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 이성욱 ( 2011-06-20 11:18:06 , Hit : 2328
 Protein synthesis: Stop the nonsense

Nature | News & Views
Adrian R. Ferré-D'Amaré1
Journal name:
Nature
Volume:
474,
Pages:
289–290
Date published:
(16 June 2011)
DOI:
doi:10.1038/474289a
Published online 15 June 2011

A subtle biochemical alteration can reprogram signals that herald the termination of protein translation into signals encoding amino acids at the level of messenger RNA — and without altering the corresponding DNA.

The amino-acid sequence of a protein is specified by combinations of 64 trinucleotides (or codons) in the corresponding messenger RNA. Of these, three codons, known as termination or nonsense codons, signal the end of protein translation. Sometimes, however, rather than stopping protein synthesis, the translation machinery decodes a termination codon as an amino acid in what is known as nonsense suppression. On page 395 of this issue, Karijolich and Yu1 report an artificial way of inducing nonsense suppression — through post-transcriptional conversion of the uridine residue in termination codons into its isomer, pseudouridine. This finding raises fundamental questions about the biochemistry of protein synthesis and has implications for treating genetic diseases.

Translation takes place in cellular organelles called ribosomes, in which each mRNA codon is matched with the anticodon of an aminoacyl-tRNA. The latter is a transfer RNA that has been loaded by its cognate aminoacyl-tRNA-synthetase enzyme with the amino acid corresponding to its anticodon. None of the tRNAs has anticodons complementary to the termination codons; normally, proteins called release factors (RF1 and RF2 in bacteria, eRF1 in eukaryotes) recognize the nonsense codons. But if a tRNA undergoes a mutation in its anticodon such that it becomes complementary to a termination codon (and if this mutant tRNA is otherwise recognized normally by its aminoacyl-tRNA synthetase and the rest of the translation machinery), it might lead to misinterpretation of the termination codon.

Indeed, such nonsense suppression by mutated tRNAs is well documented2. The findings of Karijolich and Yu1 are surprising, however, because of their significance for the mechanism by which release factors are thought to recognize termination codons, and because of the structural similarity between pseudouridine (Ψ) and uridine (U).

The crystal structures of the bacterial ribosome with its release factors caught in the act of recognizing termination codons3, 4 indicate how RF1 and RF2 recognize the U of all three termination codons (UAA, UAG or UGA): chemical groups in the backbone of these release factors seem to form hydrogen bonds with groups on the face of U that normally participate in hydrogen bonding with another nucleotide — the Watson–Crick face. Although Ψ and U differ in that the former has a carbon–carbon, rather than a carbon–nitrogen, glycosidic bond and an imine (NH) group that it projects into the major groove of the RNA, the Watson–Crick faces of these two residues are identical (Fig. 1). Thus, release factors should be insensitive to conversion of the termination codons to ΨAA, ΨAG or ΨGA.

What, then, is the property of Ψ — other than its ability to form Watson–Crick base pairs — that gives rise to nonsense suppression? Ψ binds water through its major-groove imine group, and this hydration makes Ψ-containing RNAs stiffer5. It could be that, when the release factors bind the Ψ-containing mRNA, the increased energy needed to dehydrate this modified mRNA results in nonsense suppression. Alternatively, nonsense suppression could be a consequence of the greater difficulty in unstacking the isomer-containing termination codon from the previous codon as the isomerized codon is brought into the 'reading' position on the ribosome. Regardless of the physico-chemical basis, however, the new results point to a crucial role for factors other than Watson–Crick base pairing in the recognition of termination codons.

Karijolich and Yu demonstrate nonsense suppression through pseudouridylation of termination codons both in vitro and in yeast. When the authors characterized the proteins synthesized following nonsense suppression, they uncovered another surprise. Rather than incorporating a random amino acid at the site occupied by the isomerized termination codon, the translation machinery specifically incorporates either serine or threonine at ΨAA and ΨAG, and either tyrosine or phenylalanine at ΨGA.

This observation is noteworthy because, although the two sets of amino acids have chemical commonalities (threonine and serine both have a hydroxyl group, and tyrosine and phenylalanine share a phenyl ring), the anticodons of tRNAs for the four amino acids do not show any obvious complementarity to the termination codons. Mechanistically, this implies that pseudouridylation of termination codons leads not only to a loss of recognition by release factors, but also to a gain of recognition by specific aminoacyl-tRNAs. The fidelity of normal translation is enhanced through a proofreading process in which the accuracy of codon–anticodon pairing is communicated across the ribosome to the amino-acylated (acceptor) end of tRNA. Perhaps pseudouridylation of termination codons also affects this process.

Site-specific enzymes called pseudouridine synthases produce Ψ from U residues of cellular RNAs6. Eukaryotes and archaea have a versatile class of pseudouridine synthases called H/ACA ribonucleoproteins (RNPs)7. These complexes have their four core proteins in common, but each assembles using one of many different RNAs (containing evolutionarily conserved sequence elements called H and ACA). The RNA component is called a guide RNA because it has a stretch of nucleotides complementary to the sequences that flank the uridine of the substrate RNA targeted for pseudouridylation. This sequence complementarity is necessary and sufficient for directing the H/ACA RNP to pseudouridylate a cellular RNA in vivo.

Karijolich and Yu1 used a custom-designed H/ACA guide RNA to target termination codons for pseudouridylation in their yeast experiments. The authors point out that this would also be an attractive approach to treating genetic disorders that result from premature termination of translation. Indeed, more than a third of genetic disorders and many cancers are due to mutations that introduce premature termination codons8. So, rather than having to correct the mutation at the level of DNA, all that is required would be delivery of an H/ACA guide RNA to pseudouridylate the defective mRNA.

More broadly, it is possible that nature is already using this kind of 'gene therapy' to increase the coding capacity of genomes. Karijolich and Yu have found several candidate mRNAs whose termination codons could be subjected to pseudouridylation by previously described H/ACA guide RNAs. Such mRNAs would produce a shorter protein in their unmodified state and a longer protein (ending at a second, unmodified termination codon) when the first termination codon is pseudouridylated.

References
Karijolich, J. & Yu, Y.-T. Nature 474, 395–398 (2011).

Affiliations
Adrian R. Ferré-D'Amaré is at the National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-8012, USA.







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