Multiplexed RNA structure characterization with selective 2′-hydroxyl acylation
analyzed by primer extension sequencing (SHAPE-Seq)
PNAS, 2011;vol. 108, 11063–11068
Presenter : Chih-Hao Wu
Date/Time : 2013/03/21
Commentator: Dr. Ta-Chien Tseng
Location: Room 602, Med College Building
Background:
The RNAs play several important roles in cellular processes and gene regulation. The coding sequence of
RNA makes it as a simple source of genetic information. Currently, there have been discovered several
non-coding, but functional RNAs that play central roles in maintaining, regulating, and defining the gene
expression. Many of these non-coding RNAs have been found having specific secondary and tertiary
structures essential to their function. To full understand of their structures, several technologies have been
developed. These techniques are able to characterize rapidly the structural features of RNAs within
complex RNA populations. In previous study, the authors developed a RNA structure analysis technique
called selective 2’-hydroxyl acylation analyzer by primer extension (SHAPE). SHAPE chemistry takes the
advantage of the nucleophilic reactivity of the ribose 2’-hydroxyl position which is strongly gated by the
underlying local nucleotide flexibility. In contrast, base paired or other conformationally constrained
nucleotides are unreactive. In this study, the authors combined the SHAPE chemistry and next generation
sequencing to develop the high-throughput technique, SHAPE-seq, then used it to infer the structural
information for the model RNA fold of the Bacillus subtilis RNase P specificity domain. The structure of
RNase P had been extensively characterized by SHAPE with capillary electrophoresis and X-ray
crystallography.
Objective/Hypothesis:
To test the capability of SHAPE-seq platform.
Results:
The authors generated an experimental pipeline for SHAPE-seq. To test its accuracy, the authors probed
the specificity domain of the highly conserved catalytic RNA, RNase P from B. subtitis. The result of
SHAPE-seq versus SHAPE-CE reactivities for every nucleotide of RNase P showed a high degree of
correlation between the two techniques. With bar coding, SHAPE-seq had the ability to identify structural
changes and simultaneously determine the structure of RNA molecules. The authors generated the RNA
library containing the wild type RNase P, four point mutants of RNase P, and two variants of the
Staphylococcus aureus plasmid transcriptional attenuator. The results showed that the overall reactivities
of those seven members of RNA library obtained from SHAPE-seq are similar to SHAPE-CE. These
demonstrated that SHAPE-seq is able to accurately and simultaneously infer nucleotide-resolution
structural information of a mixture of RNA species.
Conclusion:
SHAPE-seq is able to infer secondary and tertiary structural information, detect subtle conformational
change due to single nucleotide mutation, and simultaneously measure the structures of a complex pool of
different RNA molecules. This new technique gives a new way to study the centrality of RNAs and their
roles in gene regulation.