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Messenger RNA Structure Regulates Translation Initiation: A Mechanism Exploited from Bacteria to Humans
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Biochemistry

Cite this: Biochemistry 2018, 57, 26, 3537–3539
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https://doi.org/10.1021/acs.biochem.8b00395
Published June 12, 2018

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Translation initiation is a major rate-limiting step in protein synthesis and is highly regulated in all cells. Messenger RNAs (mRNAs) play a central role in directing this regulation. Through instructions that remain largely cryptic, mRNA transcripts modulate the initiation process to achieve a specific translation efficiency (TE), the amount of protein made from a given mRNA transcript. TE is precisely tuned, can vary significantly depending on cell type, varies by orders of magnitude across different transcripts, and thus constitutes an essential variable in gene expression. Understanding how mRNAs encode their own unique TEs is therefore a fundamental challenge in biology.

Classic studies have shown that mRNAs can encode TE by folding into structures that facilitate or impede translation initiation. (1) The mRNA-binding cleft of the ribosome can accommodate only single-stranded mRNA. Thus, translation initiation requires unfolding of any mRNA structures that overlap the start codon, imposing a structure-dependent energetic penalty on initiation (Figure 1). Synthetic biologists have harnessed these principles to tune the TEs of designed mRNAs over a large dynamic range.

Figure 1

Figure 1. General mechanism by which RNA structure regulates translation initiation. The mRNA start codon (empty box) and surrounding sequence must be single-stranded to be accommodated into the mRNA cleft of the ribosomal preinitiation complex (brown). Unfolding the mRNA structure imposes an energetic penalty (ΔG⧧) on translation initiation.

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Despite these classic studies, the overall importance of mRNA structure in regulating TEs of endogenous genes has remained surprisingly unclear. Until recently, it has been all but impossible to model mRNA structure with high accuracy. Studies of endogenous genes have largely relied on in silico structure predictions that have supported only a weak relationship between mRNA structure and TE. Whether this weak relationship reflected biological reality or the shortcomings of RNA structure modeling was a persistent unanswered question. In two recent studies, (2,3) our laboratories used the SHAPE-MaP RNA chemical probing strategy to determine high-confidence, experimentally supported structure models for hundreds of mRNAs, providing a unique opportunity to revisit this question.

In one of these studies, (2) we used a data set of approximately 200 SHAPE-MaP-determined mRNA structures to investigate translation regulation in the simple prokaryote Escherichia coli. We initially assumed that it would be straightforward to quantify the influence of structure on TE, but properly addressing this question necessitated a broad evaluation of the mechanism of translation initiation. The ribosomal preinitiation complex recognizes mRNAs via a two-step mechanism. First, the preinitiation complex nonspecifically binds the mRNA via a loosely defined “standby site”; second, the mRNA is unfolded and is accommodated into the mRNA-binding cleft of the ribosomal small subunit, allowing recognition of the Shine-Dalgarno sequence and the start codon (Figure 1). (4) Important details of this second accommodation step were unclear. Specifically, mRNA accommodation might be an equilibrium process wherein the mRNA has time to refold into other low-energy structures, in which case TE should depend on the equilibrium free energy of mRNA unfolding (ΔG). Alternatively, accommodation could be a non-equilibrium process during which the mRNA does not have time to refold. In this case, TE will depend on the non-equilibrium energy of mRNA unfolding (ΔG⧧) (Figure 2A). Our structural data allowed us to estimate ΔG and ΔG⧧ for each gene, which we correlated with TE measurements published by J. Weissman’s lab. Strikingly, our data indicated that TE strongly depends on start codon ΔG⧧ (non-equilibrium) but not ΔG (equilibrium). We subsequently validated this conclusion using reporter assays for 29 genes. (2)

