Site-specific K63 ubiquitinomics reveals post-initiation regulation of ribosomes under oxidative stress

Context:

Cells rely on the plasticity of their gene expression programs to respond, adapt to, and maximize their survival under a variety of stresses. While adaptation involves both transcriptional and post-transcriptional changes, translation regulation is particularly crucial in situations that demand a rapid response [1]. Stress such as hypoxia, starvation, viral infections, and heat shock lead to global translation downregulation [2], typically by phosphorylation of initiation factor eIF2a [3]. Initiation is thought to be the limiting step in mRNA translation, and therefore the principal control point during stress. However, there have been several lines of evidence that translation elongation is also limited under conditions of stress [4, 5]. Studying regulation of elongation is challenging, since almost all stresses that down-regulate elongation also inhibit initiation upstream [3], and deconvoluting the myriad cellular effects of a single stressor is not always feasible.

Oxidative stress induces rapid cellular accumulation of ubiquitinated proteins. Ubiquitin itself has seven lysine (K) residues, each of which can participate in further ubiquitination, generating poly-ubiquitin chains of different linkage types, which confer different fates upon the poly-ubiquitinated protein. K48 linkages are most common, and direct proteins for proteasomal degradation. K63 linkages, on the other hand, are the second most abundant, and have a variety of non-degradation associated cellular functions. While the authors have previously shown that K63 ubiquitination is important for cellular resistance to the oxidative stress, the mechanism by which this resistance was conferred remained unclear [6]. One reason was that available methods to globally identify sites of ubiquitination destroy information about the type of linkage.

In this preprint, the authors developed a new method called “Ub-DiGGer” to accurately and specifically identify sites of K63 ubiquitination proteome-wide in an unbiased manner. This method allowed to identify several sites of K63 ubiquitination upon the ribosome and characterize the role of this modification in the translational response to stress. The authors combined this high-resolution mapping with orthogonal studies that explored the effect of K63-ubiquitin on the ribosome – and found evidence for a new elongation-based regulatory mechanism.

 

Tools and Key Findings:

Using an unbiased method that combines selective affinity enrichment, differential isotope labelling and mass spectrometry, Back et al. identified ~1,100 sites of increased K63 ubiquitination in response to stress, mapping to ~500 yeast proteins. Their approach successfully identified a number of the previously confirmed sites of K63 ubiquitination, as well as many novel sites. In particular, the head of the 40S ribosomal subunit (which harbors the decoding center of the ribosome as well as flanks both the mRNA entry and exit sites) is enriched in sites modified by K63 ubiquitin linkages under oxidative stress, indicating a role for this linkage in translation regulation.

 

Three observations within this preprint are particularly crucial to defining the role of K63 ubiquitination in translational regulation. Firstly, although translation initiation is inhibited under oxidative stress, abrogation of this inhibition is insufficient to restore the reduced translation output under oxidative stress back to normal levels. This result indicates that multiple stages of the translation cycle are repressed under stress. Secondly, abrogation of K63 ubiquitin conjugation in a mutant incapable of inhibiting initiation causes translation output to remain unaltered in response to stress, suggesting that K63 ubiquitination is involved in control of translation post-initiation, and likely targets the elongating ribosome. Finally, the authors show that K63 ubiquitination in response to stress occurs on fully assembled polysomes, further supporting that K63 ubiquitination affects translation post initiation.  Proteomic profiling of the ribosome indicates that the inability to form K63 ubiquitin linkages results in decreased association of elongation factors with the ribosome in response to stress, indicating an inhibited elongation cycle.

Taken together, the authors demonstrate that K63 ubiquitination is important for repression of translation elongation under oxidative stress, potentially by altering the recruitment of accessory translation factors to the ribosome.

 

Why I chose this preprint:

Primarily, this preprint pioneers a powerful new method to identify ubiquitin modified lysine residues as well as the type of ubiquitin linkage simultaneously proteome-wide in an unbiased and quantitative fashion. Given the diversity of ubiquitin linkages, this tool will be invaluable in understanding the biological roles of ubiquitination of various proteins in various contexts.

On a personal note, my own interests lie in gene regulation, particularly translation, under stress. Because most stresses result in robust repression of translation initiation, deconvolution of any mechanisms regulating the translation elongation cycle remains almost impossible (although observations suggest that such regulation does in fact exist). By leveraging a novel proteomic approach and the power of yeast genetics, the authors indicate (to the best of my knowledge) one of the very few bona fide post-initiation regulatory mechanisms of translation under stress, potentially via direct modification of the ribosome.

 

Open questions and future directions:

This question is perhaps obvious, but it is worthwhile to lay out. What is the mechanism? The authors have demonstrated an enrichment of K63 ubiquitination sites on the ribosome, as well a role for K63 in the regulation of translation. However, the direct link between the two has not been established. Which of the ~80 modification sites on the ribosome contribute to translation regulation under stress? Is the effect on translation an indirect downstream consequence of modification of a different protein? Although the latter seems unlikely to me, it has not been formally ruled out. Further, it is also possible that K63 ubiquitination affects both initiation and elongation in response to stress, and the former in a manner orthogonal to canonical eIF2a phosphorylation (which was tested by the authors). Of course, answering these questions is a non-trivial endeavor, and will likely involve systematic mutational analysis of the identified modification sites on the ribosome.

Additionally, a thorough analysis of ribosome occupancy on transcripts would perhaps be able to identify which step in the elongation cycle is affected by K63 ubiquitination, as would any potential structure of the “stressed” ribosome decorated with K63 ubiquitin.

Broadly, the techniques laid out in this article, as well as the datasets described, provide an exciting opportunity to further study the biological roles of this ubiquitous yet incompletely understood protein modification. Additionally, a dozen years after the first evidence for an elongation-based translation halt under stress [7], I am excited to see evidence for this novel mechanism.

 

References:

1. de Nadal, E., G. Ammerer, and F. Posas, Controlling gene expression in response to stress. Nat Rev Genet, 2011. 12(12): p. 833-45.
2. Panas, M.D., P. Ivanov, and P. Anderson, Mechanistic insights into mammalian stress granule dynamics. J Cell Biol, 2016. 215(3): p. 313-323.
3. Liu, B. and S.B. Qian, Translational reprogramming in cellular stress response. Wiley Interdiscip Rev RNA, 2014. 5(3): p. 301-15.
4. Shalgi, R., et al., Widespread regulation of translation by elongation pausing in heat shock. Mol Cell, 2013. 49(3): p. 439-52.
5. Liu, B., Y. Han, and S.B. Qian, Cotranslational response to proteotoxic stress by elongation pausing of ribosomes. Mol Cell, 2013. 49(3): p. 453-63.
6. Silva, G.M., D. Finley, and C. Vogel, K63 polyubiquitination is a new modulator of the oxidative stress response. Nat Struct Mol Biol, 2015. 22(2): p. 116-23.
7. Shenton, D., et al., Global translational responses to oxidative stress impact upon multiple levels of protein synthesis. J Biol Chem, 2006. 281(39): p. 29011-21.

Tags: k63, translation, ubiquitin

doi: https://doi.org/10.1242/prelights.4507

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