Mechanisms of Mrna Frame Maintenance and Its Subversion During Translation of the Genetic Code

Biochimie xxx (2015) 1e7

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Biochimie

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Mini-review Mechanisms of mRNA frame maintenance and its subversion during translation of the genetic code

* Jack A. Dunkle, Christine M. Dunham

Emory University School of Medicine, Department of Biochemistry, 1510 Clifton Road NE, Suite G223, Atlanta, GA 30322, USA article info abstract

Article history: Important viral and cellular gene products are regulated by stop codon readthrough and mRNA frame- Received 11 December 2014 shifting, processes whereby the ribosome detours from the reading frame deﬁned by three nucleotide Accepted 11 February 2015 codons after initiation of translation. In the last few years, rapid progress has been made in mechanis- Available online xxx tically characterizing both processes and also revealing that trans-acting factors play important regulatory roles in frameshifting. Here, we review recent biophysical studies that bring new molecular Keywords: insights to stop codon readthrough and frameshifting. Lastly, we consider whether there may be com- Ribosome mon mechanistic themes in 1 and þ1 frameshifting based on recent X-ray crystal structures of þ1 Protein synthesis Frameshifting frameshift-prone tRNAs bound to the ribosome. © RNA structure 2015 Elsevier B.V. and Societe française de biochimie et biologie Moleculaire (SFBBM). All rights Translocation reserved.

The ribosome is a macromolecular machine that performs incorporated, at least for the most error-prone codoneanticodon protein synthesis, one of the most conserved cellular functions in pairs [5e7]. The most common translational errors are missense or all organisms. Translating the information encoded on the tRNA miscoding errors but non-initiation, stop codon readthrough messenger RNA (mRNA) template into amino acids is a highly and mRNA reading frame errors do occur, although at lower fre- complex but coordinated process with four defined stages - initi- quencies [8]. All of these types of errors likely develop from non- ation, elongation, termination and recycling (for review see Ref. canonical interactions between the mRNA codon and the tRNA [1]). Initiation is the most regulated step with the reading frame of anticodon, whose cognate interactions are required for accurate the mRNA defined by the AUG start codon. The three letter mRNA tRNA selection and movement of tRNAs through the ribosome template or codon is read by a complementary three nucleotide during elongation. anticodon of transfer RNAs (tRNAs) during the elongation phase The term “frameshift” or “recoding” refers to a shift in the three- until protein factors recognize stop codons to terminate translation. nucleotide mRNA coding sequence, resulting in a movement in Although much is known about protein synthesis, it remains un- either the 50 or the 30 direction that produces an amino acid clear how the ribosome preserves or deviates in a controlled sequence distinct from the wild-type or 0 frame sequence (Fig. 1). manner from a three nucleotide reading frame. Frameshift errors are predicted to occur rarely, ~1 out of 30,000 A feature that distinguishes the ribosome from DNA or RNA decoding events [9] while programmed frameshift events occur polymerases is that the polynucleotide template is transferred into much more frequently, in some cases 50e80% of the expressed an entirely different chemical, a polypeptide. DNA replication is protein [10]. Ribosomes that depart from the correct reading frame tightly regulated for accurate inheritance of genetic information frequently encounter stop codons that abort translation and with proofreading repair mechanisms to minimize errors [2].RNA thereby, prevent the expression of the incorrect protein sequence polymerase also has proofreading capability and in addition, mRNA [11]. Because frameshifting usually produces a truncated, non- degradation pathways that eliminate incorrectly synthesized functional polypeptide, it is more deleterious than missense er- mRNAs [3,4]. The ribosome is the most error-prone polymerase rors wherein a single incorrect amino acid is incorporated. The first with error rates estimated at 10 3e10 4 per amino acid evidence that ribosomes could decode a non three nucleotide codon was revealed over forty years ago where extragenic suppressors to insertions and deletions of the genetic code were found to be primarily located in tRNA genes, indicating that they func- * Corresponding author. Tel.: þ1 404 712 1756; fax: þ1 404 727 2738. e E-mail address: [email protected] (C.M. Dunham). tioned by altering protein synthesis [12 14]. Suppressors-of- http://dx.doi.org/10.1016/j.biochi.2015.02.007 0300-9084/© 2015 Elsevier B.V. and Societ e française de biochimie et biologie Moleculaire (SFBBM). All rights reserved.

