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

  1. Piotr Gawroński
  2. Christel Enroth
  3. Peter Kindgren
  4. Sebastian Marquardt
  5. Stanisław Karpiński
  6. Dario Leister
  7. Poul Jensen
  8. Jeppe Vinther
  9. Lars Scharff

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Abstract

mRNA secondary structure influences translation. Proteins that modulate the mRNA secondary structure around the translation initiation region may regulate translation in plastids. To test this hypothesis, we exposed Arabidopsis thaliana to high light, which induces translation of psbA mRNA encoding the D1 subunit of photosystem II. We assayed translation by ribosome profiling and applied two complementary methods to analyze in vivo RNA secondary structure: DMS-MaPseq and SHAPE-seq. We detected increased accessibility of the translation initiation region of psbA after high light treatment, likely contributing to the observed increase in translation by facilitating translation initiation. Furthermore, we identified the footprint of a putative regulatory protein in the 5′ UTR of psbA at a position where occlusion of the nucleotide sequence would cause the structure of the translation initiation region to open up, thereby facilitating ribosome access. Moreover, we show that other plastid genes with weak Shine-Dalgarno sequences (SD) are likely to exhibit psbA-like regulation, while those with strong SDs do not. This supports the idea that changes in mRNA secondary structure might represent a general mechanism for translational regulation of psbA and other plastid genes.

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  1. Version published to 10.3390/cells10020322
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    Reply to the reviewers

    Dear reviewers,

    Thank you very much for your constructive and helpful remarks and suggestions!

    We marked the changes in the manuscript in yellow.

    Our replies to the specific points:

    Reviewer #1 In the Introduction the authors need to cite earlier work in Chlamydomonas which first showed that binding of specific proteins to the psbA 5'UTR is correlated with increased translation in the light (Danon et al. 1991).

    As suggested, we added the reference to the introduction.

    Reviewer #1 The paper could be improved by testing for protein binding to the footprint region in high vs low light. An obvious candidate is HCF173.

    We agree that HCF173 is an obvious candidate, although its interaction could be mediated via additional proteins. Alice Barkan’s group has demonstrated that in maize HCF173 binds to the same region upstream of the translation initiation region (McDermott et al., 2019) where we detected a footprint (Supplemental Figure S11A-D). Furthermore, McDermott et al showed that the binding sequence is conserved. We would like to analyze this question in more detail, but we have currently in the lab no approach available to specifically isolate psbA mRNA with its bound proteins for this analysis and therefore have to postpone the answer to this question to future studies.

    Reviewer #2: **Important changes to make before full submission:** 1)It is becoming clear that the translation efficiency (TE) is often not a calculation of translational output from specific mRNAs but in fact is better to be described as ribosome association. There can be many reasons for increased ribosome association including ribosome stalling and increased translational engagement. It would be good for the authors to add a simple Western blot to demonstrate directly increased protein output from psbA during high light as compared to low light treatments. This figure could be added to Figure S1.

    We want to stress that we have chosen a condition that is well known to increase psbA translation in higher plants as shown in the literature with different methods (e.g. Chotewutmontri and Barkan, 2018; Schuster et al., 2020). The protein encoded by psbA, the D1 subunit of photosystem II, has an increased turnover in high light, i.e. a higher amount of D1 has to be produced to compensate for the increased degradation of photodamaged D1 (Mulo et al., 2012; Li et al., 2018).

