ArticleNucleic acids research2000
Better conditions for mammalian in vitro splicing provided by acetate and glutamate as potassium counterions.
Article in Nucleic acids research, 2000. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 13 papers.
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Who cites it
13 citing papers in PubMed.
- Nucleic Acid Sequence Determinants of Transcriptional Pausing by the Human Mitochondrial RNA Polymerase (POLRMT).Biochemistry · 2025Article
- Protein Cofactors Are Essential for High-Affinity DNA Binding by the Nuclear Factor κB RelA Subunit.Biochemistry · 2018Article
- Development of an in vitro pre-mRNA splicing assay using plant nuclear extract.Plant methods · 2018Article
- Pathogenic variants that alter protein code often disrupt splicing.Nature genetics · 2017Article
- Preparation of Splicing Competent Nuclear Extract from Mammalian Cells and In Vitro Pre-mRNA Splicing Assay.Methods in molecular biology (Clifton, N.J.) · 2017Article
- HnRNP A1 tethers KSRP to an exon splicing silencer that inhibits an erythroid-specific splicing event in PU.1-induced erythroleukemia.American journal of cancer research · 2015Article
- In vitro assay of pre-mRNA splicing in mammalian nuclear extract.Methods in molecular biology (Clifton, N.J.) · 2014Article
- Isolation and accumulation of spliceosomal assembly intermediates.Methods in molecular biology (Clifton, N.J.) · 2014Article
- Production of ACAT1 56-kDa isoform in human cells via trans-splicing involving the ampicillin resistance gene.Cell research · 2013Article
- Meayamycin inhibits pre-messenger RNA splicing and exhibits picomolar activity against multidrug-resistant cells.Molecular cancer therapeutics · 2009Article
- The biflavonoid isoginkgetin is a general inhibitor of Pre-mRNA splicing.The Journal of biological chemistry · 2008Article
- Muscleblind-like 1 interacts with RNA hairpins in splicing target and pathogenic RNAs.Nucleic acids research · 2007Article
- Purification and characterization of native spliceosomes suitable for three-dimensional structural analysis.RNA (New York, N.Y.) · 2002Article
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2 authors.
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Abstract
We demonstrate here that replacing potassium chloride (KCl) with potassium acetate (KAc) or potassium glutamate (KGlu) routinely enhances the yield of RNA intermediates and products obtained from in vitro splicing reactions performed in HeLa cell nuclear extract. This effect was reproducibly observed with multiple splicing substrates. The enhanced yields are at least partially due to stabilization of splicing precursors and products in the KAc and KGlu reactions. This stabilization relative to KCl reactions was greatest with KGlu and was observed over an extended potassium concentration range. The RNA stability differences could not be attributed to heavy metal contamination of the KCl, since ultrapure preparations of this salt yielded similar results. After testing various methods for altering the salts, we found that substitution of KAc or KGlu for KCl and MgAc(2)for MgCl(2)in splicing reactions is the simplest and most effective. Since the conditions defined here more closely mimic in vivo ionic concentrations, they may permit the study of more weakly spliced substrates, as well as facilitate more detailed analyses of spliceosome structure and function.
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