Tissue-specific changes in the RNA structurome mediate salinity response in Arabidopsis

Abstract

Little is known concerning the effects of abiotic factors on in vivo RNA structures. We applied Structure-seq to assess the in vivo mRNA structuromes of Arabidopsis thaliana under salinity stress, which negatively impacts agriculture. Structure-seq utilizes dimethyl sulfate reactivity to identify As and Cs that lack base pairing or protection. Salt stress refolded transcripts differentially in root vs. shoot, evincing tissue-specificity of the structurome. Both tissues exhibited an inverse correlation between salt stress-induced changes in transcript reactivity and changes in abundance, with stressrelated mRNAs showing particular structural dynamism. This inverse correlation is more pronounced in mRNAs wherein the mean reactivity of the 5'UTR, CDS and 3'UTR concertedly change under salinity stress, suggesting increased susceptibility to abundance control mechanisms in transcripts exhibiting this phenomenon, which we name "concordancy". Concordant salinity-induced increases in reactivity were notably observed in photosynthesis genes, thereby implicating mRNA structural loss in the well-known depression of photosynthesis by salt stress. Overall, changes in secondary structure appear to impact mRNA abundance, molding the functional specificity of the transcriptome under stress.

Structure-seq2 probing of RNA structure upon amino acid starvation reveals both known and novel RNA switches inBacillus subtilis

“…Using Structure-seq2 to probe the in vivo RNA structure genome-wide in B. subtilis in the presence and absence of amino acids, we saw that there is transcriptome-wide reduced DMS reactivity of RNA in response to amino acid starvation. Similarly, changes in the RNA structurome were observed in eukaryotic species where both heat stress and salinity stress promoted reduced DMS reactivity of RNA (Su et al, 2018;Tack et al, 2020). These findings suggest that there may be a universal stress response mechanism throughout the different domains of life that is manifested in changes in RNA accessibility.…”

“…When relating DMS reactivity changes with abundance changes following amino acid starvation, we saw an inverse correlation among all transcripts in which higher average DMS reactivity in the absence of amino acids corresponded to a decrease in transcript abundance. This inverse correlation found in eukaryotes (rice, Arabidopsis) (Su et al, 2018;Tack et al, 2020) and now in bacteria (B. subtilis)…”

“…In addition to structural information, Structure-seq2 provides transcript abundance, similar to RNA-seq (Su et al, 2018;Tack et al, 2018). Previously, we found that changes in transcript reactivity are inversely correlated with changes in transcript abundance following 10 min of heat shock in rice seedlings (Su et al, 2018) and after persistent salinity stress in Arabidopsis (Su et al, 2018;Tack et al, 2020). Those…”

“…S2). When +DMS replicates of each growth condition were pooled, coverage was sufficient (greater than 1 reverse transcriptase [RT] stop per A or C in the transcript) (Ding et al, 2014; Ritchey -6 Ritchey et al, 2019;Su et al, 2018;Tack et al, 2020;Waldron et al, 2019) to resolve 2602 (+AA) and 2600 (-AA) transcripts, with an overlap of 2333 transcripts. The RT stops of +DMS replicates of both +AA and -AA treatments were well-correlated for all resolvable transcripts within that condition (Supplemental Fig.…”

“…Filtered mappings were converted to .rtsc (reverse transcriptase stop count) files, replicates were merged, and per transcript coverage was calculated via StructureFold2 . For transcripts above the coverage threshold of 1 in the merged file (amounting to ≥ 1 RT stop per A or C residue in the transcript) (Ding et al, 2014;Su et al, 2018;Tack et al, 2020), we correlated raw RT stop patterns between component replicates (Supplemental Fig. S3; Supplemental Table T.1) with R (R Core Team, 2019) using the plyr package (Wickham, 2011) via the rtsc_correlation module of StructureFold2 ; this follows the same process for evaluating library replicate consistency as was done previously (Su et al, 2018;Tack et al, 2020;Waldron et al, 2019).…”