Key Points
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mRNA 5′ untranslated regions (UTRs), which serve as the entry point for the ribosome during translation, can adopt elaborate RNA secondary and tertiary structures that may regulate translation initiation in a cap-dependent or cap-independent manner.
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Complex RNA structures in 5′ UTRs, such as RNA G-quadruplexes, may serve as steric blocks to RNA structure unwinding by the scanning ribosome, eukaryotic initiation factor 4A (eIF4A) and other helicases.
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Internal ribosome entry sites (IRESs) recruit ribosomes to 5′ UTRs in a cap-independent manner. Many cellular IRESs are activated upon stress, whereas some are required in regular physiological conditions. Cellular IRESs tend to integrate or interact with RNA chaperones, upstream open reading frames and/or G-quadruplex structures for function.
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The initiation factor eIF3 can have a specialized function in translation by directly binding to structured or chemically modified 5′ UTRs in target mRNAs to mediate selective internal initiation.
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Global in vivo RNA structure probing technologies use chemical modifiers to assess the transcriptome RNA structure inside cells.
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Single-nucleotide-resolution chemical RNA structure probing is emerging to explore the folding state of the transcriptome in living cells; the resulting models for how 5′ UTR structures impact translation should be validated by compensatory mutagenesis.
Abstract
RNA molecules can fold into intricate shapes that can provide an additional layer of control of gene expression beyond that of their sequence. In this Review, we discuss the current mechanistic understanding of structures in 5′ untranslated regions (UTRs) of eukaryotic mRNAs and the emerging methodologies used to explore them. These structures may regulate cap-dependent translation initiation through helicase-mediated remodelling of RNA structures and higher-order RNA interactions, as well as cap-independent translation initiation through internal ribosome entry sites (IRESs), mRNA modifications and other specialized translation pathways. We discuss known 5′ UTR RNA structures and how new structure probing technologies coupled with prospective validation, particularly compensatory mutagenesis, are likely to identify classes of structured RNA elements that shape post-transcriptional control of gene expression and the development of multicellular organisms.
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Acknowledgements
The authors apologize to those researchers whose work was not cited owing to space limitations and thank G. W. Byeon in the Barna laboratory for data analysis and figure preparation of eukaryotic 5′ UTR lengths, the Barna laboratory members for helpful comments on the manuscript, and H.-G. Wendel, J. Kieft, P. Sarnow and M. Hentze for thoughtful suggestions. RNA research in the authors' laboratories is supported by the New York Stem Cell Foundation (M.B.), Mallinckrodt Foundation Award (M.B.), Pew Scholars Award (M.B.) and NIH grants R01HD086634 (M.B.), R21HD086730 (M.B.), R01 GM102519 (R.D.) and R35 GM122579 (R.D.). M.B. is a New York Stem Cell Foundation Robertson Investigator. K.L. is supported by an EMBO Long-Term Fellowship (ALTF 539–2015) and is the Layton Family Fellow of the Damon Runyon Cancer Research Foundation (DRG-2237-15).
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Supplementary information
Supplementary information S1 (box)
Genome-wide RNA-structure probing technologies (PDF 284 kb)
Supplementary information S2 (box)
Viral IRESs and systematic IRES discovery (PDF 4714 kb)
Supplementary information S3 (table)
Experimental probing of cellular IRES RNA structures (PDF 203 kb)
Glossary
- Secondary structures
-
Patterns of Watson–Crick base pairs (G–C, A–U and G–U) that define the double helices of an RNA.
- Tertiary structures
-
Interactions that orient RNA double helices into specific three-dimensional arrangements, often involving non-Watson–Crick base pairs.
- Ribozymes
-
RNA molecules that catalyse specific biochemical reactions.
- Riboswitches
-
Non-coding mRNA structures that sense the environmental status of a cell by directly binding to small molecule ligands, such as a metabolite or an ion. This interaction changes the RNA conformation and controls gene expression through alternative splicing, transcription or translation.
