Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells
| Autor: | Xuerong Yang, Jiefu Li, Yuanlong Liu, Huan Huan Wei, Qianyun Lu, Yang Wang, Zefeng Wang |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
General Chemical Engineering Protein domain Cell Culture Techniques RNA-binding protein Bioengineering Computational biology Biology General Biochemistry Genetics and Molecular Biology 03 medical and health sciences Protein Domains Genetics Humans Gene General Immunology and Microbiology Effector General Neuroscience Alternative splicing RNA RNA-Binding Proteins Protein engineering Alternative Splicing 030104 developmental biology HEK293 Cells RNA splicing Plasmids Transcription Factors |
| Zdroj: | Journal of Visualized Experiments. |
| ISSN: | 1940-087X |
| Popis: | The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which specifically binds the pre-mRNA target and an effector domain. Previously, we have taken advantage of the unique RNA binding mode of the PUF domain in human Pumilio 1 to generate a programmable RNA binding scaffold, which was used to engineer various artificial RBPs to manipulate RNA metabolism. Here, a detailed protocol is described to construct Engineered Splicing Factors (ESFs) that are specifically designed to modulate the alternative splicing of target genes. The protocol includes how to design and construct a customized PUF scaffold for a specific RNA target, how to construct an ESF expression plasmid by fusing a designer PUF domain and an effector domain, and how to use ESFs to manipulate the splicing of target genes. In the representative results of this method, we have also described the common assays of ESF activities using splicing reporters, the application of ESF in cultured human cells, and the subsequent effect of splicing changes. By following the detailed protocols in this report, it is possible to design and generate ESFs for the regulation of different types of Alternative Splicing (AS), providing a new strategy to study splicing regulation and the function of different splicing isoforms. Moreover, by fusing different functional domains with a designed PUF domain, researchers can engineer artificial factors that target specific RNAs to manipulate various steps of RNA processing. |
| Databáze: | OpenAIRE |
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