Date of this Version
The functional structure of all biologically active molecules is dependent on intra- and inter-molecular interactions. This is especially evident for RNA molecules whose functionality, maturation, and regulation require formation of correct secondary structure through encoded base-pairing interactions. Unfortunately, intra- and inter-molecular base-pairing information is lacking for most RNAs. Here, we marry classical nuclease-based structure mapping techniques with high-throughput sequencing technology to interrogate all base-paired RNA in Arabidopsis thaliana and identify ∼200 new small (sm)RNA–producing substrates of RNA–DEPENDENT RNA POLYMERASE6. Our comprehensive analysis of paired RNAs reveals conserved functionality within introns and both 5′ and 3′ untranslated regions (UTRs) of mRNAs, as well as a novel population of functional RNAs, many of which are the precursors of smRNAs. Finally, we identify intra-molecular base-pairing interactions to produce a genome-wide collection of RNA secondary structure models. Although our methodology reveals the pairing status of RNA molecules in the absence of cellular proteins, previous studies have demonstrated that structural information obtained for RNAs in solution accurately reflects their structure in ribonucleoprotein complexes. Furthermore, our identification of RNA–DEPENDENT RNA POLYMERASE6 substrates and conserved functional RNA domains within introns and both 5′ and 3′ untranslated regions (UTRs) of mRNAs using this approach strongly suggests that RNA molecules are correctly folded into their secondary structure in solution. Overall, our findings highlight the importance of base-paired RNAs in eukaryotes and present an approach that should be widely applicable for the analysis of this key structural feature of RNA.
© 2010 Zheng et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Zheng, Q., Ryvkin, P., Li, F., Dragomir, I., Valladares, O., Yang, J., Cao, K., Wang, L., & Gregory, B. D. (2010). Genome-Wide Double-Stranded RNA Sequencing Reveals the Functional Significance of Base-Paired RNAs in Arabidopsis. PLoS Genetics, 6 (9), http://dx.doi.org/10.1371/journal.pgen.1001141
Additional FilesFigure 1_Genome-wide double-stranded RNA sequencing.pdf (103 kB)
Figure 2_Genome-wide double-stranded RNA sequencing.pdf (177 kB)
Figure 3_Genome-wide double-stranded RNA sequencing.pdf (145 kB)
Figure 4_Genome-wide double-stranded RNA sequencing.pdf (185 kB)
Figure 5_Genome-wide double-stranded RNA sequencing.pdf (134 kB)
Figure 6_Genome-wide double-stranded RNA sequencing.pdf (118 kB)
Figure 7_Genome-wide double-stranded RNA sequencing.pdf (172 kB)
Date Posted: 14 July 2017
This document has been peer reviewed.