Menu

Close

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 November 24, 2018 https://www.biorxiv.org/content/early/2018/11/24/477661

When ribosomes take an alternative path: single-molecule sensor of translation reveals extensive heterogeneity in mRNA decoding

Selected by Lorenzo Lafranchi

Background

Translation, the process by which a ribosome reads an mRNA molecule, is a crucial and tightly regulated step in gene expression. The canonical view of translation is that a ribosome scans the mRNA from its 5’ end, beginning protein synthesis at the most upstream start codon, and continues translation until encountering an in-frame stop codon. The region enclosed between in-frame start and stop codons is referred to as open reading frame (ORF). This simplistic view of translation has been recently challenged by the development of ribosome profiling, which highlighted the heterogeneity existing in mRNA decoding. In fact, mRNA molecules can contain multiple ORFs and different sections of an mRNA can be translated. Altogether, non-canonical translation has been widely observed but the extent and underlying causes of heterogeneity in mRNA translation remain largely unexplored.

 

Key findings

Boersma and colleagues develop a fluorescence reporter, called MoonTag, for labeling nascent polypeptides. Shortly, MoonTag consists of a genetically-encoded antibody-epitope pair. A short peptide is fused as an array to a sequence of interest and upon translation is recognized and bound by the fluorescently-labeled nanobody. Live-cell imaging then enables the analysis of translation kinetics. With the MoonTag being orthogonal to the previously-published SunTag system, the authors are now able to combine the two reporters for studying translational heterogeneity at a single-molecule level.

After testing the MoonTag and proving its orthogonality to the SunTag, the authors combine the two reporters in an alternating fashion and in different reading frames into the MashTag. Since the MashTag is provided with a single start codon, in-frame with either the Sun- or the MoonTag, canonical translation generates only one of the two fluorescent signals. The observed colocalization of SunTag and MoonTag signals therefore represents ribosomes that are translating the reporter out-of-frame (OOF).To further understand OOF translation, the authors compute the theoretical trace expected from a single ribosome translating the entire array of peptides. Comparison of the experimental profiles of single OOF translation events to the theoretical trace revealed that OOF translation is mainly due to alternative start site selection near the 5’ end of the mRNA. Next, the theoretical trace was used to extract information from the measured fluorescence intensity traces about the frequency and timing of translation initiation at both canonical and alternative start sites. This analysis showed that both canonical and OOF translation occur intermittently on the majority of mRNAs. On the individual mRNA level, start site selection seems to be largely stochastic. Surprisingly, the probability of using alternative translation start sites differs between mRNAs, with OOF translation ranging from 0% to 100% of the ribosomes. These findings highlight how different mRNAs, despite carrying the same ORFs, can be heterogeneously read by the ribosomes. Further experiments show that near-cognate translation start sites both upstream or downstream the canonical start codon can be responsible for initiating OOF translation.By fusing the 5’UTR of two genes to the MashTag, the authors demonstrate that OOF translation is not restricted to exogenous sequences and suggest that alternative start site selection might be, to different extents, a widespread phenomenon on endogenous mRNAs.

Finally, the authors design a sensor for visualizing the translational paths occurring on an mRNA containing an upstream ORF (uORF). uORFs are present in thousands of mRNAs and generally repress translation of the main ORF. Different to the previously-described reporters, the uORF sensor contains two out-of-frame canonical start codons. As expected, presence of the uORF reduces the translation rate of the main ORF, but translation of both ORFs occurs for most mRNA molecules. Translation of the main ORF can both be achieved by leaky scanning of the first start codon or translation re-initiation after translation of the uORF. Interestingly, ribosomes following different paths along the mRNA co-exist on most mRNAs.In addition, the authors observed a strong temporal correlation between translation of the two ORFs that is most likely caused by a burst-like behavior of translation initiation. Despite the majority of mRNAs showing a positive correlation between translational level of the two sensors, a fraction of mRNA molecules experiences temporal bursts in choosing the different translation paths. Bursts in translation start site selectionoccur in a mRNA-specific fashion indicating that this phenomenonis not regulated in a cell-wide manner, but at the level of individual mRNA molecules.

