Long non-coding RNAs regulate the expression of cell surface receptors in plants

  1. Hemal Bhasin
  2. Hasna Khan
  3. Zachary Kileeg
  4. G. Adam Mott

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Abstract

Plants are exposed to a variety of growth, developmental, and environmental cues during their lifespan. To survive and thrive, plants have developed sophisticated ways of responding to these signals that involve regulation at the transcriptional, post-transcriptional, translational, and post-translational levels. Leucine-rich repeat receptor-like kinases are the largest family of receptor-like kinases in plants and respond to a range of external and internal stimuli. They act as crucial regulators of plant growth, development, and immunity. To fully understand LRR-RLK function, it is essential to understand how their expression is regulated under different conditions. While there have been numerous studies on post-translational regulation of LRR-RLKs through phosphorylation and ubiquitination, there is little known about the mechanisms of transcriptional and post-transcriptional regulation of LRR-RLKs. In this study, we show that natural antisense transcript long non-coding RNAs are central regulators of LRR-RLK expression at the transcriptional and post-transcriptional levels. LRR-RLK genes are almost universally associated with cis-NATs and we confirm cis-NAT expression in planta using strand-specific RT-PCR. We leverage several well-studied LRR-RLKs to demonstrate that cis-NATs regulate LRR-RLK expression and function. For cis-NATs to fine-tune LRR-RLK expression, their expression and regulatory activity must be tightly controlled and cell autonomous. Using a combination of GUS reporter assays and tissue-specific promoters, we provide evidence that cis-NATs have these characteristics, positioning them as key regulators of LRR-RLK function. We also demonstrate that the association of LRR-RLK genes with cis-NATs is conserved across much of plant evolution, suggesting that this previously unexplored regulatory mechanism serves an important and ancient purpose.

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    Referee #2

    Evidence, reproducibility and clarity

    In this ms, the authors searched using bioinformatic approaches for the presence of natural antisense RNAs (cis-NATs) linked to LRR-RLK genes, a large gene family containing many major regulators of plant growth, to detect a potential novel mechanism for their post-transcriptional regulation. They detected a large proportion of LRR-RLK genes containing cis-NATs. TO address their potential functions, they overexpressed specific cis-NATs against Bri1, CLV1 and SOBIR1 and observed phenotypes reminiscent of the respective mutant in these genes. This means that these cis-NATs can act in trans on the endogenous loci. The authors detected reduction in expression of the cognate LRR-RLK in several transgenic lines except for SOBIR1 where a minor change in protein production was found. Then, they prepare transgenic GUS lines with the promoters of the NATs and detect variable levels of expression. In addition, expressing the NATs under epidermal specific promoters but not in a control promoter induced differences in BRI1 expression specifically in the plants. Finally, they search for the presence of NATs linked to LRR-RLK loci in other plants.

    The paper is interesting but the message about NAT regulation is overstated and there are several conclusions that require major additional experiments