    Although there is a lot of evidence in the literature for good correlation of translation efficiency as determined by ribosome profiling and protein synthesis, the reviewer raised a valid concern. Ribosome pausing or even ribosome stalling could also cause increased ribosome binding and thereby increased amounts of ribosome footprints. Therefore, we analyzed ribosome pausing in selected genes including psbA and rbcL. The pattern of ribosome pausing was very similar in low and high light (new Supplemental Figure 14), which rules out any ribosome stalling at specific sites or drastic changes in ribosome pausing. To analyze if there is increased ribosome pausing, we determined the fraction of footprints at pause sites compared to the total number of footprints. We used two different pause scores as cutoffs to determine pause sites. To include as many pausing events as possible, we used a pause score of 1, i.e. everything higher than the mean ribosome density per nucleotide of the corresponding coding region (Gawronski et al., 2018). This fraction was unaltered in low and high light (new Supplemental Figure 14). With a more stringent pause score of 20 (20 times higher ribosome density than the mean), an increase of ribsome pausing in high light was detected for psbA, whereas we did not find differences between high and low light for rbcL and psaA. However, this increase in pausing at the psbA mRNA is insufficient to explain the increase in the total amounts of ribosome footprints. Additional pause scores were tested, the value for the psbA fraction with a pause score of 20 included in Supplemental Figure S14 showed the largest difference.

    Reviewer #2: **Strongly suggested additions to the manuscript to improve its significance before publication** 1)Identifying the RNA-binding protein(s) (likey HCF173 which may be in a complex with other proteins) that interacts with the 5' UTR of psbA in a highlight dependent manner would increase the significance of this study. Finding that this protein binds to other plastid transcripts with weak Shine-Delgarno sequences would also be a nice addition to this study.

    See comment to reviewer 1. McDermott et al. (2019) describe HCF173 as relatively specific for psbA. Therefore, we do not assume that other genes with weak Shine-Dalgarno sequences are regulated via HCF173 but via different proteins using a similar molecular mechanism to influence the mRNA secondary structure at the translation initiation region.

    Reviewer #2: **Strongly suggested additions to the manuscript to improve its significance before publication** 2)Mutational analysis of the RBP binding site and also to change the secondary structure around the start codon based on the new structure maps to show the effects of these various changes on protein output would really provide important new findings on how important the RBP being as compared to the RNA secondary structure changes are for regulating protein output form psbA. It could also allow the demonstration of the dependence or independence of these two features on regulating translation from chloroplast mRNAs.

    We agree with the reviewer that this would be a very interesting study. Unfortunately, it requires a larger collection of lines with mutated psbA sequences. Plastid transformation in Arabidopsis thaliana is still technically demanding and time consuming. Even in the case of Nicotiana tabacum, for which plastid transformation is well established, such a project would likely need several years. We therefore think that such a study is beyond the scope of the current manuscript.

    Reviewer #3 1.In this paper, author mentioned that DMS can modify four nucleotides under alkaline conditions. Because the chloroplast is slightly alkaline, the authors use DMS reactivity from 4 nucleotides to model RNA secondary structure. Based on Kevin Weeks' s paper, it shows that in cell-free condition, DMS has very limited ability to modify single-stranded G and U compared to A and C (Anthony M. Mustoe et al., 2019, PNAS 116: 24574. fig. 1B). In Lars B. Scharff' paper which is cited by the author, it is also mentioned that A and C is more reliable to model RNA secondary structure. The authors might need to calculate the correlation the DMS data and known RNA structure using G/U or all four nucleotides to show that DMS reactivity from G and U is also reliable to be used. Also in Fig. S3B, the reproducibility of G/U between replicates is not as good as A/C. I don' t think G and U can be used to predict RSS.

    We agree with the reviewer that DMS reactivities at G/U are less reliable than those at A/C. This was shown by Mustoe et al. (2019) and by us for chloroplast rRNAs (Gawronski et al., 2020, Plants). We included a correlation of the known 16S rRNA secondary structure and the DMS reactivities at the different nucleotides (Supplemental Figure S5A) that demonstrates that the DMS reactivities at G/U actually contain information about rRNA secondary structure. This analysis demonstrated again that the reactivities at G/U are less reliable than at A/C. Therefore, we added an analysis of the more reliable A/C for comparison with the results for all four nucleotides (Figure 1D-F, 3C-F).

    Reviewer #3 2.Is the 5'UTR the only region which has RSS change? If not, how do RSS changes in other region contribute to translation?

