Abstract
BackgroundThe understanding of RNA structure is a key feature toward the comprehension of RNA functions and mechanisms of action. In particular, non-coding RNAs are thought to exert their functions by specific secondary structures, but an efficient annotation on a large scale of these structures is still missing.ResultsBy using a novel high-throughput method, named chemical inference of RNA structures, CIRS-seq, that uses dimethyl sulfate, and N-cyclohexyl- N'-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate to modify RNA residues in single-stranded conformation within native deproteinized RNA secondary structures, we investigate the structural features of mouse embryonic stem cell transcripts. Our analysis reveals an unexpected higher structuring of the 5′ and 3′ untranslated regions compared to the coding regions, a reduced structuring at the Kozak sequence and stop codon, and a three-nucleotide periodicity across the coding region of messenger RNAs. We also observe that ncRNAs exhibit a higher degree of structuring with respect to protein coding transcripts. Moreover, we find that the Lin28a binding protein binds selectively to RNA motifs with a strong preference toward a single stranded conformation.ConclusionsThis work defines for the first time the complete RNA structurome of mouse embryonic stem cells, revealing an extremely distinct RNA structural landscape. These results demonstrate that CIRS-seq constitutes an important tool for the identification of native deproteinized RNA structures.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-014-0491-2) contains supplementary material, which is available to authorized users.
Highlights
The understanding of RNA structure is a key feature toward the comprehension of RNA functions and mechanisms of action
We present here a new method, named chemical inference of RNA structures followed by massive parallel sequencing (CIRS-seq), that allows genome-wide investigation of native deproteinized RNA secondary structures by exploiting the capacity of dimethyl sulfate (DMS) and Ncyclohexyl-N’-(2-morpholinoethyl)carbodiimide metho-ptoluenesulfonate (CMCT) to react with RNA unpaired bases
To perform transcriptome-wide probing of RNAs in their native deproteinized conformation, we lysed mouse embryonic stem cell (ESC) in an isotonic buffer, and treated the lysate with Proteinase K to unmask regions of RNAs bound by proteins, without affecting the RNA structure
Summary
The understanding of RNA structure is a key feature toward the comprehension of RNA functions and mechanisms of action. While for certain small RNAs the in vitro folding landscape recapitulates well the in vivo one [19,20,21], long RNAs often exhibit rugged folding landscapes that lead in vitro to the prevalence of kinetically trapped intermediates and misfolded structures [22,23,24]. Comparative analysis of in vivo and in vitro probing data on human telomerase RNA revealed that while the 3′-terminal small nucleolar RNA (snoRNA)-like domain folds into comparable structures in the two conditions, the 5′ template domain exhibits very different foldings [26]. The non-treated (NT) signal at each position is subtracted from the DMS and CMCT signals to obtain the raw reactivity profile at base resolution. After scaling each data point above the 90th percentile to the 90th percentile, reactivity at each position is divided by the 90th percentile (90% Winsorising) to obtain the normalized reactivity
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