 

What I like about this work

Ribosome profiling and proteogenomics studies suggested that larger portions of the genome are translated in comparison to what was expected based on the textbook view of translation. These methods highlighted an unforeseen proteome diversity, possibly due to the flexibility of mRNA-translating ribosomes. Despite these findings, translational heterogeneity has never been directly visualized. Boersma and colleagues developed an elegant strategy to dissect the complexity of translational dynamics at a single-molecule level. With these new tools in hand they answer different open questions on how ribosomes initiate translation and they show that alternative translation is a surprisingly common event.

 

Future directions

Although the system presented in the paper seems difficult to multiplex, it would be interesting to fuse the MashTag to several endogenous 5’ UTRs; similar to what the authors already did in the paper, but with larger numbers. Collecting more information would help in defining sequences prone to OOF translation.

“What is the role and importance of OOF translation for cellular physiology?” is an important question arising from the study. OOF translation could be important for controlling translation of the main ORF, generate functional alternative proteins, or simply represent errors in translational initiation. Since translation is energetically costly for a cell, the first two explanations seem to be the most likely. Nevertheless, this open question is worth to be addressed experimentally.

 

Questions to the authors

How stable are the products of the MashTag? Is there a difference between “canonical” product and OOF-translated products?

Would it be possible to perform ribosome profiling after harringtonine treatment to identify the alternative initiation sites?

Can the overall level of OOF translation in a cell be measured using the MashTag reporter? Can this system be implemented in a genetic or chemical screen to identify factors controlling OOF translation?

 

Posted on: 18th December 2018

Read preprint (No Ratings Yet)




  • Have your say

    Your email address will not be published. Required fields are marked *

    This site uses Akismet to reduce spam. Learn how your comment data is processed.

    Sign up to customise the site to your preferences and to receive alerts

    Register here

    Also in the cell biology category:

    Blue light induces neuronal-activity-regulated gene expression in the absence of optogenetic proteins

    Kelsey M. Tyssowski, Jesse M. Gray



    Selected by Zheng-Shan Chong

    Mutations in the Insulator Protein Suppressor of Hairy Wing Induce Genome Instability

    Shih-Jui Hsu, Emily C. Stow, James R. Simmons, et al.



    Selected by Maiko Kitaoka

    1

    Multi-immersion open-top light-sheet microscope for high-throughput imaging of cleared tissues

    Adam K. Glaser, Nicholas P. Reder, Ye Chen, et al.



    Selected by Tim Fessenden

    1

    ATAT1-enriched vesicles promote microtubule acetylation via axonal transport

    Aviel Even, Giovanni Morelli, Chiara Scaramuzzino, et al.



    Selected by Stephen Royle

    1

    HIV-1 Gag specifically restricts PI(4,5)P2 and cholesterol mobility in living cells creating a nanodomain platform for virus assembly

    C. Favard, J. Chojnacki, P. Merida, et al.



    Selected by Amberley Stephens

    Hepatocyte-specific deletion of Pparα promotes NASH in the context of obesity

    Marion Regnier, Arnaud Polizzi, Sarra Smati, et al.



    Selected by Pablo Ranea Robles

    Mitochondrial biogenesis is transcriptionally repressed in lysosomal lipid storage diseases

    King Faisal Yambire, Lorena Fernandez-Mosquera, Robert Steinfeld, et al.



    Selected by Sandra Franco Iborra

    1

    Thyroid hormone regulates distinct paths to maturation in pigment cell lineages

    Lauren Saunders, Abhishek Mishra, Andrew J Aman, et al.



    Selected by Hannah Brunsdon

    1

    Kinesin-6 Klp9 plays motor-dependent and -independent roles in collaboration with Kinesin-5 Cut7 and the microtubule crosslinker Ase1 in fission yeast

    Masashi Yukawa, Masaki Okazaki, Yasuhiro Teratani, et al.



    Selected by I. Bouhlel

    A pair of E3 ubiquitin ligases compete to regulate filopodial dynamics and axon guidance

    Nicholas P Boyer, Laura E McCormick, Fabio L Urbina, et al.



    Selected by Angika Basant

    1

    SorCS1-mediated Sorting of Neurexin in Dendrites Maintains Presynaptic Function

    Luis Filipe Ribeiro, Ben Verpoort, Julie Nys, et al.



    Selected by Carmen Adriaens

    1

    ENDOSOMAL MEMBRANE TENSION CONTROLS ESCRT-III-DEPENDENT INTRA-LUMENAL VESICLE FORMATION

    Vincent Mercier, Jorge Larios, Guillaume Molinard, et al.



