Massive parallel-sequencing-based hydroxyl radical probing of RNA accessibility

Hydroxyl Radical Footprinting (HRF) is a tried-and-tested method for analysis of the tertiary structure of RNA and for identification of protein footprints on RNA. The hydroxyl radical reaction breaks accessible parts of the RNA backbone, thereby allowing ribose accessibility to be determined by detection of reverse transcriptase termination sites. Current methods for HRF rely on reverse transcription of a single primer and detection by fluorescent fragments by capillary electrophoresis. Here, we describe an accurate and efficient massive parallel-sequencing-based method for probing RNA accessibility with hydroxyl radicals, called HRF-Seq. Using random priming and a novel barcoding scheme, we show that HRF-Seq dramatically increases the throughput of HRF experiments and facilitates the parallel analysis of multiple RNAs or experimental conditions. Moreover, we demonstrate that HRF-Seq data for the Escherichia coli 16S rRNA correlates well with the ribose accessible surface area as determined by X-ray crystallography and have a resolution that readily allows the difference in accessibility caused by exposure of one side of RNA helices to be observed

SHAPE selection (SHAPES) enrich for RNA structure signal in SHAPE sequencing-based probing data

Selective 2′ Hydroxyl Acylation analyzed by Primer Extension (SHAPE) is an accurate method for probing of RNA secondary structure. In existing SHAPE methods, the SHAPE probing signal is normalized to a no-reagent control to correct for the background caused by premature termination of the reverse transcriptase. Here, we introduce a SHAPE Selection (SHAPES) reagent, N-propanone isatoic anhydride (NPIA), which retains the ability of SHAPE reagents to accurately probe RNA structure, but also allows covalent coupling between the SHAPES reagent and a biotin molecule. We demonstrate that SHAPES-based selection of cDNA–RNA hybrids on streptavidin beads effectively removes the large majority of background signal present in SHAPE probing data and that sequencing-based SHAPES data contain the same amount of RNA structure data as regular sequencing-based SHAPE data obtained through normalization to a no-reagent control. Moreover, the selection efficiently enriches for probed RNAs, suggesting that the SHAPES strategy will be useful for applications with high-background and low-probing signal such as in vivo RNA structure probing

mRNA decay of most arabidopsis miRNA targets requires slicer activity of AGO₁

MicroRNAs (miRNAs) are key posttranscriptional regulators of gene expression in animals and plants. They guide RNA-induced silencing complexes to complementary target mRNA, thereby mediating mRNA degradation or translational repression. ARGONAUTE (AGO) proteins bind directly to miRNAs and may catalyze cleavage (slicing) of target mRNAs. In animals, miRNA target degradation via slicing occurs only exceptionally, and target mRNA decay is induced via AGO-dependent recruitment of deadenylase complexes. Conversely, plant miRNAs generally direct slicing of their targets, but it is unclear whether slicer-independent mechanisms of target mRNA decay also exist, and, if so, how much they contribute to miRNA-induced mRNA decay. Here, we compare phenotypes and transcript profiles of ago1 null and slicer-deficient mutants in Arabidopsis (Arabidopsis thaliana). We also construct conditional loss-of-function mutants of AGO1 to allow transcript profiling in true leaves. Although phenotypic differences between ago1 null and slicer-deficient mutants can be discerned, the results of both transcript profiling approaches indicate that slicer activity is required for mRNA repression of the vast majority of miRNA targets. A set of genes exhibiting up-regulation specifically in ago1 null, but not in ago1 slicer-deficient mutants was also identified, leaving open the possibility that AGO1 may have functions in gene regulation independent of small RNAs