    Translation initiation in plastids is mainly influenced by the secondary structure of the translation initiation region, especially at the cis-elements required for the recognition of the start codon. In addition, we have analyzed different other regions, e.g. the coding regions, the coding regions without the sequences next to the start codon, the end of the coding region, and the complete 5’ UTR (Supplemental Figure S14). We added a more detailed analysis of the changes of secondary structure of the coding region of those genes we focus on (Supplemental Figure S16). This shows that the secondary structure changes of the complete coding region correlate negatively with translation efficiency (see also Supplemental Figure S14G). A similar observation was made in E. coli and explained to be caused by differences in translation initiation, which are mainly influenced by the secondary structure of the translation initiation region (Mustoe et al., 2018).

    Reviewer #3 3.In Fig. 2A and 2B, the DMS reactivities seem very similar under low light and high light. Why did the authors obtain significantly different RNA secondary structure? Are the parameter of low light and high light the same when modelling RNA structure?

    The parameters for the RNA secondary structure predictions in Figure 2 are not identical (see Figure legend). For all structure predictions, the DMS reactivities were used as constrains, but only for the high light structure the sequence of the RNA binding protein’s footprint was forced to be single-stranded. These structure predictions are included to illustrate the mRNA structures in the presence and absence of an RNA binding protein. These structures are based on the observation that the two halves of the stem loop structure have different DMS reactivities in response to high light. The sequence including the protein footprint has lower DMS reactivities in both low and high light. This is in agreement with both a double-stranded sequence as well as a protein-bound sequence. In contrast, the other half of the stem loop, the sequence including the cis-elements of the translation initiation region, has increased DMS reactivities in high light, indicating that it is single-stranded. This suggests that there is protein binding in high light preventing the formation of the inhibitory stem loop.

    Reviewer #3 4.In Fig. S12, the correlationship between HL and LL in ribo-seq and RNAseq is high, which means no significant changes upon light change. In this paper, psbA should have translation change under high light conditions. I suggest the authors to label the dot representing psbA.

    Thank you very much for this suggestion! We marked psbA in the correlation plots (Supplemental Figure 12). The changes in the transcript levels are really minor, whereas for some genes the translation efficiency changes (see Figure 4 and Supplemental Figure S13).

    Reviewer #3 5.I suggest to use plants at the same stage for DMS-MaPseq and SHAPE probing.

    The different plant material was chosen because of the different requirements during probing. In this context, we would like to point out that observing the same changes in the translation initiation region in response to high light in different developmental stages is a stronger confirmation than observing the same response at the same developmental stage. This indicates that the response is not specific for a developmental stage.

    Reviewer #3 6.In Huang's paper (Jianyan Huang et al., 2019, Cell Reports 29: 4186-4199), there are many differential express genes under high light for 0.5hr. However, in the RNAseq data here, the correlation between high light and low light conditions is very high (Fig. S12). Why? Also, it would be nice if the authors could label several DEG whose expression change under high light treatment in Fig. S12?

    Supplemental Figure S12 contains only plastid-encoded RNAs, whereas Huang et al. (2019) focused on nuclear-encoded mRNAs. We clarified the figure legend of Supplemental Figure S12 by adding “of the plastid-encoded genes”. The values for the individual genes can be seen in Supplemental Figure S13.

    Reviewer #3 7.For the MNase footprint method, is the as-SD region the only region show enrichment under high light conditions? Besides, please provide the detailed method of MNase footprint. Does it work for RNA footprinting?

    The used methods are described under “Ribosome profiling (Ribo-seq)” and “Processing of Ribo-seq and RNA-seq reads” in Material and Methods. The approach was very similar to the one used for ribosome profiling with the difference that also smaller read lengths were included in the analysis (18-40 nt instead of 28-40 nt). We did this, because many plastid RNA binding proteins have footprints that are smaller than a ribosomal footprint. The described footprint is the only one detected near the translation initiation region of psbA. Binding of HCF173 was detected by the Barkan group in the same region using a RIP-Seq Analysis combined with RNase I digestion (McDermott et al., 2019), which confirms that our approach is working. We added a reference to the method section in the results part to clarify which approach was chosen.

