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Multi-color single molecule imaging uncovers extensive heterogeneity in mRNA decoding

Sanne Boersma, Deepak Khuperkar, Bram M.P. Verhagen, Stijn Sonneveld, Jonathan B. Grimm, Luke D. Lavis, Marvin E. Tanenbaum

Preprint posted on December 13, 2018 https://www.biorxiv.org/content/early/2018/11/24/477661

and

Live-cell single RNA imaging reveals bursts of translational frameshifting

Kenneth R Lyon Jr, Luis U Aguilera, Tatsuya Morisaki, Brian Munsky, Timothy J Stasevich

Preprint posted on November 24, 2018 https://www.biorxiv.org/content/early/2018/11/24/478040

The secret shifty life of mRNA – evidence for non-stochastic ribosomal frameshifts and out-of-frame translation in cells.

Selected by Nicola Stevenson

Background

During protein synthesis, mRNA codon sequences, consisting of three base pairs, are read by the ribosomes and translated into the appropriate amino acid sequence. In classical models of translation, ribosomes bind a ‘start codon’ with sequence AUG and then the next triplet of base pairs denotes the next amino acid and so on. This is the open reading frame (ORF). However, in reality the situation is far more complicated and mRNA molecules are a lot more versatile than originally thought. For example, mRNAs may contain multiple start sites or leaky stop codons to produce different length proteins. As codons are composed of three base pairs, it is also possible for ribosomes to read the mRNA in three different reading frames, taking a different base within one codon as position one. This results in three different proteins. To use words as an example ….APEGODEN… could be read as APE-GOD or PEG-ODE or EGO-DEN depending on where you start, completely changing the meaning each time. This frameshifting strategy has been used by many viruses to expand their protein repertoire without increasing the mass of their genetic material.

To date, whilst frameshifting has been observed in vitro, it has not been observed in living cells due to technical limitations. This is necessary to ascertain to what extent it actually occurs in vivo and under what parameters. Now two studies by Lyon et al and Boersma et al, presented here, have managed to address this issue by developing mRNA transcripts encoding two different tags in different reading frames to permit live tracking of protein translation in each frame.

 

Key findings – Lyon et al

The multiframe tag produced by Lyon et al encodes FLAG epitopes in the 0 frame (detected by Fab) and SunTag epitopes in the -1 frame (labelled with scFv). When all ribosomes use the 0 frame, polysomes appear green, when all translate the -1 frame they are blue and if there is a mixture of reading frames the colours mix. Upstream of the multiframe tag they also insert the HIV-1 frameshift sequence (FSS) to see what affect this has on translation. In bulk assays this sequence has been shown to induce translation in the -1 frame 5-10% of the time but it is unknown how this is achieved. In the hands of Lyon et al the FSS sequence did indeed cause ~8% of individual translation sites to frameshift in cells but within these sites between 30-100% of ribosomes were frameshifted. This demonstrates that frameshifting happens on a small subset of RNA but with high probability. Further experiments revealed that it preferentially occurs in higher-order complexes or ‘translation factories’ and that the FSS sequence itself promotes the formation of these factories. It also appears that frameshifting occurs in bursts on a single mRNA molecule that can last for several rounds of translation. Ribosomes that frameshift are paused for longer at the frameshifting sequence, inducing ribosomal traffic jams that can maintain the production of frameshifted protein despite global inhibition of translation.

 

Key findings – Boersma et al

Boersma et al generate a similar mRNA reporter encoding SunTag and their newly developed MoonTag in different reading frames to monitor reading frame choice. To aid imaging, the authors also anchor the mRNA to the plasma membrane and the tags are downstream of a gene of interest. Translation from a transcript encoding both tags but with a start codon in frame with MoonTag was found to produce frequent, brief bursts of SunTag signal with an intensity consistent with the full transcript being translated in that frame. As there were no known frameshift sequences at this site the authors conclude this is due to an alternative start site being used at the 5’ end of the transcript. These experiments therefore provide a readout of alternative start site selection. Using this approach, the authors found that 66% of mRNA molecules produced translation products in both frames suggesting multiple translation start tags were used intermittently on most mRNAs. However, there was huge variability (0-100%) in the number of ribosomes producing the out of frame product from an mRNA molecule. The probability of using alternative start sites is therefore distinct among different mRNAs and the mRNAs produced from a single gene are heterogeneous, as was also found by Lyon et al. Out of frame start site selection also seemed to occur in bursts. The introduction of 5’ UTRs from the RPL12 gene, but not the HMBS gene, upstream of the MoonStart site increased out of frame translation demonstrating translation initiation is influenced by 5’ UTR sequences and that alternative start site selection may be widespread on endogenous mRNAs.

 

Conclusions

I chose to highlight these papers because of the remarkable finding that mRNA molecules created from the same gene are actually heterogeneous in their translation potential inside cells. Whether through frameshifting or the use of alternative start sites, it is clear that only a subset of mRNA molecules will produce out of frame translation products with any regularity and even then, this occurs in bursts of activity. This implies a remarkable level of selection and temporal regulation which has not been observed before and dramatically increases the potential of the genome for diversifying proteins. Although Lyon et al and Boersma et al interpret their findings as frameshifting and alternative start site selection respectively, the burst-type kinetics and frequencies are very similar which is interesting. Of course the development of a technique to watch translational frameshifts live at the single molecule level also makes this worth highlighting as this has the potential to accelerate progress in the field.

The most obvious future question for me now is whether mRNAs with high levels of translational heterogeneity are modified in some way or bound to different proteins to promote this, for example by driving their incorporation into translation factories. The mechanisms of temporal regulation will also be interesting to investigate.

Tags: frameshift, moontag, mrna, reading frame, start site, suntag, translation

Posted on: 18th December 2018

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