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Optoribogenetic control of regulatory RNA molecules

Sebastian Pilsl, Charles Morgan, Moujab Choukeife, Andreas Möglich, Günter Mayer

Preprint posted on 8 July 2020 https://www.biorxiv.org/content/10.1101/2020.07.07.191379v1

Article now published in Nature Communications at http://dx.doi.org/10.1038/s41467-020-18673-5

Lights on, gene on! Optogenetic control of short regulatory RNAs.

Selected by Angika Basant

Background:

Optogenetics in cell and developmental biology has been applied to regulate gene expression at the transcriptional level and to post-translationally control protein activity, stability and localisation. Readily adaptable, optogenetic control of genes at the level of mRNA transcripts has thus far been challenging. Such a technique would allow spatiotemporal and reversible modulation of gene knock-down, with potentially benign dosage of light as the only external input. In addition to “on-demand” gene silencing, this method would also permit detailed, mechanistic investigation of transcript regulation as a whole.

Many small regulatory RNA molecules exercise control of mRNA stability and translation. microRNAs (miRs) and short interfering RNA (siRNA) are found endogenously while short hairpin RNA (shRNA) are artificial RNA molecules that can also silence genes via RNA interference.

This study uses an RNA aptamer i.e. a nucleotide sequence with a unique 3D structure, that is bound by a bacterial photoreceptor PAL in the presence of blue light (1). This aptamer can, in principle, be incorporated into the sequence of any shRNA or miR that targets a gene of interest. PAL binding is anticipated to block gene silencing by the linked short regulatory RNA.

Scheme 1 from Pilsl et al., 2020 made available under a CC-BY-NC-ND license.

Key findings:

The authors incorporate the native targeting sequence of a well-characterised microRNA miR-21 into the coding sequence of luciferase or eGFP reporters. In HEK293 cells stably expressing mCherry-PAL, they co-express these targetable reporters along with a plasmid expressing the miR-21-aptamer sequence. In the dark, reporter gene expression was not detectable. In the presence of blue light however, miR-21 activity was blocked and luciferase or eGFP expression could be seen.

These findings were extended to an shRNA-aptamer targeting an eGFP reporter. Here they also optimise the hinge region present between the aptamer hairpin and dsRNA targeting sequence to find marked differences based on the nucleotides chosen. For example, adenine and uridine upstream of the aptamer domain were more effective in blocking shRNA activity. The authors however warn that these observations may vary with specific shRNAs.

Finally, by constructing an shRNA-aptamer fusion molecule against cyclin B1 and CDK-1 they were able to show light-dependent changes in cell-cycle arrest, suggesting that their tool is compatible with biologically relevant functional assays.

What I like about this preprint:

This preprint describes a creative application of optogenetics to gene silencing that promises to be modular and widely applicable. I also enjoyed reading the short, clearly written text accompanying this well-designed study.

Questions for the authors:

  1. This is a small technical point. I notice that the mCherry-PAL intensity in cells seems to reduce in the presence of light. Is this due to photobleaching alone? Perhaps a western blot over the course of the experiment could clarify that bulk PAL levels remain constant throughout.
  2. Could you observe whether cyclin B1 and Cdk1 protein levels can be reversibly restored over multiple light-dark cycles? If so, at what timescales could functional recovery be detected?
  3. I am curious why you use the term “near-arbitrary” when referring to the application of your tool. What restrictions do you anticipate would prevent this from being used to target some mRNA?

References:

  1. Weber et al., Nature Chem Bio 2019

 

Posted on: 5 August 2020

doi: https://doi.org/10.1242/prelights.23735

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Author's response

Günter Mayer shared

Thank you for your interest in our work and for preparing the nice post on our study.

We are happy to provide further information and answers to the questions you have.

1. We cannot explain this observation on a molecular level yet, but believe that it correlates with the fusing mCherry to PAL.

2. We did not test the reversibility of cell cycle control using the shRNA constructs targeting cyclin B1 and CDK1. In principle we demonstrate reversibility using luciferase as reporter gene and, thus, do believe that this feature might also apply to other shRNA-PAL aptamer constructs.

3. We believe that our tool will be useful to address many mRNA molecules of interest. By using the term “near-arbitary”, we take into account that some siRNAs may not be efficiently adaptable to our system or our approach might also be limited by the common restrictions of siRNA technology, e.g., accessibility of target sites.

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