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    Referee #3

    Evidence, reproducibility and clarity

    RNA can fold into secondary and tertiary structure through base-pairing. RNA structure plays a crucial role in gene functions and regulations, including transcription, processing, translation and decay. Plants acclimate to fluctuating light conditions to optimize photosynthesis and minimize photodamage. Translational regulation is known to be a strategy of these acclimations. It reported that translation of psbA, encoding the D1 reaction center protein of Photosystem II, is increased under high light condition. The light-controlled psbA translation has been intensively studied and was suggested to be related with redox/thiol signals, the ATP status, and some certain proteins. In this ms, Gawroński et al. explored the possible link between RNA secondary structure and translational efficiency. They adopted DMS-MaPseq and SHAPE-seq methods to profile the RNA secondary structure in 5UTR of psbA under low light and high light conditions. The results showed that the DMS and SHAPE activities of Shine-Dalgarno (SD) sequence, star codon and as-SD region are higher under high light condition than that under low light control, indicating that the psbA translation initiation region becomes more single-strandeness and accessible under high light condition. MNase-digestion and DMS activity analysis suggested that protein binding might cause the change of RNA secondary structure of psbA translation initiation region. In addition, the authors probed the RNA secondary structure of the translation initiation region of rbcL that encodes the large subunit of Rubisco and found no change in RNA structure of rbcL, while the translation of rbcL is also increased under high light condition. To address the question that RNA structure changes is related with high light-dependent translational activation of psbA but not rbcL, plastome-wide translational efficiency and RNA structure were analyzed. The results showed that a significant correlation between the RNA secondary changes and translational efficiency changes in the chloroplast-coded mRNAs with week SDs (such as psbA), but not with strong SDs (such as rbcL).

    The light-dependent translational activation of psbA is critical for maintaining photosynthetic homeostasis. Also, the molecular mechanism of RSS's impact on translation is still exclusive The topic of this study is very important. However, this study just described the phenomenon of RNA secondary structure changes in translational initiation region, but does not give further evidence to validate the effect of RNA secondary changes on the translational activation of psbA under high light condition. Besides, the evidence of protein binding causing RNA structure changes is week and unclear. In addition, there is much room for improvement for this work

    1.In this paper, author mentioned that DMS can modify four nucleotides under alkaline conditions. Because the chloroplast is slightly alkaline, the authors use DMS reactivity from 4 nucleotides to model RNA secondary structure. Based on Kevin Weeks' s paper, it shows that in cell-free condition, DMS has very limited ability to modify single-stranded G and U compared to A and C (Anthony M. Mustoe et al., 2019, PNAS 116: 24574. fig. 1B). In Lars B. Scharff' paper which is cited by the author, it is also mentioned that A and C is more reliable to model RNA secondary structure. The authors might need to calculate the correlation the DMS data and known RNA structure using G/U or all four nucleotides to show that DMS reactivity from G and U is also reliable to be used. Also in Fig. S3B, the reproducibility of G/U between replicates is not as good as A/C. I don' t think G and U can be used to predict RSS.

    2.Is the 5'UTR the only region which has RSS change? If not, how do RSS changes in other region contribute to translation?

    3.In Fig. 2A and 2B, the DMS reactivities seem very similar under low light and high light. Why did the authors obtain significantly different RNA secondary structure? Are the parameter of low light and high light the same when modelling RNA structure?

    4.In Fig. S12, the correlationship between HL and LL in ribo-seq and RNAseq is high, which means no significant changes upon light change. In this paper, psbA should have translation change under high light conditions. I suggest the authors to label the dot representing psbA.