either a simple stem loop or a more complex pseudoknot structure proximal to the mRNA entrance tunnel [22e24]. How the structured mRNA region influences the elongation process is unknown but it is thought to act synergistically with the slippery sequence to direct translation into a new frame [22]. In a subtle twist on this theme, the Murine Leukemia Virus (MLV) Pol protein is expressed by stop codon readthrough of the gag gene [25] (Fig. 1A). To determine the molecular basis of mRNA recoding required for Pol expression in MLV, the 30 pseudoknot was characterized by NMR [26]. Two conformational populations of the pseudoknot formed at physiological pH; their relative abundance could be influenced by either changing the pH or mutating the RNA to favor one state over the other. These two states were proposed to represent stop codon readthrough permissive or restrictive states that the ribosome encounters during translation. The relative abundance of each state also correlated with the extent of frameshifting in vivo. For example, the pseudoknot adopts the readthrough permissive state 6% of the time, which corresponds to the in vivo stop codon readthrough rate [26,27]. This study provides Fig. 1. Examples of recoding events in viral and cellular genes. A) MLV stop codon compelling evidence for how cis-acting RNA elements direct the readthrough required for expression of the pol gene. B) An example of 1 frame- frame, but several important issues remain. First, it is unclear if the shifting on the slippery sequence (blue) of the IBV 1a/1b gene (note wild-type structure solved in the absence of the ribosome recapitulates the sequence is 50-U UUA AAC-30) and C) on the slippery sequence (blue) of the dnaX conformations of the pseudoknot RNA when it is in the vicinity of gene. D) þ1 frameshifting by frameshift suppressor tRNASufA6 on hisD3018 mRNA. the mRNA entrance channel [26]. Second, since the proposed mechanism requires that the ribosome encounter one of the two frameshift tRNAs do not cause a translational ‘error’, but rather they conformational states during reading of the mRNA, this intercon- return the mRNA back to the 0 frame to allow wild-type protein version between the two states is required to be slower than the expression. The ability of the ribosome to decode in alternate rate of translation. Future experiments to address how conforma- mRNA frames, that is, þ1or1 frames (mRNA movement towards tional switching affects frameshifting in the context of the ribo- 0 0 the 5 or 3 ends), provided evidence the genetic code was not some are required to better understand this phenomenon. immutable. Though frameshifting in viruses was demonstrated to be one way to regulate the expression of essential proteins in a 2. Pre-steady state kinetic investigations of viral ¡1 defined ratio and in some retroviruses, to maximize limited ge- frameshifting nomes, this regulatory mechanism also occurs in bacteria and eukaryotes, demonstrating frameshifting as a universal gene Infectious Bronchitis Virus (IBV) undergoes high levels of 1 expression mechanism [15e18]. frameshifting at the 1a/1b gene containing a conserved slippery Genetic and molecular biology approaches have provided a sequence of the type 50-U UUA AAC-30 (where the spacings separate wealth of data regarding the identification and causes of frame- three nucleotide codons in the 0 frame) (Fig. 1B). The IBV frameshift shifting but a defined molecular mechanism remains elusive. For signal was the first recoding signal discovered containing a 30 example, where is the slippery sequence of the mRNA located on pseudoknot structure that promotes frameshifting [23].Frame- the ribosome when a frameshift occurs and what step of the shifting by the IBV signal can be recapitulated in the context of both elongation cycle does the frameshift affect? Are certain tRNAs bacterial and eukaryotic ribosomes implying functional conservation prone to frameshifting and do tRNA modifications play a role in the of ribosomal features required for this type of recoding [28]. stability of the mRNA-tRNA pair and thus canonical decoding Recently, pre-steady state kinetic analysis of an Escherichia coli properties? Can a concise model be developed to explain both viral in vitro translation system revealed the mechanism of the IBV and cellular frameshifts and can this model be extended to explain frameshift signal using radiolabeled aminoacyl groups on tRNAs to frameshifting in both directions on the mRNA? Here, we report on monitor the nascent peptide chain elongation, along with fluores- recent breakthroughs using biophysical and structural approaches cent tRNAs or ribosomes to separately uncover the dynamics [29] to dissect the molecular mechanisms of þ1 and 1 frameshifting. (Fig.2A and B). Unexpectedly, the amino acid incorporation rate These studies explore the different origins of the shift and provide during reading of the slippery sequence was found to be unaffected. mechanistic insights that reveal potential commonalities between However, both the rate of peptide bond formation for two over- the different types of frameshifting. lapping downstream amino acids, corresponding to the 0 and 1 frame and proximal to the slippery sequence, were substantially 1. mRNA conformational plasticity in the recoding of slower. Surprisingly, the incorporation of the 1 frame Val (codon retrovirus messages GUU; first G nucleotide is from the AAG codon of the slippery sequence) occurred at a faster rate than the 0 frame Phe (codon UUU; Programmed frameshifting was first identified in the Rous Sar- after the AAG slippery sequence codon), indicating movement into coma Virus (RSV) and explained how the gag structural gene fol- the 1 frame was the preferred elongation path (Fig. 1B). The overall lowed by a pol enzymatic gene in a 1 reading frame produces a slower rate of incorporation of either residue was coincidental with single Gag-Pol fusion polyprotein [19]. Programmed frameshifts the IBV pseudoknot reaching the edge of mRNA entrance tunnel were subsequently discovered in diverse viruses where mRNA where unfolding would be required for the single stranded mRNA to shifting was required to control the expression levels of Gag and Pol enter. These studies demonstrated that the IBV frameshift occurs during viral replication [20e22]. A typical 1 recoding mRNA either during translocation of the codon following the slippery signal consists of a ‘slippery’ nucleotide sequence usually defined as sequence or during accommodation of the downstream tRNA, again a run of the same nucleotides, followed by a base-paired mRNA, following the slippery sequence. The possibility that a shift into

