title: RNA-protein analysis using a conditional CRISPR nuclease.
abstract: 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.
authors.affiliation: Department of Molecular and Cell Biology, and Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, USA.
authors.forename: Ho Young
authors.initials: HY
authors.lastname: Lee
authors.forename: Rachel E
authors.initials: RE
authors.lastname: Haurwitz
authors.forename: Alex
authors.initials: A
authors.lastname: Apffel
authors.forename: Kaihong
authors.initials: K
authors.lastname: Zhou
authors.forename: Brian
authors.initials: B
authors.lastname: Smart
authors.forename: Craig D
authors.initials: CD
authors.lastname: Wenger
authors.forename: Stephen
authors.initials: S
authors.lastname: Laderman
authors.forename: Laurakay
authors.initials: L
authors.lastname: Bruhn
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Doudna
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D015153:Blotting, Western; D064130:CRISPR-Associated Proteins; D004722:Endoribonucleases / Q000378:metabolism*; D006367:HeLa Cells; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D007093:Imidazoles; D055029:Inverted Repeat Sequences / Q000235:genetics*; D013058:Mass Spectrometry; D035683:MicroRNAs / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods; D040901:Proteomics / Q000379:methods*; D016601:RNA-Binding Proteins / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D017423:Sequence Analysis, RNA
chemical_list: D001426:Bacterial Proteins; D064130:CRISPR-Associated Proteins; D007093:Imidazoles; D035683:MicroRNAs; D016601:RNA-Binding Proteins; C029899:imidazole; C552242:Csy4 endoribonuclease, Pseudomonas aeruginosa; D004722:Endoribonucleases