Three recent papers begin to explore the dynamics of miRNA translation repression and mRNA decay at the single-molecule resolution. One paper contradicts the others.
Our understanding of the regulation on mRNA molecules has advanced a lot thanks to single-molecule imaging. Single-molecule FISH (smFISH) and live imaging showed us new things about transcription, nuclear export, transport, sub-cellular localization, intercellular transfer, translation and degradation of mRNAs at the single cell and single molecule resolution. Yet one of the main regulatory elements on mRNA biology remained un-explored by these methods – micro RNAs (miRNAs).
miRNAs are 22 nucleotides-long small non-coding RNAs. They are transcribed as longer RNAs and processed by a series of steps that I won’t go into. The mature miRNA associates with Argonaute proteins, as part of a protein complex that binds to mRNAs. This binding – mediated by RNA-RNA hybridization of the miRNA to “miRNA binding site” on the mRNA – leads to translation silencing and mRNA degradation. The mechanism is fairly understood at the global level: i.e. when looking at whole cell populations, or whole mRNA population of single cells by biochemical methods. But what happens spatially and temporally at the single-molecule level is not known.
Three recent preprints, from the labs of Rob Singer , Tim Stasevich and Jeff Chao, provide the first look at how mRNAs are affected by miRNAs at the single molecule level.
Three approaches to the same problem:
The first preprint, led by postdoc Hotaka Kobayashi (update: published in Nat comm), explored how miRNAs affect translation and degradation by using smFISH. He constructed a reporter system with a bi-directional promoter: one side transcribes a luciferase gene as control and the other side the Suntag protein, which is used to visualize translation: it is composed of 24 GCN4 peptides in tandem that can be identified by a specific antibody co-translationally. The 3’UTR contains 8 sites for miR-21 binding. Hotaka then used smFISH combined with immunofluorescence (FISH-IF) to visualize single mRNAs along with Suntag translation and Argonaute binding to the mRNA. Since he initially saw a major reduction in mRNA numbers – presumably by miRNA-mediated degradation, he prepared a new construct (Fig 1A) with an anti-decay sequence: A114-N40 instead of a poly-A cleavage site. This is a clever way to protect the mRNA from deadenylation-mediated decay, but may affect the translation-decay communication.
The second preprint, led by grad student Charlotte Cialek, used a different approach (update June 2022: published at Nat comm). She designed a construct to express an mRNA with three parts: the ORF includes 10 consecutive FLAG tags which are recognized by a Cy3-tagged Fab; the 3’UTR contains 24xMS2 stem loops to visualize the mRNA; and 15xBoxB stem loops that are used to tether a protein of interest (hence the name they gave the system: translation & tethering or TnT) (Fig 1B). In this paper, she tethered GFP-Argonaute protein, but in principle it could be anything of interest. Unlike the first paper, this biosensor was used for live imaging and thus enables tracking of single mRNAs for up to 12 hours(!). Later, they also made a construct with 3x miRNA binding sites for miR-26-5p instead of Ago tethering.
The third preprint, led by postdoc Pratik Dave, took a different approach. First, they inserted an iron response element (IRE) in the 5’UTR, upstream to the coding sequence of Suntag-luciferse, and put MS2 in the 3’UTR. There is little translation in the absence of iron, and when iron is added, translation is induced. They then added the TREAT element in the 3’UTR upstream to MS2, which allows imaging of mRNA decay – the element stops the exonuclease Xrn1, so the MS2 fragment remains and can be measured compared to the upstream part. Here was a very interesting result – inducing translation also induced faster mRNA decay. This is very interesting. We know that translation is linked to decay, but this is a strong evidence for a causal link at the single molecule level. Very cool result. Future research should look at how is it that mRNAs that are highly translated (e.g. actin, GAPDH) are also very stable mRNAs. Finally, they added 3x Let7 miRNA sites in the 3’UTR (Fig 2) to measure the effect of the miRNA on translation and decay.
miRNA effect on translation & decay
The papers by Singer & Stasevich showed that miRNA binding or Ago tethering leads to a reduced percentage of translated mRNAs as well as a reduced number of ribosomes per mRNA. Furthermore, both showed a negative correlation between Ago binding (either tethered or endogenous, visualized by FISH-IF) and mRNA translation status. Singer also showed that the Ago-containing complex binds preferentially to translating mRNAs and that there is a timeline by which Ago associates with the mRNA already upon nuclear export. Translation repression occurs within 30 minutes but mRNA decay occurs about 1 hour after nuclear export.
Contrary to these two papers, Chao’s paper showed that the miRNA had no effect on translation – not on the fraction of translating mRNAs, nor on the number of ribosomes per mRNA.
How can we explain these contradictory results?
Well, first of all, Singer used an anti-decay tail that prevented mRNA decay in translation experiments, and did not measure translation when poly-A tail was present. Thus, we cannot really tell how translation and decay are manifesting in this system simultaneously. So we cannot compare it to Chao’s et al experiments.
Second, Stasevich’s paper does not include any data on mRNA decay, except for two rare cases in their live imaging analysis. In fact, they claim (and I hope I understand correctly) that Ago tethering does not induce mRNA decay at least for several hours, and that sometimes there is re-initiation of translation. So again, it is difficult to compare the claims on mRNA decay.
Each paper used a different miRNA binding site. Singer used 8 copies, while Stasevich & Chao – 3 copies. The distance of the miRNA binding sites from the stop codon or from the poly-A tail might also play a role, I guess.
The experiments with Ago tethering (15 copies!) show accumulation of the mRNA in P-body like structures. P bodies are known to repress translation but (based on Chao’s & Singer’s earlier work) do not induce degradation. However, since Ago is artificially tethered to the mRNA, this could be due to a LLPS created from the high concentration of Ago on a single mRNA. I think a missing piece of information here is whether the construct with miRNA binding sites – instead of Ago tethering – also accumulates in P-bodies or not. If not, then maybe reducing the number of tethered Ago will also eliminate the P-body localization. Another idea to test: What happens if Ago2 & β-gal compete on the boxB in the same cell, or if they use an inducible degron to get rid of Ago? Would the mRNA be released from the P-bodies and resume translation?
Back to our problem: Is let-7, the miRNA used by Chao’s group even related to translation? Apparently yes. It was shown >15 years ago to repress translation in human cells, it was shown to repress translation in a cell-free system, and this review suggests that translation regulation is a major function of miRNAs, even a pre-requisite for mRNA decay. So why doesn’t it work? My guess is that the IRE somehow bypasses the effect of miRNA on translation.
I think it will be very interesting if Chao will compare to an IRE-less construct, or if Singer/Stasevich will introduce an IRE to their system.
Overall, these are three very interesting papers, with lots of data in them (I only discussed some of it here) and amazing technological achievements. Go read these papers.
Update 16 June 2022: In the journal version of their paper, Stasevich et al added experiments with Ago2 mutants that suggest a direct role of Ago2 in P-body accumulation and translation inhibition.
Imaging translational control by Argonaute with single-molecule resolution in live cells. Charlotte A. Cialek, Tatsuya Morisaki, Ning Zhao, Taiowa A. Montgomery, Timothy J. Stasevich . Nat comm 13:3345 2022
Single-molecule imaging of microRNA-mediated gene silencing in cells. Hotaka Kobayashi, Robert H. Singer. Nat comm 13:1435 2022
Single-molecule imaging reveals the coupling of translation and mRNA decay. Pratik Dave, Esther Griesbach, Gregory Roth, Daniel Mateju, Jeffrey A. Chao. bioRxiv 2021.06.07.447377v1
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