The Conservation and Function of RNA Secondary Structure in Plants

Abstract

RNA transcripts fold into secondary structures via intricate patterns of base pairing. These secondary structures impart catalytic, ligand binding, and scaffolding functions to a wide array of RNAs, forming a critical node of biological regulation. Among their many functions, RNA structural elements modulate epigenetic marks, alter mRNA stability and translation, regulate alternative splicing, transduce signals, and scaffold large macromolecular complexes. Thus, the study of RNA secondary structure is critical to understanding the function and regulation of RNA transcripts. Here, we review the origins, form, and function of RNA secondary structure, focusing on plants. We then provide an overview of methods for probing secondary structure, from physical methods such as X-ray crystallography and nuclear magnetic resonance (NMR) imaging to chemical and nuclease probing methods. Combining these latter methods with high-throughput sequencing has enabled them to scale across whole transcriptomes, yielding tremendous new insights into the form and function of RNA secondary structure.

Keywords: RNA secondary structure; RNA-binding proteins; high-throughput sequencing; microRNAs; posttranscriptional regulation; small interfering RNAs.

Figure 1. Methods for probing RNA secondary structure

A schematic representation of the nuclease- and chemical-based probing techniques for empirically determining secondary structure. RNA can either be probed in a native state bound by RNA binding proteins (orange ovals) or deproteinated through extraction protocols or proteinase K treatement. (A) PARS assigns structure by the sites of transcript cleavage (green triangles), whereas (B) dsRNase/ssRNase-seq and PIP-seq both work by complete digestion. (C–D) Chemical probing works through reagents that preferentially adduct to nucleotides in a single-stranded confirmation, forming covalent modifications in either a (C) nucleotide-biased (green hexagons) or (D) unbiased (blue hexagons) manner. While multiple cleavage sites and covalent modifications are represented in this schematic, it is worth noting that PARS and the chemical probing techniques work with single-hit stoichiometry, with one cut/modification site interrogated per sequencing read.

Figure 2. Structural patterns in mRNAs

This is an example of a folded Arabidopsis thaliana mRNA. The displayed secondary structure profiles are representative of metagene patterns at start codons (dark red) miRNA target sites (orange), stop codons (red), constitutive intron (purple), retained intron (green), and cassette exon (blue) splice donor sites.

Figure 3