Processing bodies (P-bodies, PBs) are aggregates of mRNA degradation proteins, suggested 20 years ago to be sites of mRNA degradation. Later work provided evidence to the contrary, and the function of PBs remained controversial. A new pre-print from Bin Wu’s lab brings us back to this debate, with new evidence for a role in degradation. Bonus – how phototoxicity affects the outcome. (Note – updated with published paper).
PBs were identified as sites of mRNA decay in yeast by Roy Parker in 2003. He found that PBs are aggregates of mRNA decay factors (or, what today we call membraneless organelles formed by liquid-liquid phase separation) and used MS2-tagged mRNA and to show it also localizes to PBs and that mRNA fragments accumulate in PBs.
However, later work from Parker lab and others, showed that mRNA decay (of a few select mRNAs) is not affected in mutants that do not form PBs. Furthermore, shuttling of mRNAs from PBs to polysomes suggested a role in storage, not decay. Then, it turned out that the MS2 system in yeast, particularly in over-expressed genes, can create mRNA fragments that accumulate in aggregates. These can be mis-interpreted when studying mRNA localization – particularly to PBs.
In mammalian cells, Yaron Shav-Tal suggested a dual role for PBs, whereas single-molecule imaging from Jeff Chao using his TREAT construct, provided strong evidence against degradation in PBs.
When measuring mRNA decay by bulk analysis (by biochemical methods such as Northern blot or RNA-seq) we lose the spatial information. We gain spatial information when using single molecule imaging, but then we rely on a stochastic event – i.e. we cannot predict which mRNA will be degraded, and typically only a few are degraded within the timeframe of the experiment. So it is technically challenging to track an mRNA to the point it is being degraded. This is particularly true to freely diffusing mRNAs. It is easier when mRNAs are tethered (e.g. Marvin Tanenbaum’s interesting work on NMD), but this is an artificial system. In addition, we measure disappearance of the signal – which can be for reasons other than decay.
So, PhD student Lauren Blake from Bin Wu’s lab at Johns Hopkins University developed RIDR (Rapid Inducible Decay of RNA) – a clever way to induce rapid degradation of a specific mRNA species. The system is simple enough – fuse an mRNA decay factor and mRNA binding protein to two proteins that can be brought together by chemically inducible dimerization. Here, she fused the mRNA decay protein (related to nonsense-mediated decay, NMD) SMG7 (C-terminus region) to FKBP−Rapamycin Binding domain (FRB) and MS2 coat protein (MCP) to FKBP. Dimerization is induced by addition of rapamycin at low concentrations. The mRNA is tagged with multiple MS2 stem loops (Figure 1, left). She first characterized and optimized the system using an ectopically expressed gene (mCherry-MS2). Using this system, in combination with RNA polymerase II inhibitor (DRB, to prevent addition of nascent mRNAs) she can see robust mRNA degradation within two hours (compared to six hours with siRNA, and ~30% with DRB alone after 9 hours). A control mRNA which is untagged is not affected.
Figure 1: On the left – scheme of the RIDR system. On the right – FISH-IF image of a MEF-MBS cell, 30min Rapa+DRB. mPolR2A FISH spots in blue randomely disperese, ACTB-MBS granules in magenta are co-localized with DCP1a (PBs) in green. (From Blake et al 2023 bioRxiv).
The more interesting biology appeared when they looked at an endogenous mRNA – β-actin mRNA in mouse embryonic fibroblasts derived from the MBS mouse: where both alleles in all cells have 24xMS2 stem loops. Although the expression level of β-actin is 25-fold higher compared to their mCherry reporter, they could still see 95% degradation within two hours. Amazing!
They also saw that within minutes, many of the mRNAs accumulate in PBs (Figure 1, right) – which become larger but fewer (probably fusion events) over time. The mRNA is then depleted from both PBs and cytoplasm. What is the role of the PBs? Is the mRNA degraded in them or stored, then degraded outside? Is there advantage to the high concentration of decay factors in PBs?
