RNA SHAPE analysis in living cells

Abstract

RNA structure plays important roles in practically every facet of gene regulation, but the paucity of structural probes that function in vivo has limited current understanding. Here we design, synthesize, and demonstrate two novel chemical probes that enable selective 2’-hydroxyl acylation for accurate RNA structural analysis in living cells. RNA structure in embryonic stem cells and several other species is read out at single nucleotide resolution, revealing tertiary contacts and RNA-protein interactions.

Determination of in vivo RNA structure in low-abundance transcripts

“…The synthesis of the SHAPE reagent NAI was performed according to the literature 17 . All manipulations involving synthesis and use of NAI were conducted in a chemical fume hood and with reagents from Sigma-Aldrich.…”

“…We demonstrate that both DMS and SHAPE chemistries permit in vivo RNA structural probing in Arabidopsis thaliana, an important model plant species and eukaryote. Notably, we employ the in vivo SHAPE reagent, 2-methylnicotinic acid imidazolide (NAI) 17 , and present the first examples of in vivo SHAPE probing in plants. We use the RT-based method ( Either DMS/SHAPE-RT (Steps 1-3) or DMS/ SHAPE-LMPCR can be used (all steps).…”

“…Recently, in vivo SHAPE reagents were developed and have been used to probe the highly abundant 5S rRNA in bacteria, yeast, fly and mammalian cells 17 . DMS methylates the N1 of adenine and the N3 of cytosine on the Watson-Crick base pairing face of unstructured regions such as loops, bulges and mismatches 12,14 , whereas SHAPE reagents acylate the 2 0 -hydroxyl group on the ribose sugar of unstructured regions of all four nucleotides 17,18 . Methylation or acylation chemistry is detected by reverse transcription (RT) stops one nucleotide before the modified nucleotide 12,14,[17][18][19] .…”

“…In cellular systems, structures of high-abundance RNAs such as rRNA can be assessed in vivo by a DMS/SHAPE-RT approach 11,12,14,17 . However, the vast majority of RNAs is of low abundance in vivo and cannot be explored by an RT-based approach.…”

The Elaborate Architecture of Rna

“…To obtain pairing information for every letter of RNA inside the cell, Chang and Spitale adapted a structure-probing technique called SHAPE 6 . This allowed them to deduce the structures of more than 19,000 RNAs in mouse embryonic stem cells, an effort they published earlier this year 7 .…”