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Rationalization and Prediction of Selective Decoding of Pseudouridine-Modified Nonsense and Sense Codons

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Rationalization and Prediction of Selective Decoding of Pseudouridine-Modified Nonsense and Sense Codons Downloaded from rnajournal.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press HYPOTHESIS Rationalization and prediction of selective decoding of pseudouridine-modified nonsense and sense codons MARC PARISIEN,1,3 CHENGQI YI,1,3 and TAO PAN1,2,4 1Department of Biochemistry and Molecular Biology, 2Institute for Biophysical Dynamics, University of Chicago, Chicago, Illinois 60637, USA ABSTRACT A stop or nonsense codon is an in-frame triplet within a messenger RNA that signals the termination of translation. One common feature shared among all three nonsense codons (UAA, UAG, and UGA) is a uridine present at the first codon position. It has been recently shown that the conversion of this uridine into pseudouridine (C) suppresses translation termination, both in vitro and in vivo. Furthermore, decoding of the pseudouridylated nonsense codons is accompanied by the incorporation of two specific amino acids in a nonsense codon-dependent fashion. C differs from uridine by a single N1H group at the C5 position; how C suppresses termination and, more importantly, enables selective decoding is poorly understood. Here, we provide molecular rationales for how pseudouridylated stop codons are selectively decoded. Our analysis applies crystal structures of ribosomes in varying states of translation to consider weakened interaction of C with release factor; thermodynamic and geometric considerations of the codon-anticodon base pairs to rank and to eliminate mRNA-tRNA pairs; the mechanism of fidelity check of the codon-anticodon pairing by the ribosome to evaluate noncanonical codon-anticodon base pairs and the role of water. We also consider certain tRNA modifications that interfere with the C-coordinated water in the major groove of the codon-anticodon mini-helix. Our analysis of nonsense codons enables prediction of potential decoding properties for C-modified sense codons, such as decoding CUU potentially as Cys and Tyr. Our results provide molecular rationale for the remarkable dynamics of ribosome decoding and insights on possible reprogramming of the genetic code using mRNA modifications. Keywords: pseudouridine; nonsense codon; molecular modeling; ribosome; tRNA INTRODUCTION own and the preceding phosphate groups (Arnez and Steitz 1994). Pseudouridine (C) is a C5-glycoside rotation isomer of In a recent study, Yu and colleagues demonstrated that uridine (Fig. 1A). It is found in transfer and ribosomal the substitution of uridine in all three nonsense codons RNA (tRNA, rRNA) throughout the three kingdoms of with C suppresses translational termination, both in vitro life and in spliceosomal small nuclear RNA (snRNA) in and in Saccharomyces cerevisiae (Karijolich and Yu 2011). eukaryotes. Although C is the most abundant RNA mod- Furthermore, they showed that C-modified nonsense co- ification (Hamma and Ferre-D’Amare 2006), it remains to dons are selectively decoded as specific amino acids (Table 1). be determined whether C is also present in messenger RNA In particular, CAA and CAG are read as serine and thre- (mRNA). The primary chemical change made by the U-to-C onine, whereas CGA is read as phenylalanine and tyrosine. conversion is the addition of a hydrogen bond donor Decoding for unmodified mRNA codons is accom- through the N1H group. In an RNA helix, this hydrogen plished in multiple steps and is under strict surveillance bond donor is in the major groove and can anchor a water by the ribosome. mRNA codons are usually recognized by molecule to bridge the interactions of this N1H group to its tRNAs featuring complementary Watson-Crick sequences and sometimes wobble base pairs at the third codon posi- tion. Watson-Crick and certain wobble base pairs enable 3These authors contributed equally to this work. cognate tRNAs to thermodynamically outcompete other 4 Corresponding author. tRNA species whose sequences would introduce mismatched E-mail [email protected]. Article published online ahead of print. Article and publication date are base pairs with the mRNA codon. Further, the mRNA-tRNA at http://www.rnajournal.org/cgi/doi/10.1261/rna.031351.111. base pairs in the A-site of a ribosome are probed in a process RNA (2012), 18:00–00. Published by Cold Spring Harbor Laboratory Press. Copyright Ó 2012 RNA Society. 1 Downloaded from rnajournal.