    Selected by Nicola Stevenson

    1

    Microtubules stabilize intercellular contractile force transmission during tissue folding



    Selected by Ivana Viktorinová

    Synthetic pluripotent bacterial stem cells

    Sara Molinari, David L. Shis, James Chappell, et al.



    Selected by Lorenzo Lafranchi

    A DNA-based voltmeter for organelles

    Anand Saminathan, John Devany, Kavya S Pillai, et al.



    Selected by Robert Mahen

    1

    Central spindle microtubules are strongly coupled to chromosomes during both anaphase A and anaphase B

    Che-Hang Yu, Stefanie Redemann, Hai-Yin Wu, et al.



    Selected by Federico Pelisch

    1

    Also in the molecular biology category:

    Blue light induces neuronal-activity-regulated gene expression in the absence of optogenetic proteins

    Kelsey M. Tyssowski, Jesse M. Gray



    Selected by Zheng-Shan Chong

    Slide-seq: A Scalable Technology for Measuring Genome-Wide Expression at High Spatial Resolution

    Samuel G Rodriques, Robert R Stickels, Aleksandrina Goeva, et al.

    AND

    High-density spatial transcriptomics arrays for in situ tissue profiling

    Sanja Vickovic, Goekcen Eraslan, Johanna Klughammer, et al.



    Selected by Carmen Adriaens

    Optical determination of absolute membrane potential

    Julia R. Lazzari-Dean, Anneliese M.M. Gest, Evan Miller



    Selected by James Marchant

    MicroRNA-mediated control of developmental lymphangiogenesis

    Hyun Min Jung, Ciara Hu, Alexandra M Fister, et al.



    Selected by Rudra Nayan Das

    Microfluidic protein isolation and sample preparation for high resolution cryo-EM

    Claudio Schmidli, Stefan Albiez, Luca Rima, et al.



    Selected by David Wright

    A DNA-based voltmeter for organelles

    Anand Saminathan, John Devany, Kavya S Pillai, et al.



    Selected by Robert Mahen

    1

    Structures of the Otopetrin Proton Channels Otop1 and Otop3

    Kei Saotome, Bochuan Teng, Che Chun (Alex) Tsui, et al.



    Selected by David Wright

    Cell growth dilutes the cell cycle inhibitor Rb to trigger cell division

    Evgeny Zatulovskiy, Daniel F. Berenson, Benjamin R. Topacio, et al.



    Selected by Zaki Ahmad

    1

    Distinct ROPGEFs successively drive polarization and outgrowth of root hairs

    Philipp Denninger, Anna Reichelt, Vanessa Aphaia Fiona Schmidt, et al.



    Selected by Marc Somssich

    Inactive USP14 and inactive UCHL5 cause accumulation of distinct ubiquitinated proteins in mammalian cells

    Jayashree Chadchankar, Victoria Korboukh, Peter Doig, et al.



    Selected by Mila Basic

    Bacteriophage resistance alters antibiotic mediated intestinal expansion of enterococci

    Anushila Chatterjee, Cydney N Johnson, Phat Luong, et al.



    Selected by Yasmin Lau

    On-site ribosome remodeling by locally synthesized ribosomal proteins in axons

    Toshiaki Shigeoka, Max Koppers, Hovy Ho-Wai Wong, et al.



    Selected by Srivats Venkataramanan

    MRE11-RAD50-NBS1 activates Fanconi Anemia R-loop suppression at transcription-replication conflicts

    Emily Yun-Chia Chang, James P Wells, Shu-Huei Tsai, et al.



    Selected by Katie Weiner

    1

    Super-resolution Molecular Map of Basal Foot Reveals Novel Cilium in Airway Multiciliated Cells

    Quynh Nguyen, Zhen Liu, Rashmi Nanjundappa, et al.



    Selected by Robert Mahen

    Atlas of Subcellular RNA Localization Revealed by APEX-seq

    Furqan M Fazal, Shuo Han, Pornchai Kaewsapsak, et al.

    AND

    Proximity RNA labeling by APEX-Seq Reveals the Organization of Translation Initiation Complexes and Repressive RNA Granules

    Alejandro Padron, Shintaro Iwasaki, Nicholas Ingolia



    Selected by Christian Bates

    Unlimited genetic switches for cell-type specific manipulation

    Jorge Garcia-Marques, Ching-Po Yang, Isabel Espinosa-Medina, et al.



    Selected by Rafael Almeida

    1

    Close