Figure 2

Figure 2. Non-equilibrium mRNA unfolding regulates translation in E. coli and humans. (A) In E. coli, the ribosomal preinitiation complex (brown) first binds the mRNA nonspecifically. Initiation is subsequently governed by mRNA unfolding and accommodation into the mRNA cleft of the preinitiation complex, depending on the non-equilibrium free energy of unfolding the mRNA structure (ΔG⧧). The dependence on non-equilibrium mRNA unfolding implies that initiation is governed by a kinetic competition between accommodation of the mRNA into the mRNA-binding site of the ribosome (kacc) and ribosome dissociation (koff); once dissociated, the preinitiation complex will rapidly bind another mRNA. The mRNA start codon and Shine-Dalgarno sequence are indicated by an empty box and light blue line, respectively. (B) In humans, translation initiation typically proceeds via the scanning mechanism. The preinitiation complex is recruited to the mRNA 5′-cap and subsequently scans along the 5′-untranslated region (5′-UTR) for a start codon. If the 5′-UTR contains a uORF (orange), the preinitiation complex must leak past the uORF to initiate at the primary open reading frame (blue). Otherwise, translation will prematurely initiate and terminate at the uORF. The initiation probability at each start codon (PuORF and PORF) is regulated by the non-equilibrium free energy of unfolding the start codon structure (note that this nomenclature differs slightly from that used in ref (3)).

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These data clearly indicated that mRNA structure tunes gene TE in E. coli. Moreover, the finding that TE depends on non-equilibrium mRNA unfolding has broad implications for our understanding of bacterial translation. First, as has been previously theorized, our finding implies that translation initiation is governed by a kinetic competition between mRNA unfolding and dissociation of the ribosome preinitiation complex. Free ribosome complexes are scarce. Because any individual mRNA comprises a small fraction of the cellular mRNA pool, a dissociated ribosome will most likely bind and initiate on another mRNA (Figure 2A). (4) Second, a dependence of TE on RNA unfolding kinetics provides new insight into the function of RNA regulatory motifs such as riboswitches and thermosensors, which regulate translation in response to small molecule binding or changes in temperature. Kinetic competition provides a consistent explanation for how these motifs modulate ribosome initiation, and thus TE, despite only modest observed changes in equilibrium stability.

In a second study, (3) we examined translation of the human gene SERPINA1, which encodes the α-1-antitrypsin protein. Deficiency of α-1-antitrypsin is clinically linked to lung, liver, and inflammatory diseases. SERPINA1 is a remarkably complex gene that has 11 different splicing isoforms, all of which change the sequence of the 5′-untranslated region (5′-UTR) but not the protein coding sequence. Each isoform contains different combinations of up to three upstream open reading frames (uORFs), which reduce gene TE by causing premature initiation during the scanning process of eukaryotic translation initiation. Under this model, only ribosome complexes that manage to “leak” past uORFs successfully initiate translation at the primary ORF (Figure 2B). While uORFs are found upstream of approximately 50% of human genes, much about their function remains unknown. Interestingly, existing models of leaky scanning inadequately explained the TE variation of different SERPINA1 isoforms, prompting us to explore whether 5′-UTR structure affects uORF function. We used SHAPE-MaP experiments to model the structures of each SERPINA1 isoform and, using the exact same non-equilibrium model that we worked out in E. coli, estimated the free energy (ΔG⧧, annotated simply as ΔG in the original paper) required to unfold structure around the start codon of each uORF and the primary ORF. Remarkably, consideration of the start codon ΔG⧧ dramatically improved the predictive power of the leaky scanning model for the eukaryotic SERPINA1 isoforms (Figure 2B). We subsequently validated this conclusion by selectively disrupting structures and observing corresponding changes in TE. (3)

Our analysis of SERPINA1 strongly implies that mRNA structure regulates start codon recognition of both the primary ORF and any uORFs during eukaryotic scanning via a mechanism similar to that uncovered in prokaryotes. This finding is surprising on two levels. First, the canonical model of eukaryotic scanning posits that 5′-UTR structures are requisitely unfolded as the mRNA is threaded through the mRNA-binding cleft of the ribosome preinitiation complex. (1) However, if all mRNA structure is unfolded, it is unclear how RNA structure could tune start codon recognition, as we have observed and mechanistically validated. Thus, for SERPINA1, and potentially many other mRNAs, our findings suggest that this canonical model needs to be revised. Very stable stem loops in a 5′-UTR are known to stall the scanning process. Nevertheless, we speculate that, in specific contexts, stable RNA structures and 5′-UTR sequences occluded by antisense oligonucleotides (5) might be bypassed (or scanned over) without unfolding during the scanning process. Second, the mechanism of translation initiation and regulation is significantly more complex in eukaryotes than in prokaryotes. It is therefore striking that both domains of life appear to exploit the simple regulatory strategy of using mRNA structure to tune TE.

