High-Throughput Analysis Reveals Rules for Target RNA Binding and Cleavage by AGO2.
Mol Cell
; 75(4): 741-755.e11, 2019 08 22.
Article
in En
| MEDLINE
| ID: mdl-31324449
ABSTRACT
Argonaute proteins loaded with microRNAs (miRNAs) or small interfering RNAs (siRNAs) form the RNA-induced silencing complex (RISC), which represses target RNA expression. Predicting the biological targets, specificity, and efficiency of both miRNAs and siRNAs has been hamstrung by an incomplete understanding of the sequence determinants of RISC binding and cleavage. We applied high-throughput methods to measure the association kinetics, equilibrium binding energies, and single-turnover cleavage rates of mouse AGO2 RISC. We find that RISC readily tolerates insertions of up to 7 nt in its target opposite the central region of the guide. Our data uncover specific guidetarget mismatches that enhance the rate of target cleavage, suggesting novel siRNA design strategies. Using these data, we derive quantitative models for RISC binding and target cleavage and show that our in vitro measurements and models predict knockdown in an engineered cellular system.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
RNA-Induced Silencing Complex
/
RNA, Small Interfering
/
Argonaute Proteins
/
Models, Chemical
Type of study:
Prognostic_studies
Limits:
Animals
Language:
En
Journal:
Mol Cell
Journal subject:
BIOLOGIA MOLECULAR
Year:
2019
Document type:
Article
Affiliation country:
United States