They developed a mathematical model to simulate the decay kinetics with various parameters. The model that fit best their data indeed suggest that decay occurs both inside and outside PBs, but the decay inside PBs is faster. Knocking down XRN1 – the 5’-3’ exonuclease, showed accumulation of the induced MS2-tagged mRNA, but not control mRNA, in PBs, suggesting degradation occurs inside PBs. Another cool result – when they knocked down DDX6, essential for PBs formation, the mRNA did not accumulate in granules and decay slower than the control.
All the work so far was done mostly on fixed cells at various time points. What happens when we go to live imaging? Surprisingly, in live imaging the mRNA granules persisted in PBs and did not disappear up to two hours. What’s going on?
The answer was phototoxicity. When they reduced the laser power to the bare minimum that allowed single molecule tracking, the mRNA disappeared from PBs, as seen by the fixed cells experiments. They suspected the phototoxicity induced an oxidative stress, so they induced an exogenous oxidative stress using low laser power – and again found accumulation in PBs without degradation.
This phototoxic effect may explain why other labs, which did single molecule tracking, did not detect mRNA decay in PBs – and it is an important lesson to remember when doing imaging. The excitation light is not inert.
I think this is a good paper, but is missing some controls. More important: it leaves the reader with open questions and a taste for more.
Controls:
- They show just two control mRNAs, none of them an NMD target. I think providing a couple more examples (maybe also γ-actin mRNA) would strengthen the claim for specificity and alleviate any concern of toxicity of SMG7C or Rapa.
- They show stress granules (SG) when oxidative stress is chemically induced, but what about the phototoxicity? Are they also formed?
- Also, why doesn’t β-actin mRNA accumulate in SG during the stress? I would expect it to go there (when SMG7C is not taking it to PBs).
- There are multiple papers looking at SG mRNA transport. Maybe look at one or two as controls. In yeast, it is known that deleting XRN1 causes the formation of multiple PBs. The number/size of PBs in the XRN1 KD is not calculated which is too bad.
Biological questions:
- Feedback to transcription: I showed, years ago, that (in yeast) mRNA decay is connected to transcription. Others later showed a similar connection in other systems (termed mRNA buffering by some). I think this system is excellent to look at any feedback between cytoplasmic decay to transcription, and it will be fairly easy to do: just don’t add DRB. You can then measure the transcription sites during induced degradation. If there is buffering, then once degradation is induced, there should be a burst of transcription. Looking at their various KD systems (XRN1, DDX6 etc…) will provide info on PBs vs cyto decay and transcription. Does decay of β-actin mRNA also affect the half-life/transcription of other actin genes or actin regulator genes? Should be very cool.
- Role of PBs in vivo still unclear. The DDX6 KD experiment just opened a whole lot of questions: If the decay rate of untagged control mRNAs (e.g. GAPDH) is not affected by DDX6 KD (i.e. no PBs) – then what IS the role of PBs, really? Why would they exist at steady state if the bulk of mRNAs still decays at the cytoplasm – and only if you force the mRNA to PBs it degrades there faster. Assuming at steady-state there is a small % of mRNAs in PBs (of each transcript, of some genes) – then how is it decided which specific molecules go to PBs and degrade (or not) there?
- What is the biological effect of RIDR-ing a specific transcript? I mean – within two hours the cell does not have β-actin mRNAs. Does this affect the actin filaments? Which β-actin mRNA molecules go first to PBs – is it just distance from PB or also function (e.g. cytoplasmic mRNA vs ER-bound or focal adhesion-bound)? What would happen in ZBP1K/O cells?
I can think of other uses for this system, but I’ll stop here.
Update: none of my comments/questions were addressed in the print version. I hope some of it will be answered in later papers.
Blake L.A, Lia Y, Inoue T and Wu B (2023) A Rapid Inducible RNA Decay system reveals fast mRNA decay in P-bodies bioRxiv 2023.04.26.538452
The print version: Blake, L.A., Watkins, L., Liu, Y. Inoue T and Wu B. (2024) A rapid inducible RNA decay system reveals fast mRNA decay in P-bodies. Nat Commun 15, 2720 .
Recommended review:
Dave & Chao (2020) Insights into mRNA degradation from single-molecule imaging in living cells Curr. Opin. Struct. Biol. 65:89-95
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