RNA–protein analysis using a conditional CRISPR nuclease
| العنوان: | RNA–protein analysis using a conditional CRISPR nuclease |
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| المؤلفون: | Stephen Laderman, Kaihong Zhou, Rachel E. Haurwitz, Laurakay Bruhn, Brian Phillip Smart, Craig D. Wenger, Alex Apffel, Jennifer A. Doudna, Ho Young Lee |
| المصدر: | Proceedings of the National Academy of Sciences. 110:5416-5421 |
| بيانات النشر: | Proceedings of the National Academy of Sciences, 2013. |
| سنة النشر: | 2013 |
| مصطلحات موضوعية: | Proteomics, Sequence analysis, Blotting, Western, CRISPR-Associated Proteins, Molecular Sequence Data, Endoribonuclease, RNA-binding protein, Computational biology, Biology, Protein Engineering, Mass Spectrometry, Bacterial Proteins, Cleave, Endoribonucleases, Humans, CRISPR, Genetics, Nuclease, Multidisciplinary, Base Sequence, Sequence Analysis, RNA, Inverted Repeat Sequences, Imidazoles, High-Throughput Nucleotide Sequencing, RNA-Binding Proteins, RNA, Protein engineering, Biological Sciences, MicroRNAs, biology.protein, HeLa Cells |
| الوصف: | RNA-binding proteins control the fate and function of the transcriptome in all cells. Here we present technology for isolating RNA–protein partners efficiently and accurately using an engineered clustered regularly interspaced short palindromic repeats (CRISPR) endoribonuclease. An inactive version of the Csy4 nuclease binds irreversibly to transcripts engineered with a 16-nt hairpin sequence at their 5′ ends. Once immobilized by Csy4 on a solid support, contaminating proteins and other molecules can be removed by extensive washing. Upon addition of imidazole, Csy4 is activated to cleave the RNA, removing the hairpin tag and releasing the native transcript along with its specifically bound protein partners. This conditional Csy4 enzyme enables recovery of specific RNA-binding partners with minimal false-positive contamination. We use this method, coupled with quantitative MS, to identify cell type-specific human pre-microRNA-binding proteins. We also show that this technology is suitable for analyzing diverse size transcripts, and that it is suitable for adaptation to a high-throughput discovery format. |
| تدمد: | 1091-6490 0027-8424 |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::1f7c397af1ea89edcde8832daa30e11a https://doi.org/10.1073/pnas.1302807110 |
| حقوق: | OPEN |
| رقم الأكسشن: | edsair.doi.dedup.....1f7c397af1ea89edcde8832daa30e11a |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 10916490 00278424 |
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