- Peptidyl transferase centre
-
The site in the large ribosomal subunit that catalyses peptide bond formation and peptide release; it is composed entirely of RNA.
- Pseudoknots
-
RNA tertiary structures formed by base pairing of a single-stranded loop of a hairpin with a complementary sequence outside that hairpin.
- Upstream open reading frames
-
(uORFs). Small ORFs located in the 5′ UTR of some mRNAs. Translation of uORFs can regulate the translation of the downstream ORF.
- Kozak sequence
-
A favourable sequence (GCCRCCAUGG in mammals, where R is a purine) surrounding the translation start codon (AUG); also called the Kozak consensus or Kozak context.
- GC content
-
Percentage of guanine and cytosine nucleotides in an RNA molecule. G–C base pairs are more stable than A–U base pairs and can form more stable RNA structures.
- Free energy
-
(ΔG). The stability of an RNA secondary structure, estimated as the sum of the free energies assigned to all loops and base pair stacks of a folded RNA, based on a computational folding algorithm.
- 5′ cap
-
A modified guanine nucleotide, m7GpppN (where m7G is 7-methylguanosine, p is a phosphate group and N is any base), located at the 5′ end of eukaryotic mRNAs.
- Ribonucleoprotein
-
(RNP). A complex of proteins in association with an RNA (RNP) or mRNA (mRNP).
- Ribosome profiling
-
Sequencing of the RNA fragments protected by ribosomes, providing a quantitative signature of the translated mRNAs at a given time.
- RNA G-quadruplex
-
(RG4). Extremely stable RNA structure formed in G-rich regions by the stacking of at least two G-tetrads, each of them forming a square-shaped structure by non-Watson–Crick interactions between two or more layers of paired G-quartets.
- Toeprinting
-
Nucleotide-resolution assay that uses primer extension inhibition induced by the complex of a protein, or the ribosome, bound to an mRNA, to report its position on the mRNA.
- In-line probing
-
Nucleotide-resolution structure probing method that uses the natural instability of RNA: the 2′ hydroxyl of each nucleotide can attack its phosphodiester backbone at a rate dependent on local structure.
- No-go decay
-
An mRNA and translation quality-control mechanism that recognizes and degrades mRNAs following prolonged ribosome stalling during translation.
- Nonsense-mediated decay
-
An RNA surveillance mechanism that recognizes and degrades mRNAs containing premature termination codons to prevent their translation.
- Selective 2′-hydroxyl acylation analysed by primer extension
-
(SHAPE). SHAPE and in vivo click SHAPE (icSHAPE) use cell-permeable reagents to acylate the 2′-OH of accessible, single-stranded RNA at all four nucleotide bases.
- Mutate-and-map
-
Two-dimensional expansion of chemical probing that infers RNA base pairing by mutating each nucleotide and detecting increased chemical accessibility at other nucleotides.
- m6A switches
-
mRNA sequences that adopt a different secondary structure depending on N6-adenosine methylation.
- Dimethyl sulfate
-
(DMS). A cell-permeable reagent that methylates adenine and cytosine nucleotides that are not protected by base pairing. Modification sites stall or induce a mutation during primer extension by reverse transcriptase, and sequencing the resulting complementary DNA indicates the folding state of a nucleotide in an RNA.
- Psoralen
-
A natural plant product that intercalates into DNA and RNA and reversibly crosslinks interacting RNA duplex molecules at UpA motifs upon irradiation with ultraviolet light.
- Sequence covariation
-
Nucleotide substitutions that differ between two or more homologous genes but retain the potential for RNA base pairing in each sequence.
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Leppek, K., Das, R. & Barna, M. Functional 5′ UTR mRNA structures in eukaryotic translation regulation and how to find them. Nat Rev Mol Cell Biol 19, 158–174 (2018). https://doi.org/10.1038/nrm.2017.103
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DOI: https://doi.org/10.1038/nrm.2017.103
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