exon 19/intron 19 boundary (AGAA//GTAA→ATAG//GTAA) parental cells (which contain four alleles) CRISPR/Cas9 editing with homology-directed repair (HDR) disrupt production full length TOP2β/180. Gene-edited clones were identified and verified quantitative polymerase chain (qPCR) Sanger sequencing, respectively. Characterization gene-edited clones, one or all TOP2ß/180 alleles mutated, revealed partial complete loss TOP2ß mRNA/protein, The protein correlated decreased XK469-induced damage resistance growth inhibition assays. Partial mitoxantrone also noted clone modified. No cross-resistance etoposide mAMSA clones. Results role sensitivity/resistance differential activity TOP2-targeted agents. Significance Statement Data indicated introduce resulted disruption expression human leukemia depending number edited alleles. Edited partially resistant XK469, while lacking mAMSA. import

Language: Английский

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Substrate diversity of NSUN enzymes and links of 5-methylcytosine to mRNA translation and turnover

Life Science Alliance, Journal Year: 2024, Volume and Issue: 7(9), P. e202402613 - e202402613

Published: July 10, 2024

Maps of the RNA modification 5-methylcytosine (m 5 C) often diverge markedly not only because differences in detection methods, data depth and analysis pipelines but also biological factors. We re-analysed bisulfite sequencing datasets from five human cell lines seven tissues using a coherent m C site calling pipeline. With resulting union list 6,393 sites, we studied distribution, enzymology, interaction with RNA-binding proteins molecular function. confirmed tRNA:m methyltransferases NSUN2 NSUN6 as main mRNA “writers,” further showed that rRNA:m methyltransferase NSUN5 can modify mRNA. Each enzyme recognises features strongly resemble their canonical substrates. By analysing proximity between sites footprints proteins, identified new candidates for functional interactions, including helicases DDX3X, involved translation, UPF1, an decay factor. found lack HeLa cells affected both steady-state levels of, UPF1-binding to, target mRNAs. Our studies emphasise emerging diversity writers readers effect on

Language: Английский

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Structure of the Nmd4-Upf1 complex supports conservation of the nonsense-mediated mRNA decay pathway between yeast and humans

PLoS Biology, Journal Year: 2024, Volume and Issue: 22(9), P. e3002821 - e3002821

Published: Sept. 27, 2024

The nonsense-mediated mRNA decay (NMD) pathway clears eukaryotic cells of mRNAs containing premature termination codons (PTCs) or normal stop located in specific contexts. It therefore plays an important role gene expression regulation. precise molecular mechanism the NMD has long been considered to differ substantially from yeast metazoa, despite involvement universally conserved factors such as central ATP-dependent RNA-helicase Upf1. Here, we describe crystal structure Upf1 bound its recently identified but yet uncharacterized partner Nmd4, show that Nmd4 stimulates ATPase activity and this interaction contributes elimination substrates. We also demonstrate a region critical for with is metazoan SMG6 protein, another major factor. involved UPF1 mutations affect levels endogenous human Our results support universal conservation eukaryotes.

Language: Английский

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A novel regulatory pathway recognizes and degrades transcripts with long 3′ untranslated regions

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: March 13, 2024

Quantitative control of gene expression is fundamental to cellular function, and post transcriptional regulation a consequential, additional layer over protein output. Within mammalian mRNAs, the 3′ untranslated region (3′UTR) acts as hub regulatory control, typically mediated by sequence elements within 3′UTR. We have found that from transcripts with long 3′UTRs strongly repressed compared those short 3′UTRs, this phenomenon appears be independent The repression increases 3′UTR length substantial; reporters 2,000 nucleotide are >10 fold 400 3′UTRs. Conversely, increasing coding has no effect on expression, demonstrating it length, not transcript elicits repression. Reporters different show difference in transcription rates nor translation efficiency but clear differences mRNA half lives nucleocytoplasmic distribution, indicating accelerated RNA decay. However, degradation does involve nonsense decay (NMD) pathway other canonical pathways. In order identify trans factors involved decay, we quantified proteins bound identified 100 differentially associated factors. Knockdowns two associating proteins, nuclear export DDX39B ZC3H11A, indicate they play role mRNAs This study establishes novel dependent feature which potentially regulates many genes.

Language: Английский

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Modulation of NBAS-Related Functions in the Early Response to SARS-CoV-2 Infection

International Journal of Molecular Sciences, Journal Year: 2023, Volume and Issue: 24(3), P. 2634 - 2634

Published: Jan. 30, 2023

Upon infection, severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is predicted to interact with diverse cellular functions, such as the nonsense-mediated decay (NMD) pathway, suggested by identification of core NMD factor upframeshift-1 (UPF1) in SARS-CoV-2 interactome, and retrograde transport from Golgi endoplasmic reticulum (ER) through reticulum-Golgi intermediate compartment (ERGIC), where coronavirus assembly occurs. Here, we investigated expression localization neuroblastoma-amplified sequence (NBAS) protein, a UPF1 partner for at ER, participating also transport, its functional partners, early time points after infection human lung epithelial cell line Calu3. We found significant decrease DExH-Box Helicase 34 (DHX34), suppressor morphogenetic effect on genitalia 5 (SMG5), SMG7 6 h post-infection, followed increase these genes UPF2 9 post-infection. Conversely, NBAS other coding factors were not modulated. Known substrates related stress (Growth Arrest Specific 5, GAS5; transducin beta-like 2, TBL2; DNA damage-inducible transcript 3, DDIT3) increased infected cells, possibly result alterations pathway direct infection. that unconventional SNARE ER 1, USE1 (p31) Zeste White 10 homolog, ZW10, partners function, significantly over cells. Co-localization proteins did change within 24 nor it differ versus non-infected cells 1 infection; similarly, co-localization p31 was altered this short frame. Finally, both co-localize S N proteins. Overall, data are preliminary evidence an interaction between NBAS-related functions deserving further investigation.

Language: Английский

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