    1. There are no experiences with mutant NATs to confirm a potential linkage to the regulation of LRR-RLK complementary transcript. Overexpressing a NAT may lead to several artefacts, including the artificial silencing of the endogenous loci (expressing a complementary RNA). Hence, it cannot be concluded that this regulation occurs in planta only based on overexpressing lines. The use of rdr6 mutants or similar may also serve to discard potential alternative NAT regulations.
    2. Complementing a bri1 mutant (or in other RLK) with construct expressing the BRI1 locus with or without the NAT (e.g. with or without its promoter) is a much cleaner manner to show NAT regulation. CRISPR or other manipulation may allow to create mutants in the NAT alone to see the effects on its cis-target as well as its eventual trans action on other LRR-RLK (as apparently acts in trans).
    3. The trans-NAT experiments will minimally require genome-wide RNAseq studies to see the level of cross-talk of a trans NAT. Even though the phenotype is related, there may be very general misregulation triggered by the NAT. A minimum is to pass all studied RLK in all the lines to define certain "specificity" of action.
    4. To propose a translational regulation for SOBIR1 with its present data is an overstatement for me. Is the antisense nuclear or accumulate in ribosomes? Can the NAT lead to a change in the recruitment into ribosomes (without changing mRNA levels). There may be indirect effects that can explain these changes in protein accumulation
    5. The GUS plants need to be shown in detail, with a more precise tissular localisation and compared to the cognate LRR-RLK. Can BRI1 expression be monitored in TRANS-NAT BRI1 vs wt using in situ or BRI1-GFP fusions. TO show the expression patterns of the antisense and the target will further support the proposed specificity regulation. What happens with other LRR-RLKs? Kinetics of expression along development in trans NATs and wt will be a possibility
    6. The epidermal connection is interesting but there is any evidence about the "cell specificity" expression of BRI1 or the differential expression of BRI1 in cells containing or not the NATs?
    7. The extrapolation to crops might be interesting if some features are conserved (e.g. BRI1 homologs contain NATs of the same size or similar cell-specific regulation). As many genes have NATs, this evolutionary argument of the presence of NATs is not convincing to conclude about a regulatory mechanism permitting "to engineer improved crop performances".

    Significance

    The paper in its present form is very limited and the major strength is the occurrence of similar phenotypes when NATs are overexpressed and the mutant cognate mRNAs. Alternative explanations of the reported data are possible and should be discarded and better presented. This paper does not advance in NAT regulatory mechanisms to be relevant for a broad audience in life sciences. However, the subject of LRR-RLK regulation is of great interest to the plant community as several critical growth regulators act through LRR-RLKs.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    The authors investigate the role of antisense transcription (cis-NATs) at Leucine-rich repeat receptor-like kinases (LRR-RLK) genes in plants. They find that many LRR-RLK genes are associated with cis-NATs, through data-mining and RT-PCR in Arabidopsis. For functional studies, they selected three cis-NATs for over-expression, from the BRI1, CLV1 and SOBIR genes, where over-expression has phenotypic consequences for the plants. They use reporter gene assays to study cisNAT expression is regulated across development. The authors examine the relationship between cis-NATs and LRR-RLKs in tomato and rice, where they also detect a high fraction of LRR-RLKs associated with cis-NATs.

    Major comments:

    • Over-expression of cis-NATs is used to support a functional role. Here, the experimental design leaves open if the effect is explained by a trans-acting role on the corresponding sense RNA (as the authors interpret), or, if the over-expression constructs trigger co-suppression of the corresponding sense RNAs. The authors should distinguish these possibilities prior to publication in a journal. It makes a big difference. Co-suppression would be a very different mechanism that is independent of the RNA functions the authors propose. The authors need to rule it out.

    Minor comments:

    • The authors use older resources to examine antisense transcription. In some sense, this makes it even more impressive that so many LRR-RLK genes are associated with antisense transcription, but it would be nice to include more recent data support the conclusions. In particular Araport11 is not considered a high-quality annotation (PMID: 34266383). The manuscript could benefit from the integration of some more recent genome-annotations, or genome browser screenshots for some the genes (e.g. BRI1, CLV1 and SOBIR) that shows some of the more recent methods (reviewed in PMID: 36259932). A lot of these data are even accessible on the TAIR website.

    Significance

    General assessment:

    The authors observe cisNATs at LRR-RLK genes. These findings hint at a contribution of cisNATs to LRR-RLK regulation that has not received much attention yet. For functional characterization, the authors rely on over-expression of cis-NATs. Here, I am sceptical, this the results could also fit a model where cisNAT OE may trigger siRNA formation and silencing of the endogenous locus (TGS/co-suppression).

    Advance: The manuscript uncovers cisNATs as potential large-scale regulators of LRR-RLK genes.

    Audience: Plant scientists interested in gene expression and cellular roles of LRR-RLK genes.

    Reviewer expertise: Plant lncRNA, genome annotation, plant gene expression, epigenetics.

  4. Version published to 10.1101/2024.04.22.590565v1 on bioRxiv