Determination of in vivo RNA structure in low-abundance transcripts

Abstract

RNA structure plays important roles in diverse biological processes. However, the structures of all but the few most abundant RNAs are presently unknown in vivo. Here we introduce DMS/SHAPE-LMPCR to query the in vivo structures of low-abundance transcripts. DMS/ SHAPE-LMPCR achieves attomole sensitivity, a 100,000-fold improvement over conventional methods. We probe the structure of low-abundance U12 small nuclear RNA (snRNA) in Arabidopsis thaliana and provide in vivo evidence supporting our derived phylogenetic structure. Interestingly, in contrast to mammalian U12 snRNAs, the loop of the SLIIb in U12 snRNA is variable among plant species, and DMS/SHAPE-LMPCR determines it to be unstructured. We reveal the effects of proteins on 25S rRNA, 5.8S rRNA and U12 snRNA structure, illustrating the critical importance of mapping RNA structure in vivo. Our universally applicable method opens the door to identifying and exploring the specific structure-function relationships of the multitude of low-abundance RNAs that prevail in living cells.

High-throughput mutate-map-rescue evaluates SHAPE-directed RNA structure and uncovers excited states

“…In some cases, the resulting models have disagreed with accepted structures (Quarrier et al 2010;Kladwang et al 2011c;Hajdin et al 2013;Sükösd et al 2013;Rice et al 2014), motivating efforts to estimate uncertainty (Kladwang et al 2011c;Ramachandran et al 2013), to incorporate alternative mapping strategies (Cordero et al 2012a;Kwok et al 2013), and to integrate mapping with systematic mutagenesis (mutate-and-map [M 2 ]) (Kladwang and Das 2010;Kladwang et al 2011a,b;Cordero et al 2014). Even these improvements do not provide routes to validation through independent experiments, and structure inferences remain under question (Wenger et al 2011).…”

Comparison of SHAPE reagents for mapping RNA structures inside living cells

“…DMS has been used both inside and outside the cell to study RNA structure, RNA-protein interactions, and even RNA structure changes that result in ligand binding (Kwok et al 2013;Rouskin et al 2014;Fang et al 2015). Although very useful, DMS suffers from the drawback of having nucleotide specificities, limiting its resolution as a general RNA structure probe.…”

The predominant circular form of avocado sunblotch viroid accumulates in planta as a free RNA adopting a rod-shaped secondary structure unprotected by tightly bound host proteins

“…3a). Importantly, essentially the same conformation was obtained when NMIA was replaced by NAI, the alternative acylating reagent used for in vivo SHAPE [26,40,41] (Fig. 3b).…”

“…Between the remaining two members of Avsunviroidae, the eggplant latent viroid (ELVd) [35] and ASBVd [11], we chose the latter because despite its natural host being a woody plant, the isolate of this viroid, which we have been working with over the years (GenBank accession number J02020 with the substitution C213fiU), accumulates in vivo to levels comparable to the 5S ribosomal RNA [12,21], much higher than those reached by ELVd in eggplant. There are modifications more sensitive than conventional SHAPE [40,46], but some entail additional steps, particularly single-stranded adapter ligation, which limit the reliability of the results. These approaches, however, particularly SHAPE-MaP [46], may become indispensable with viroids reaching low titres in vivo.…”

“…Moreover, this reagent has been previously applied to determine the structure in planta of a chloroplast mRNA of Arabidopsis thaliana [40], thus indicating that NAI can traverse the membrane of this organelle. In vivo SHAPE of the mc ASBVd (+) form showed that it accumulates in chloroplasts as a free RNA uncovered by tightly bound host protein(s) which, like viral coat proteins, might exert a protecting role.…”