    5.I suggest to use plants at the same stage for DMS-MaPseq and SHAPE probing.

    6.In Huang's paper (Jianyan Huang et al., 2019, Cell Reports 29: 4186-4199), there are many differential express genes under high light for 0.5hr. However, in the RNAseq data here, the correlation between high light and low light conditions is very high (Fig. S12). Why? Also, it would be nice if the authors could label several DEG whose expression change under high light treatment in Fig. S12?

    7.For the MNase footprint method, is the as-SD region the only region show enrichment under high light conditions? Besides, please provide the detailed method of MNase footprint. Does it work for RNA footprinting?

    Significance

    see above

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    Referee #2

    Evidence, reproducibility and clarity

    This study uses multiple high-throughput sequencing approaches to probe the secondary structure of the chloroplasitc psbA mRNA during low and high light treatments. They are able to demonstrate a shift in secondary structure around the start codon of this mRNA in response to the high light treatment as compared to under low light conditions. This structural shift is also accompanied by an RBP binding even that may also be involved in regulating the translation from this mRNA in response to high light. I think this study is very interesting and timely. However, I think determining the relative contributions of the secondary structure and RBP binding changes to potential increases in protein outputs from this mRNA in response to high light would improve this manuscript. I also think directly looking at protein levels through a straight-forward Western blot to show increase psbA protein in response to high light treatment is an important addition to this study. I outline my few suggested experimental additions for this manuscript below.

    Important changes to make before full submission:

    1)It is becoming clear that the translation efficiency (TE) is often not a calculation of translational output from specific mRNAs but in fact is better to be described as ribosome association. There can be many reasons for increased ribosome association including ribosome stalling and increased translational engagement. It would be good for the authors to add a simple Western blot to demonstrate directly increased protein output from psbA during high light as compared to low light treatments. This figure could be added to Figure S1.

    Strongly suggested additions to the manuscript to improve its significance before publication

    1)Identifying the RNA-binding protein(s) (likey HCF173 which may be in a complex with other proteins) that interacts with the 5' UTR of psbA in a highlight dependent manner would increase the significance of this study. Finding that this protein binds to other plastid transcripts with weak Shine-Delgarno sequences would also be a nice addition to this study.

    2)Mutational analysis of the RBP binding site and also to change the secondary structure around the start codon based on the new structure maps to show the effects of these various changes on protein output would really provide important new findings on how important the RBP being as compared to the RNA secondary structure changes are for regulating protein output form psbA. It could also allow the demonstration of the dependence or independence of these two features on regulating translation from chloroplast mRNAs.

    Significance

    This study definitely focuses on a research topic that is currently of interest and highly timely.

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    Referee #1

    Evidence, reproducibility and clarity

    This manuscript addresses the regulation of chloroplast translation, an important topic in chloroplast biology. The authors show that specific changes in the secondary structure of the 5'UTR of the psbA mRNA involving the Shine-Dalgarno sequence and the AUG initiation codon can be correlated with changes in translational efficiency during a low light to high light shift. Based on indirect evidence they propose that this may be caused by binding of specific proteins to this region. They also show that this correlation appears to be valid to some extent for other mRNAs with a weak SD sequence. The technical quality of this manuscript is excellent and the manuscript is clearly written.

    Additional remarks

    In the Introduction the authors need to cite earlier work in Chlamydomonas which first showed that binding of specific proteins to the psbA 5'UTR is correlated with increased translation in the light (Danon et al. 1991). The paper could be improved by testing for protein binding to the footprint region in high vs low light. An obvious candidate is HCF173.

    Significance

    This work provides valuable new insights into the molecular mechanisms involving the psbA 5'UTR in the initiation of chloroplast translation.

    This work will be of interest to a wide audience interested in the mechanisms of translational regulation.

    My expertise is in chloroplast biogenesis and in assembly and regulation of the photosynthetic apparatus

  6. Version published to 10.1101/2020.06.11.145870v1 on bioRxiv