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press Parisien et al. ciently suppressed as translation stops, they are also selectively decoded as just two amino acids (Karijolich and Yu 2011). Although the decoding pattern of the modified nonsense codons includes Watson-Crick type C/A base pairs, e.g., those in tRNASer, the presence of C in the first codon posi- tion also permits decoding by tRNAs with U36 at their third anticodon position, e.g., those in tRNAThr. This opens up the possibility for a mRNA-tRNA mismatched C/U base pair. Furthermore, the presence of C leads to additional changes in base-pairing rules in the second or even in the third codon- anticodon pairs, e.g., A/G mismatches in the case of CAA/ CAG decoding at the second position and in the case of CGA decoding at the third position (Table 1). No rational expla- nation was provided for these experimental observations. Here, we provide a molecular explanation of how the three C-modified nonsense codons are suppressed as ter- mination codons and, more importantly, are selectively de- coded as specific amino acids. Taking advantage of the large number of available crystal structures of ribosomes in vary- ing states of translation, we provide molecular models on selective decoding, A-site probing, thermodynamic and geo- metric considerations, and influence of certain tRNA mod- ifications on selective decoding of C-modified nonsense codons. Because no structures of eukaryotic ribosomes con- taining both mRNA and tRNA are available, our analysis had to rely on the structures of bacterial ribosomes. Due to the involvement of many interaction partners, it was not pos- FIGURE 1. Structural comparison of U and C and proposed role of sible to predict the precise fraction of the two amino acids decreased recognition by release factors. (A) Chemical structure of U that selectively decode C-containing nonsense codons. and C. Partial charge is labeled for each atom and calculated dipole Despite these caveats, our results provide insights for the moments of the two bases (debye, or D) are shown with arrays. (B) remarkable dynamics of ribosome decoding and lead to pre- Recognition of U in a nonsense codon by RF1 (PDB code: 3D5A) and RF2 (2WH3). (C) Comparison of attractive dipole moment between dictions of potential decoding rules for C-modified sense the release factor protein and the nucleobase, when either a U or C codons which should be useful in rational reprogramming (assuming C occupies the same position as U) is present in the active of the genetic codes using RNA modifications. site of RF1. The extra water molecule present only with C-modified nonsense codons is also shown. RESULTS AND DISCUSSION termed ‘‘the A-site test’’ to ensure the fidelity of the decoded C has to escape recognition by release factors codon (Ogle et al. 2001; Schuwirth et al. 2005; Selmer et al. 2006). Specifically, the A-site test is performed by a small Nonsense codons in the A-site of ribosome are normally ribosomal subunit via A1493 and A1492 of helix 44 in the recognized by release factors, thereby triggering dissociation 16S rRNA (Thermus thermophilus numbering is used here- after), which form an extensive network of hydrogen bonds in the minor groove with the mRNA-tRNA base pairs at the TABLE 1. Amino acids incorporated in C-containing nonsense codons first and second codon position, and by G530 of loop 530 at the third codon position (Ogle et al. 2001). Such minor- Stop Incorporation Putative tRNA groove interactions are universally conserved and serve as codon AA (%) anticodon a key step in the fidelity control of the decoding process 59-CAA ser ;50 59-IGA, 59-CGA, 59-UGA, 59-GCU (Lescoute and Westhof 2006). Many tRNAs are extensively thr ;50 59-IGU, 59-CGU, 59-UGU modified at nucleotide 37, the immediate 39 nucleotide to the 59-CAG ser ;90 59-IGA, 59-CGA, 59-UGA, 59-GCU third anticodon nucleotide which reads the first position of thr ;10 59-IGU, 59-CGU, 59-UGU 59-CGA tyr ;80 59-GUA the mRNA codon. Modification at nucleotide 37 could also phe ;20 59-GAA influence the accuracy to decode the first codon nucleotide. When the first position of the nonsense codons is modi- Corresponding tRNA anticodons for all tRNA isoacceptors are also shown. (I) inosine. fied from U to C, the modified codons are not only effi- 2 RNA, Vol. 18, No. 3 Downloaded from rnajournal.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press Recoding of pseudouridine-modified codons of ribosomal subunits and releasing the newly synthesized The weaker affinity derived from altered dipoles and peptide chain. In order to allow base-pairing to anticodons water-mediated base geometry could enable some tRNA and subsequent incorporation of amino acids, C-modified species to compete for the binding of the modified non- nonsense codons must first escape recognition by the release sense codon. factors. In yeast, all three nonsense codons are recognized by eRF1 which forms a functional complex with eRF3, while in tRNA abundance cannot explain the specificity prokaryotes, two proteins (RF1 and RF2) recognize the non- of amino acid incorporation sense codons. Due to the lack of structural information of eRF1 in the context of nonsense codon recognition in the One trivial explanation for the specific incorporation of ribosome, our analysis was performed with prokaryotic re- certain amino acids for the C-modified nonsense codon lease factors (RF1 and RF2) where rich structural informa- (Table 1) is that decoding might be governed by an in vivo tion is available (Korostelev et al.
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