Mini-Review: Ribosome Profiling
Introduction
Ribosome Profiling (Ribo-seq) is a deep-sequencing technique developed by Nicholas Ingolia and Jonathan Weissman that facilitates a genome-wide view of translation in vivo (1). This technique is based on the principle that translating ribosomes cover a short stretch of mRNA (~30 nucleotides) that is resistant to RNase degradation (2) commonly referred to as the ribosome protected fragment (RPF). Ribosomes are ‘frozen’ in place using translation inhibitors while RNases can be added to destroy any unprotected mRNA leaving only the RPFs behind (Figure 1). By isolating and sequencing these RPFs the exact position of these ribosomes on any mRNA transcript can be determined at nucleotide resolution (3). Ribosome Profiling thus provides researchers with a “snapshot” of translation offering insights into the mechanism of protein synthesis and co-translational processes (1,4). As translation is a highly regulated step, mRNA abundance – as measured by transcriptomics – is not an accurate reflection of protein synthesis. Ribosome profiling on the other-hand is useful for this purpose (5).
Figure 1: An Overview of Ribosome Profiling from Cell lysis to sequencing
How is it done
Ribosome profiling begins with the harvesting and lysis of cells under conditions that are expected to capture in vivo translation. Depending on factors such as the organism the cells/tissue are derived from and the objective of the study cell lysate preparation will vary. If investigating translation initiation sites in eukaryotes for example, cells may be pre-treated with translation initiation inhibitors such as lactimidomycin or harringtonine (6,7). Whereas, to target translation elongation cycloheximide is commonly included in the lysis buffer (8). Following harvesting and lysis, cell lysates undergo nuclease digestion. It is at this point that RPFs are generated. After nuclease digestion, monosome isolation and purification, RPFs are size-selected using denaturing polyacrylamide gel electrophoresis (PAGE). After this RPFs undergo linker ligation, rRNA depletion, reverse transcription and polymerase chain reaction (PCR) amplification. Though, the way these steps are carried out vary across different lab-groups.
Biological Insights
One of the most noteworthy outcomes of ribosome profiling was the discovery of RPFs in the 5’ UTR (untranslated region) indicating translation upstream of the main coding region (4). These upstream translation regions are known as upstream open reading frames (uORFs). uORFs act as tissue-specific cis-regulatory elements and can inhibit downstream translation in a number of ways (9). In their landmark study, Ingolia et al. (1) found that one quarter of 5’UTRs showed significant translational activity. Ribosome profiling has now contributed to the identification of several genes with uORF regulation (10) some of which are discussed in greater detail in our Translatomics in Action: uORF detection post. Like uORF detection, identifying translation initiation sites is a powerful way of uncovering alternative reading frames (11) as many genes have multiple sites of initiation. In the model halophilic archaeon Haloferax volcanii, for example, 160 novel TISs were identified (12) in a single ribosome profiling study. Other areas of translational biology that have exploited the application of ribosome profiling include research on stop codon readthrough (13) and ribosome stalling (3) Refer to our Translatomics in Action posts for a more detailed description of the many biological insights ribosome profiling can provide.
Applications
Translation is a process carried out by all living cells and so the applications of ribosome profiling are plentiful. Ribosome profiling can be used to show how external stress factors such as temperature (14) oxidative stress, and drug treatment (10) impact the synthesis of proteins. A change in temperature for example can cause an upregulation in the translational efficiency of heat shock proteins (14), while their transcription levels remain stable. This is because ribosome profiling enables the capturing of rapid cellular responses to external stimuli. By directly comparing these rapid changes in protein synthesis with steady state transcription, quantification of in vivo translation efficiency can be determined (5). A useful application of ribosome profiling is its utilisation to explore new ways for industrial exploitation of various cells. One such example of this is the advancement of biotherapeutic development with Kallehague et al. (15) presenting the first comprehensive genome-wide view of translation in Chinese Hamster Ovarian (CHO) cells in response to the introduction of a recombinant mRNA for monoclonal antibody (mAb) production. Another is the use of ribosome profiling by Li et al. (16) to study the relationship between translation efficiency and codon usage to optimise bacterial vectors for expression of recombinant proteins. Both of these papers are discussed in greater detail here.
Apart from industrial applications, ribosome profiling will also continue to play a vital role in uncovering mechanisms of disease as translation is often adversely affected in neurological diseases such as Huntingtin disease (17) as well as various cardiovascular diseases (18) and cancers (19). Ribosome profiling can also be used to study the interaction between infectious agents and their hosts or investigate the implications antibiotics play in affecting bacterial translation. Ribosome profiling was used for this very purpose to uncover that macrolide-mediated inhibition of translation does not occur primarily by blocking the peptide exit channel of the ribosome as previously thought, but instead inhibits effective peptide bond formation for certain amino acid sequences (20,21). Novel insights such as this will contribute to the development of newer and more effective antibiotics.
Future Perspectives
Originally carried out in yeast, ribosome profiling has since been applied to mammalian cells, plant cells, bacterial cells, archaeal cells, parasites such as Plasmodium falciparum and Trypanosoma brucei amongst many others (5). Ribosome profiling can essentially accelerate our understanding of complex biological processes happening within the cell and in turn can be utilised to explore almost any cellular process that is implicated by translation.