We emphasize that high-quality RNA structural data are an absolute prerequisite for understanding the functions of mRNA structure. In both studies, we repeated our analyses using “no-data” (in silico only) structure predictions and observed inconclusive relationships between mRNA structure and TE. Native mRNAs have highly diverse and complex structures that are very challenging to predict. These structures may not be well-conserved or uniquely stable compared to expectations of “random” RNA. Nonetheless, idiosyncratic RNA structures that overlap translation start sites play fundamental roles in tuning the TE of each mRNA.

Our analyses are, of course, not perfect. SHAPE-MaP-guided calculations of RNA folding energies include several approximations. (2) In addition, mRNA unfolding is likely manipulated by physical interactions with the ribosome and other proteins, whereas our studies approximate mRNA unfolding as if it occurs in isolation. Our studies thus far have assumed that mRNAs fold into a single structure, whereas many mRNAs likely fold into an ensemble of structures. Tackling such complexities will yield further insight into the mechanisms through which mRNA structure influences translation initiation.

In summary, our studies paint an emerging portrait of start codon structural accessibility as a critical and pervasive regulator of translation initiation. This mechanism is simple, does not require mRNA structure to be particularly well-defined or well-conserved, and notably modulates expression of genes in both prokaryotes and eukaryotes. More broadly, we expect that this general mechanism extends to other RNA-mediated biological processes, with seemingly unremarkable RNA structures likely playing broad roles in tuning interactions between RNA and diverse ligands (Figure 3).

Figure 3

Figure 3. mRNAs tune translation via idiosyncratic structures with varied unfolding energies. The brown box indicates the gene start (AUG) region, and arcs illustrate RNA base pairs. Note that this mechanism likely applies to other RNA-mediated processes in which accessibility to a binding partner is tuned by preexisting RNA structure.

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Author Information

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  • Corresponding Authors
  • Authors
    • Meredith Corley,  Department of Biology and , University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States
    • Alain Laederach,  Department of Biology and , University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States
  • Funding

    A.M.M. is supported by an Arnold O. Beckman postdoctoral fellowship. A.L. is supported by Grants R01 HL111527 and R01 GM101237. K.M.W. is supported by Grants R01 AI068462 (RNA structure methods development) and R35 GM122532 (RNA function discovery).

  • Notes
    The authors declare the following competing financial interest(s): K.M.W. is an advisor to and holds equity in Ribometrix, to which SHAPE-MaP technologies have been licensed.

Acknowledgments

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We thank J. Weissman and his lab for publicly sharing their data, and acknowledge the many authors that we could not cite due to space limitations. The authors thank Chase Weidmann for helpful discussions.

References

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This article references 5 other publications.

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Cite this: Biochemistry 2018, 57, 26, 3537–3539
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https://doi.org/10.1021/acs.biochem.8b00395
Published June 12, 2018

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  • Abstract

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    Figure 1

    Figure 1. General mechanism by which RNA structure regulates translation initiation. The mRNA start codon (empty box) and surrounding sequence must be single-stranded to be accommodated into the mRNA cleft of the ribosomal preinitiation complex (brown). Unfolding the mRNA structure imposes an energetic penalty (ΔG⧧) on translation initiation.

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    Figure 2

    Figure 2. Non-equilibrium mRNA unfolding regulates translation in E. coli and humans. (A) In E. coli, the ribosomal preinitiation complex (brown) first binds the mRNA nonspecifically. Initiation is subsequently governed by mRNA unfolding and accommodation into the mRNA cleft of the preinitiation complex, depending on the non-equilibrium free energy of unfolding the mRNA structure (ΔG⧧). The dependence on non-equilibrium mRNA unfolding implies that initiation is governed by a kinetic competition between accommodation of the mRNA into the mRNA-binding site of the ribosome (kacc) and ribosome dissociation (koff); once dissociated, the preinitiation complex will rapidly bind another mRNA. The mRNA start codon and Shine-Dalgarno sequence are indicated by an empty box and light blue line, respectively. (B) In humans, translation initiation typically proceeds via the scanning mechanism. The preinitiation complex is recruited to the mRNA 5′-cap and subsequently scans along the 5′-untranslated region (5′-UTR) for a start codon. If the 5′-UTR contains a uORF (orange), the preinitiation complex must leak past the uORF to initiate at the primary open reading frame (blue). Otherwise, translation will prematurely initiate and terminate at the uORF. The initiation probability at each start codon (PuORF and PORF) is regulated by the non-equilibrium free energy of unfolding the start codon structure (note that this nomenclature differs slightly from that used in ref (3)).

