Secondary Structure of Chloroplast mRNAs In Vivo and In Vitro

Abstract

mRNA secondary structure can influence gene expression, e.g., by influencing translation initiation. The probing of in vivo mRNA secondary structures is therefore necessary to understand what determines the efficiency and regulation of gene expression. Here, in vivo mRNA secondary structure was analyzed using dimethyl sulfate (DMS)-MaPseq and compared to in vitro-folded RNA. We used an approach to analyze specific, full-length transcripts. To test this approach, we chose low, medium, and high abundant mRNAs. We included both monocistronic and multicistronic transcripts. Because of the slightly alkaline pH of the chloroplast stroma, we could probe all four nucleotides with DMS. The structural information gained was evaluated using the known structure of the plastid 16S rRNA. This demonstrated that the results obtained for adenosines and cytidines were more reliable than for guanosines and uridines. The majority of mRNAs analyzed were less structured in vivo than in vitro. The in vivo secondary structure of the translation initiation region of most tested genes appears to be optimized for high translation efficiency.

Light-dependent translation change of Arabidopsis psbA correlates with RNA structure alterations at the translation initiation region

“…Furthermore, the 111 observed DMS probing for helix 33 of the 16S rRNA corresponds nicely with the rRNA structure previously 112 described for plastid ribosomes (Ahmed et al, 2017) (Supplemental Figure S5) and is similar for low and high 113 light conditions (Supplemental Figure S3C, S5B). The structure signals for guanosines and uridines in vivo 114 were, as expected (Mustoe et al, 2019; Gawroński et al, 2020), weaker than those for adenosines and 115 cytidines, but still informative compared to the in vitro, protein-free control (Supplemental Figure S5A). In 116 5 addition, the reproducibility of the probing of adenosines and cytidines was better than for guanosines and 117 uridines (Supplemental Figure S3B).…”

“…Indeed, translational regulation is a major 53 3 determinant of gene expression in plastids (Barkan, 2011; Sugiura, 2014; Sun and Zerges, 2015; Zoschke and 54 Bock, 2018). The intrinsic mRNA features that determine the efficiency of start codon recognition in plastids 55 of higher plants, and hence the efficiency of translation initiation, are well characterized: a) Shine-Dalgarno 56 sequences hybridize to the anti-Shine-Dalgarno sequence at the tail of the 16S rRNA and thereby position 57 the start codon so that it can bind to the initiator tRNA; b) local minima of mRNA secondary structure around 58 the start codon make it accessible for the ribosome, whereas other AUGs are masked by folded RNA (Hirose 59 and Sugiura, 2004; Scharff et al, 2011; Zhang et al, 2012; Scharff et al, 2017;Gawroński et al, 2020). 60Compared to the intrinsic mRNA features determining the efficiency of translation initiation, we understand 61 much less about the molecular mechanisms regulating translation in plastids.…”

“…However, 92 recently, it was demonstrated that under alkaline conditions DMS can probe also guanosines and uridines 93 (Mustoe et al, 2019). As the chloroplast stroma is slightly alkaline, all four nucleotides of chloroplast RNA 94 can be probed, although the probing of adenosines and cytidines is more reliable (Gawroński et al, 2020). 95High DMS probing at a nucleotide indicates a single-stranded confirmation.…”

Light-Dependent Translation Change of Arabidopsis psbA Correlates with RNA Structure Alterations at the Translation Initiation Region

“…Raw DMS reactivities from DMS− were subtracted from DMS+ samples and all negative values set to 0. Next, DMS reactivities were normalized separately for G/U and A/C by dividing the reactivities by the mean reactivity of the most highly reactive nucleotides (90th–99th percentile) of each transcript followed by 99% winsorization to remove extremely high values, as described earlier [ 27 ]. For structure prediction, RNA sequences were folded by the Fold program from RNAstructure (version 6.2) [ 54 ] with normalized DMS reactivities for all nucleotides used as soft constrains.…”

“…Indeed, translational regulation is a major determinant of gene expression in plastids [ 21 , 22 , 23 , 24 ]. The intrinsic mRNA features that determine the efficiency of start codon recognition in plastids of higher plants, and hence, the efficiency of translation initiation, are well characterized: (a) Shine-Dalgarno sequences hybridize to the anti-Shine-Dalgarno sequence at the tail of the 16S rRNA and thereby position the start codon so that it can bind to the initiator tRNA; (b) local minima of mRNA secondary structure around the start codon make it accessible for the ribosome, whereas other AUGs are masked by folded RNA [ 6 , 8 , 25 , 26 , 27 , 28 ].…”