RNA architecture influences plant biology

Abstract

The majority of the genome is transcribed to RNA in living organisms. RNA transcripts can form astonishing arrays of secondary and tertiary structures via Watson-Crick, Hoogsteen, or wobble base pairing. In vivo, RNA folding is not a simple thermodynamic event of minimizing free energy. Instead, the process is constrained by transcription, RNA-binding proteins, steric factors, and the microenvironment. RNA secondary structure (RSS) plays myriad roles in numerous biological processes, such as RNA processing, stability, transportation, and translation in prokaryotes and eukaryotes. Emerging evidence has also implicated RSS in RNA trafficking, liquid-liquid phase separation, and plant responses to environmental variations such as temperature and salinity. At molecular level, RSS is correlated with splicing, polyadenylation, protein synthesis, and miRNA biogenesis and functions. In this review, we summarize newly reported methods for probing RSS in vivo and functions and mechanisms of RSS in plant physiology.

Keywords: RNA secondary structure; RNA trafficking; miRNA; phase separation; polyadenylation; salinity stress; splicing; translation.

Fig. 1.

In vitro and in vivo methods for RNA structure probing. (A) In vitro enzymatic or chemical probing of RSS. RNA is extracted from plants and then subjected to RNase S1, RNase V1, or chemical (DMS or SHAPE reagents) treatment. RNase S1 is specific for ssRNA digestion, resulting in higher coverage of dsRNA regions in RNA sequencing. In contrast, RNase V1 treatment results in higher coverage of ssRNA regions in RNA sequencing. Chemical treatment modifies single-stranded regions of RNA, resulting in higher mismatch rates or reverse transcription stops in modified sites. (B) In vivo chemical probing of RSS. Plants are treated with chemicals such as DMS or SHAPE reagents, which can modify nucleotides in single-stranded regions under native conditions. Modified RNA is extracted and sequenced. The modified sites are pinpointed as mismatches or reverse transcription stops. Bioinformatic analysis such as the DREEM algorithm is used to analyze heterogeneity of RNA.

Fig. 2.

RSS impacts multiple molecular and biological processes. (A) RSS regulates alternative splicing of pre-mRNAs. (B) RNA folding between poly(A) signal and poly(A) sites may faciliate polyadenylation. (C) RSS is correlated with translation efficiency. (D) RSS regulates miRNA biogenesis. CHR2, an ATPase subunit of SWI2/SNF2 complexes; SE, a subunit of plant Microprocessor. CHR2 remodels RSS of pri-miRNAs via SE to inhibit miRNA production. (E) RSS of mRNAs impacts miRNA targeting efficiency. (F) RNA conformations change in response to different temperatures to modify translation or degradation of transcripts. The purple box refers that single-strandedness in the targets flanking the miRNA binding sites promotes RISC occupancy. (G) Cloverleaf-like RNA structures promote RNA trafficking. (H) G-quadruplex (GQ) triggers phase separation.

Fig. 3.

The structural patterns across splice sites are associated with splicing events. (A) The structural patterns of the transcriptome across splice sites in vivo. The regions ~40 nt upstream of the 5′ splice sites display significantly more flexible structure in spliced transcripts than those of unspliced transcripts in vivo. (B) The structural patterns of nuclear RNA across splicing sites and branch sites in vivo. –1 nt and –2 nt upstream of 5′ splice sites and the branch point tend to be more single-stranded in spliced transcripts than in unspliced transcripts. The models are derived from Ding et al. (2014), Deng et al. (2018), and Liu et al. (2019, Preprint).

Fig. 4.

RSS across poly(A) sites is correlated with polyadenylation. (A) The folding of the mRNA 3′ region facilitates polyadenylation through juxtaposing the poly(A) signal and cleavage sites that are otherwise too far apart for the optimal distance in human cells. (B) The structural patterns across poly(A) sites for the transcriptome in vivo. RSS from –15 nt to –2 nt upstream of the poly(A) sites is more structured, while the region from –1 nt to +5 nt across the poly(A) sites shows more single-strandedness. (C) The structural patterns of nuclear RNA across poly(A) sites in vivo. RSS from –28 nt to –17 nt upstream of and from –4 nt to +1 nt across both the constitutive and alternative poly(A) sites show more single-strandedness. The models are derived from Ding et al. (2014), Wu and Bartel, 2017 and Liu et al. (2019, Preprint).

Fig. 5.

RSS is associated with translation efficiency of transcripts. (A) Different structural patterns of transcripts with high and low translation efficiency. The single-stranded region upstream of start codons is enriched in high translation efficiency transcripts but lacks low translation efficiency transcripts. The CDSs show a three-nucleotide periodicity in vivo, and the periodicity is intensified in high translation efficiency transcripts. (B) Average structuredness in UTRs and CDSs. The CDSs are more folded than UTRs in vivo. The models are derived from Ding et al. (2014) and Deng et al. (2018).