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    Figure 3

    Figure 3. mRNAs tune translation via idiosyncratic structures with varied unfolding energies. The brown box indicates the gene start (AUG) region, and arcs illustrate RNA base pairs. Note that this mechanism likely applies to other RNA-mediated processes in which accessibility to a binding partner is tuned by preexisting RNA structure.

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  • References

    This article references 5 other publications.

    1. 1
      Kozak, M. (2005) Regulation of translation via mRNA structure in prokaryotes and eukaryotes. Gene 361, 13– 37,  DOI: 10.1016/j.gene.2005.06.037
      1
      Regulation of translation via mRNA structure in prokaryotes and eukaryotes
      Kozak, Marilyn
      Gene (2005), 361 (), 13-37CODEN: GENED6; ISSN:0378-1119. (Elsevier B.V.)
      A review. The mechanism of initiation of translation differs between prokaryotes and eukaryotes, and the strategies used for regulation differ accordingly. Translation in prokaryotes is usually regulated by blocking access to the initiation site. This is accomplished via base-paired structures (within the mRNA itself, or between the mRNA and a small trans-acting RNA) or via mRNA-binding proteins. Classic examples of each mechanism are described. The polycistronic structure of mRNAs is an important aspect of translational control in prokaryotes, but polycistronic mRNAs are not usable (and usually not produced) in eukaryotes. Four structural elements in eukaryotic mRNAs are important for regulating translation, (i) the m7G cap; (ii) sequences flanking the AUG start codon; (iii) the position of the AUG codon relative to the 5' end of the mRNA; and (iv) secondary structure within the mRNA leader sequence. The scanning model provides a framework for understanding these effects. The scanning mechanism also explains how small open reading frames near the 5' end of the mRNA can down-regulate translation. This constraint is sometimes abrogated by changing the structure of the mRNA, sometimes with clin. consequences. Examples are described. Some mistaken ideas about regulation of translation that have found their way into textbooks are pointed out and cor.
    2. 2
      Mustoe, A. M., Busan, S., Rice, G. M., Hajdin, C. E., Peterson, B. K., Ruda, V. M., Kubica, N., Nutiu, R., Baryza, J. L., and Weeks, K. M. (2018) Pervasive regulatory functions of mRNA structure revealed by high-resolution SHAPE probing. Cell 173, 181– 195,  DOI: 10.1016/j.cell.2018.02.034
      2
      Pervasive regulatory functions of mRNA structure revealed by high-resolution SHAPE probing
      Mustoe, Anthony M.; Busan, Steven; Rice, Greggory M.; Hajdin, Christine E.; Peterson, Brant K.; Ruda, Vera M.; Kubica, Neil; Nutiu, Razvan; Baryza, Jeremy L.; Weeks, Kevin M.
      Cell (Cambridge, MA, United States) (2018), 173 (1), 181-195.e18CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      MRNAs can fold into complex structures that regulate gene expression. Resolving such structures de novo has remained challenging and has limited our understanding of the prevalence and functions of mRNA structure. We use SHAPE-MaP expts. in living E. coli cells to derive quant., nucleotide-resoln. structure models for 194 endogenous transcripts encompassing approx. 400 genes. Individual mRNAs have exceptionally diverse architectures, and most contain well-defined structures. Active translation destabilizes mRNA structure in cells. Nevertheless, mRNA structure remains similar between in-cell and cell-free environments, indicating broad potential for structure-mediated gene regulation. We find that the translation efficiency of endogenous genes is regulated by unfolding kinetics of structures overlapping the ribosome binding site. We discover conserved structured elements in 35% of UTRs, several of which we validate as novel protein binding motifs. RNA structure regulates every gene studied here in a meaningful way, implying that most functional structures remain to be discovered.
    3. 3
      Corley, M., Solem, A., Phillips, G., Lackey, L., Ziehr, B., Vincent, H. A., Mustoe, A. M., Ramos, S. B. V., Weeks, K. M., Moorman, N. J., and Laederach, A. (2017) An RNA structure-mediated, posttranscriptional model of human α-1-antitrypsin expression. Proc. Natl. Acad. Sci. U. S. A. 114, E10244– E10253,  DOI: 10.1073/pnas.1706539114
