Purification of Eukaryotic Exoribonucleases Following Heterologous Expression in Bacteria and Analysis of Their Biochemical Properties by In Vitro Enzymatic Assays

Tomecki, Rafał; Drążkowska, Karolina; Krawczyk, Antonina O.; Kowalska, Katarzyna; Dziembowski, Andrzej

doi:10.1007/978-1-4939-2214-7_25

Cited by 8 publications

(6 citation statements)

References 23 publications

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“…Initially, 10 cycles of PCR product joining in the absence of primers was performed, based on the complementarity between fragments of CBP_R1 and CBP_F2 primers, followed by addition of CBP_F1-CBP_R2 primer pair and normal PCR, which eventually resulted in synthesis of the product corresponding to two-piece CBP80-CBP20 operon, which was purified as above. Final PCR products encompassing Cdc33 ORF and Cbp80-Cbp20 operon were inserted by SLIC into Bam HI/ Xho I sites of pET28M N-6xHis-SUMO vector ( 23 ). Escherichia coli MH1 strain ( E. coli araD lacX74 galU hsdR hsdM rpsL ) was transformed with SLIC products.…”

Section: Methodsmentioning

confidence: 99%

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A cap 0-dependent mRNA capture method to analyze the yeast transcriptome

Nowacka

Latoch

Izert

et al. 2022

Nucleic Acids Research

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Analysis of the protein coding transcriptome by the RNA sequencing requires either enrichment of the desired fraction of coding transcripts or depletion of the abundant non-coding fraction consisting mainly of rRNA. We propose an alternative mRNA enrichment strategy based on the RNA-binding properties of the human IFIT1, an antiviral protein recognizing cap 0 RNA. Here, we compare for Saccharomyces cerevisiae an IFIT1-based mRNA pull-down with yeast targeted rRNA depletion by the RiboMinus method. IFIT1-based RNA capture depletes rRNA more effectively, producing high quality RNA-seq data with an excellent coverage of the protein coding transcriptome, while depleting cap-less transcripts such as mitochondrial or some non-coding RNAs. We propose IFIT1 as a cost effective and versatile tool to prepare mRNA libraries for a variety of organisms with cap 0 mRNA ends, including diverse plants, fungi and eukaryotic microbes.

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Section: Methodsmentioning

confidence: 99%

“…vector (23). Escherichia coli MH1 strain (E. coli araD lacX74 galU hsdR hsdM rpsL) was transformed with SLIC products.…”

Section: Cdc33 and Cbp80-cbp20 Cloningmentioning

confidence: 99%

A cap 0-dependent mRNA capture method to analyze the yeast transcriptome

Nowacka

Latoch

Izert

et al. 2022

Nucleic Acids Research

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“…Methyltransferase gene (gene symbol: orf1ab) from coronavirus SARS-CoV-2 was purchased from Invitrogen Thermo Scientific with restriction sites for BamHI and XhoI endonucleases, compatible with the cloning sites of pET28_SUMO expression vector. 54 The vector included the sequence encoding 6xHis-Sumo tag attached at 5' end to nsp14 insert by ligation. Nsp 14 methyltransferase (nsp14) was overexpressed as a fusion protein with His-tagged Sumo in BL21 (DE3) RIL E.coli (Invitrogen) Procaryotic system.…”

Section: Methodsmentioning

confidence: 99%

Identification and Evaluation of Potential SARS-CoV-2 Antiviral Agents Targeting mRNA Cap Guanine N7-Methyltransferase

Kasprzyk¹,

Spiewla²,

smietanski³

et al. 2021

Preprint

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SARS-CoV-2, the cause of the currently ongoing COVID-19 pandemic, encodes its own mRNA capping machinery. Insights into this capping system may provide new ideas for therapeutic interventions and drug discovery. In this work, we employ a previously developed Py-FLINT screening approach to study the inhibitory effects of compounds against the cap guanine N7-methyltransferase enzyme, which is involved in SARS-CoV-2 mRNA capping. We screened five commercially available libraries (7039 compounds in total) to identify 83 inhibitors with IC50 < 50 μM, which were further validated using RP HPLC and dot blot assays. Novel fluorescence anisotropy binding assays were developed to examine the targeted binding site. The inhibitor structures were analyzed for structure-activity relationships in order to define common structural patterns. Finally, the most potent inhibitors were tested for antiviral activity on SARS-CoV-2 in a cell based assay<br>

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“…cDNA obtained from total RNA isolated from HEF 293 Flp-In T-REx cells was used as a template to amplify the ORF coding for the full-length human DXO wild type by PCR with the primer pair hDXO1For-hDXO1Rev (0.2 M both) and using Phusion High-Fidelity DNA Polymerase (F530, Thermo Fisher Scientific) with 1× HF buffer, 0.2 mM dNTP mix and 3% DMSO. The PCR product corresponding to the hDXO wild-type ORF was gel-purified using the Gel-Out kit (023-50, A&A Biotechnology) and inserted by SLIC into BamHI/XhoI sites of the pET28M N-6xHis-TEV vector (30), giving rise to the phDXOwt construct. Escherichia coli strain MH1 (araD lacX74 galU hsdR hsdM rpsL) was used for transformation with SLIC products.…”

Section: Recombinant Human Dcp2 (Hdcp2) and Hdxo Cloning Overproducti...mentioning

confidence: 99%

2′-O-Methylation of the second transcribed nucleotide within the mRNA 5′ cap impacts the protein production level in a cell-specific manner and contributes to RNA immune evasion

Drążkowska

Tomecki

Warmiński

et al. 2022

Nucleic Acids Research

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In mammals, m7G-adjacent nucleotides undergo extensive modifications. Ribose of the first or first and second transcribed nucleotides can be subjected to 2′-O-methylation to form cap1 or cap2, respectively. When the first transcribed nucleotide is 2′-O-methylated adenosine, it can be additionally modified to N6,2′-O-dimethyladenosine (m6Am). Recently, the crucial role of cap1 in distinguishing between ‘self’ and ‘non-self’ in mammalian cells during viral infection was revealed. Here, we attempted to understand the impact of cap methylations on RNA-related processes. Therefore, we synthesized tetranucleotide cap analogues and used them for RNA capping during in vitro transcription. Using this tool, we found that 2′-O-methylation of the second transcribed nucleotide within the mRNA 5′ cap influences protein production levels in a cell-specific manner. This modification can strongly hamper protein biosynthesis or have no influence on protein production levels, depending on the cell line. Interestingly, 2′-O-methylation of the second transcribed nucleotide and the presence of m6Am as the first transcribed nucleotide serve as determinants that define transcripts as ‘self’ and contribute to transcript escape from the host innate immune response. Additionally, cap methylation status does not influence transcript affinity towards translation initiation factor eIF4E or in vitro susceptibility to decapping by DCP2; however, we observe the resistance of cap2-RNA to DXO (decapping exoribonuclease)-mediated decapping and degradation.

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Purification of Eukaryotic Exoribonucleases Following Heterologous Expression in Bacteria and Analysis of Their Biochemical Properties by In Vitro Enzymatic Assays

Cited by 8 publications

References 23 publications

A cap 0-dependent mRNA capture method to analyze the yeast transcriptome

A cap 0-dependent mRNA capture method to analyze the yeast transcriptome

Identification and Evaluation of Potential SARS-CoV-2 Antiviral Agents Targeting mRNA Cap Guanine N7-Methyltransferase

2′-O-Methylation of the second transcribed nucleotide within the mRNA 5′ cap impacts the protein production level in a cell-specific manner and contributes to RNA immune evasion

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