encounter mRNAs containing stem loops or pseudoknots, ribosomal translocation or E-site tRNA ejection are compromised, depending upon the relative strength of the structured mRNA [42]. Translation factor EF-G catalyzes translocation of mRNA-tRNA pairs through the ribosome, hydrolyzing GTP in the process. To dissect the sub-steps of the translocation process, fluorescently labeled EF- G, ribosomes or tRNAs were used by Caliskan et al. to demonstrate that the ribosome adopts two intermediate translocation states called POST1 and POST2 during movement of the slippery sequence, before adopting the canonical post-translocation state, POST3. These experiments indicate that the ribosomes commit to the frameshifting pathway early in the translocation process and Fig. 2. Selected probes used in recent biophysical studies of 1 programmed frame- that movement of the ribosome into the POST2 then POST3 state shifting. A) Multiple conformational changes of ribosome components are necessary occurs more rapidly for ribosomes that switch to the 1 reading for mRNA-tRNA translocation during the elongation cycle. Opening and closing of the L1 arm is presumed to be associated with mRNA-tRNA translocation. The small ribo- frame. These experiments further provide convincing evidence that somal subunit body (b), head (h) and platform (pl) domains can move independently frameshifting occurs during translocation and suggests that the of one another while rotation of the small subunit (blue) relative to the large subunit ‘mechanical’ model best approximates how the IBV 1a/1b mRNA (gray) and an orthogonal swiveling of the head domain are necessary for mRNA-tRNA undergoes high levels of frameshifting (Fig. 3D) [30,34]. translocation (30S shoulder domain closing is not shown). B) Radiolabeled methionine along with fluorescently labeled (purple star) tRNA (P-site tRNA is orange; A-site tRNA dnaX ¡ is yellow), EF-G and ribosomal proteins (S13, purple) were used to follow the mech- 3. Single molecule FRET analysis of the bacterial 1 anistic sub-steps of 1 programmed frameshifting on the Infectious Bronchitis Virus frameshift signal (IBV) pseudoknot frameshift signal [29]. C) Fluorescent and fluorescence quencher oligonucleotides were hybridized to 16S rRNA helix 44 and 23S rRNA Helix 101 to The E. coli dnaX gene exhibits high levels of 1 frameshifting follow small subunit rotation. tRNA or elongation factor arrival during translation of the dnaX frameshift signal was also followed by fluorescence [46]. D) Qin et al. used required for expression of the g subunit of DNA polymerase III multiple fluorescently labeled ribosomal proteins (S6 and L9 depicted in purple) to while the 0 frame expresses the DNA polymerase III t subunit [43]. identify conformational states of the ribosome while programmed with the dnaX As with the IBV frameshift signal, the dnaX gene contains a slippery frameshift signal [45]. E) A study by Kim et al. used smFRET between tRNA and ri- sequence located upstream of a base-paired RNA element. How- bosomal protein L1 to follow peptide bond formation and tRNA translocation on the ever, there are three differences between the mRNAs: the dnaX dnaX frameshift signal [44]. structured RNA is likely a stem loop rather than a pseudoknot; the dnaX mRNA contains an internal ShineeDalgarno-like sequence the 1 frame occurs during translocation was previously suggested upstream