      3
      An RNA structure-mediated, post-transcriptional model of human α-1-antitrypsin expression
      Corley, Meredith; Solem, Amanda; Phillips, Gabriela; Lackey, Lela; Ziehr, Benjamin; Vincent, Heather A.; Mustoe, Anthony M.; Ramos, Silvia B. V.; Weeks, Kevin M.; Moorman, Nathaniel J.; Laederach, Alain
      Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (47), E10244-E10253CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Chronic obstructive pulmonary disease (COPD) affects over 65 million individuals worldwide, where α-1-antitrypsin deficiency is a major genetic cause of the disease. The α-1-antitrypsin gene, SERPINA1, expresses an exceptional no. of mRNA isoforms generated entirely by alternative splicing in the 5'-untranslated region (5'-UTR). Although all SERPINA1 mRNAs encode exactly the same protein, expression levels of the individual mRNAs vary substantially in different human tissues. We hypothesize that these transcripts behave unequally due to a posttranscriptional regulatory program governed by their distinct 5'-UTRs and that this regulation ultimately dets. α-1-antitrypsin expression. Using whole-transcript selective 2'-hydroxyl acylation by primer extension (SHAPE) chem. probing, we show that splicing yields distinct local 5'-UTR secondary structures in SERPINA1 transcripts. Splicing in the 5'-UTR also changes the inclusion of long upstream ORFs (uORFs). We demonstrate that disrupting the uORFs results in markedly increased translation efficiencies in luciferase reporter assays. These uORF-dependent changes suggest that α-1-antitrypsin protein expression levels are controlled at the posttranscriptional level. A leaky-scanning model of translation based on Kozak translation initiation sequences alone does not adequately explain our quant. expression data. However, when we incorporate the exptl. derived RNA structure data, the model accurately predicts translation efficiencies in reporter assays and improves α-1-antitrypsin expression prediction in primary human tissues. Our results reveal that RNA structure governs a complex post-transcriptional regulatory program of α-1-antitrypsin expression. Crucially, these findings describe a mechanism by which genetic alterations in noncoding gene regions may result in α-1-antitrypsin deficiency.
    4. 4
      de Smit, M. H. and van Duin, J. (2003) Translational standby sites: how ribosomes may deal with the rapid folding kinetics of mRNA. J. Mol. Biol. 331, 737– 743,  DOI: 10.1016/S0022-2836(03)00809-X
      4
      Translational Standby Sites: How Ribosomes May Deal with the Rapid Folding Kinetics of mRNA
      De Smit, Maarten H.; Van Duin, Jan
      Journal of Molecular Biology (2003), 331 (4), 737-743CODEN: JMOBAK; ISSN:0022-2836. (Elsevier)
      We have previously shown that stable base-pairing at a translational initiation site in Escherichia coli can inhibit translation by competing with the binding of ribosomes. When the base-pairing is not too strong, this competition is won by the ribosomes, resulting in efficient translation from a structured ribosome binding site (RBS). We now re-examine these results in the light of RNA folding kinetics and find that the window during which a folded RBS is open is generally much too short to recruit a 30 S ribosomal subunit from the cytoplasm. We argue that to achieve efficient expression, a 30 S subunit must already be in contact with the mRNA while this is still folded, to shift into place as soon as the structure opens. Single-stranded regions flanking the structure may constitute a standby site, to which the 30 S subunit can attach non-specifically. We propose a steady-state kinetic model for the early steps of translational initiation and use this to examine various quant. aspects of standby binding. The kinetic model provides an explanation of why the earlier equil. competition model predicted implausibly high 30 S-mRNA affinities. Because all RNA is structured to some degree, standby binding is probably a general feature of translational initiation.
    5. 5
      Liang, X.-H., Shen, W., Sun, H., Migawa, M. T., Vickers, T. A., and Crooke, S. T. (2016) Translation efficiency of mRNAs is increased by antisense oligonucleotides targeting upstream open reading frames. Nat. Biotechnol. 34, 875– 880,  DOI: 10.1038/nbt.3589
      There is no corresponding record for this reference.