Despite all that has been answered by this technique, important questions remain such as the function of novel, short, and alternate translated regions identified thus far by ribosome profiling. More specialised alterations of ribosome profiling will enhance its functionality in complex systems. Alterations include the analysis of molecularly defined subsets of ribosomes such as those with protein modifications or those associated with specific factors (5). Experimental advancements like these along with more sophisticated tools tailored specifically for analysing ribosome profiling data will open the door for transformative advances which will enable major new insights into translation, even in systems once thought to be well characterised.
References
1. Ingolia NT, Ghaemmaghami S, Newman JRS, Weissman JS. Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science (80- ). 2009;324(5924):218–23.
2. Steitz JA. Polypeptide chain initiation: nucleotide sequences of the three ribosomal binding sites in bacteriophage R17 RNA. Nature. 1969;224(5223):957–64.
3. Ingolia NT. Ribosome profiling: new views of translation, from single codons to genome scale. Nat Rev Genet. 2014;15(3):205–13.
4. Ingolia NT, Hussmann JA, Weissman JS. Ribosome profiling: global views of translation. Cold Spring Harb Perspect Biol. 2019;11(5):a032698.
5. Brar GA, Weissman JS. Ribosome profiling reveals the what, when, where and how of protein synthesis. Nat Rev Mol Cell Biol. 2015;16(11):651-664. doi:10.1038/nrm4069
6. Ingolia NT, Lareau LF, Weissman JS. Ribosome profiling of mouse embryonic stem cells reveals the complexity and dynamics of mammalian proteomes. Cell. 2011;147(4):789–802.
7. Iwasaki S, Ingolia NT. The growing toolbox for protein synthesis studies. Trends Biochem Sci. 2017;42(8):612–24.
8. Eastman G, Smircich P, Sotelo-Silveira JR. Following ribosome footprints to understand translation at a genome wide level. Comput Struct Biotechnol J. 2018;16:167–76.
9. Zhang H, Wang Y, Wu X, Tang X, Wu C, Lu J. Determinants of genome-wide distribution and evolution of uORFs in eukaryotes. Nat Commun. 2021;12(1):1–17.
10. Andreev DE, O’Connor PBF, Loughran G, Dmitriev SE, Baranov P V, Shatsky IN. Insights into the mechanisms of eukaryotic translation gained with ribosome profiling. Nucleic Acids Res. 2017;45(2):513–26.
11. Ingolia NT. Ribosome footprint profiling of translation throughout the genome. Cell. 2016;165(1):22–33.
12. Gelsinger DR, Dallon E, Reddy R, Mohammad F, Buskirk AR, DiRuggiero J. Ribosome profiling in archaea reveals leaderless translation, novel translational initiation sites, and ribosome pausing at single codon resolution. Nucleic Acids Res. 2020;48(10):5201–16.
13. Loughran G, Chou M-Y, Ivanov IP, Jungreis I, Kellis M, Kiran AM, et al. Evidence of efficient stop codon readthrough in four mammalian genes. Nucleic Acids Res. 2014;42(14):8928–38.
14. Zhang Y, Xiao Z, Zou Q, Fang J, Wang Q, Yang X, et al. Ribosome profiling reveals genome-wide cellular translational regulation upon heat stress in Escherichia coli. Genomics Proteomics Bioinformatics. 2017;15(5):324–30.
15. Kallehauge TB, Li S, Pedersen LE, Ha TK, Ley D, Andersen MR, et al. Ribosome profiling-guided depletion of an mRNA increases cell growth rate and protein secretion. Sci Rep. 2017;7(1):1–12.
16. Li G-W, Oh E, Weissman JS. The anti-Shine–Dalgarno sequence drives translational pausing and codon choice in bacteria. Nature. 2012;484(7395):538–41.
17. Eshraghi M, Karunadharma PP, Blin J, Shahani N, Ricci EP, Michel A, et al. Mutant Huntingtin stalls ribosomes and represses protein synthesis in a cellular model of Huntington disease. Nat Commun. 2021;12(1):1–20.
18. Doroudgar S, Hofmann C, Boileau E, Malone B, Riechert E, Gorska AA, et al. Monitoring cell-type–specific gene expression using ribosome profiling in vivo during cardiac hemodynamic stress. Circ Res. 2019;125(4):431–48.
19. Hsieh AC, Liu Y, Edlind MP, Ingolia NT, Janes MR, Sher A, et al. The translational landscape of mTOR signalling steers cancer initiation and metastasis. Nature. 2012;485(7396):55–61.
20. Kannan K, Kanabar P, Schryer D, Florin T, Oh E, Bahroos N, et al. The general mode of translation inhibition by macrolide antibiotics. Proc Natl Acad Sci. 2014;111(45):15958–63.
21. Kannan K, Vázquez-Laslop N, Mankin AS. Selective protein synthesis by ribosomes with a drug-obstructed exit tunnel. Cell. 2012;151(3):508–20.
22. Brar GA, Weissman JS. Ribosome profiling reveals the what, when, where and how of protein synthesis. Nat Rev Mol Cell Biol. 2015;16(11):651-664. doi:10.1038/nrm4069