RNA molecules perform many crucial functions
in nearly every cellular pathway. Critical to controlling RNA function
is its ability to fold into complex three-dimensional structures. Effort
has been focused on understanding the structural components of RNA.
Traditionally this has been done by one-RNA-at-a-time experiments.
However, these conventional methods have now been married with
genome-wide technologies to provide a systems view of RNA structure.
Miles Kubota, Dalen Chan, and Robert C. Spitale, University of
California, Irvine, CA, USA, describe the use of chemical methods to
probe RNA structure and how these methods have been merged with deep
sequencing. They discuss how a combination of these two methods has
revealed new understanding of RNA structure and function, particularly
within a living cell, and what future challenges for transcriptome-wide
RNA structure probing are.
Various interatomic bindings determine how an RNA molecule folds. In the
selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE)
methode, a chemical reagent (the SHAPE probe) is mixed into a solution
containing many thousands of RNA molecules. The SHAPE probe can
chemically attack and modify every base of every RNA in the solution;
the position of a modified base is then detected as a stop in an
optimized primer extension reaction, followed by electrophoretic
fragment separation. The speed with which the probe modifies the
individual bases is an indication of how flexible the backbone of the
RNA is at each base position. This flexibility is highly correlated with
(but not wholly determined by) whether or not the base is paired to one
of its complements. In SHAPE-Seq, SHAPE is extended by bar-code based
multiplexing combined with RNA sequencing to be used in a
high-throughput fashion.
In hydroxyl radical cleavage, hydroxyl radicals damage the RNA depending
on the local structure. Coupled with the high-throughput analytical
capabilities of capillary electrophoresis this allows to quickly
evaluate the structural variation of longer RNA sequences than was
previously possible using standard gel electrophoresis. Additionally,
the Multiplexed ·OH Cleavage Analysis, which uses anchored iron-radical
cleavage for 3-D structure probing and modeling, has been married with
deep sequencing. This makes it possible to measure RNA structure,
transcriptome-wide, in three dimensions.
RNA structure: Merging chemistry and genomics for a holistic perspective
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