of the slippery sequence that enhances the frameshift from cryo-EM studies of the 80S bound to the IBV frameshift signal efficiency [10]; and the dnaX slippery sequence is 50-A AAA AAG-30 (not containing the slippery sequence) where eukaryotic elongation rather than the canonical 1 viral mRNA sequence of 50-X XXY YYZ factor 2 (bacterial EF-G) was modeled bound [30]. (where X, Y and Z represent different nucleotides) (Fig. 1C). Three Additional kinetic studies reveal no change in the A-site incor- separate groups have recently reported on how the dnaX mRNA poration rate of fluorescent Lys-tRNALys suggesting the 1 shift promotes 1 frameshifting using single molecule Forster€ resonance follows this step. These data argue against the ‘9Å’, ‘simultaneous energy transfer (smFRET) approaches [44e46]. slippage’ and the ‘three tRNA’ models which posit that viral 1 Chen et al. used smFRET to address the timing of the frameshift frameshifting occurs during tRNA accommodation at the second event using fluorescent donor and quencher dyes attached to rRNA codon of the slippery sequence, i.e. with tRNALys decoding AAG in of both the 30S and the 50S subunits (Fig. 2C) [46]. The ability to the IBV 1a/1b gene [31e33] (Figs. 1B and 3). Of the two remaining monitor the entire elongation cycle over multiple amino acid in- models, the ‘dynamic’ and ‘mechanical’ models [30,34,35] propose corporations, including decoding of the slippery sequence, provided frameshifting occurs during translocation of the second and third unprecedented mechanistic insights. The placement of the dyes codons (1a/1b gene codons UUA and AAG that encode for Leu and allowed for monitoring of the relative subunit movements, which Lys residues, respectively) (Fig. 3C and D). An additional possibility cycle from a non-rotated state (EF-Tu-aminoacyl-tRNA-GTP ternary is that the process of tRNA decoding downstream of the slippery complex delivery, accommodation and peptide bond formation), to sequence at either Val (1 frame; GUU codon) or Phe (0 frame; a rotated state (EF-G binding and translocation and tRNA and 30S UUU codon) plays a role in re-setting the reading frame. To test the head domain movement towards the E site), and back to a non- latter, varying concentrations of Val-tRNAVal or Phe-tRNAPhe were rotated state (backward 30S body/platform rotation and 30S head added to programmed ribosome complexes which would be pre- domain swivel), approximating one round of elongation. These dicted to influence frameshift efficiencies. No effect on the frame- studies revealed that 75% of ribosomes undergo a 1 frameshift and shift efficiencies was observed, strongly suggesting that the reading have a 10-fold longer pause following decoding of the second codon frame was changed during movement of the preceding codon, that of the slippery sequence (Lys7) as compared to the 25% of ribosomes is, translocation of the slippery sequence UUA AAG from the P and A that remain in the 0 frame (Fig. 2C). The longer pause is indicative of sites, to the E and P sites of the ribosome, respectively. ribosomes in a rotated state waiting for EF-G to bind and translocate Translation is a dynamic process wherein the ribosome adopts the mRNA-tRNA pairs. Analysis of the behavior of these two pop- multiple intermediate states in a stepwise manner to move the ulations demonstrated that pausing in the rotated state at the Lys7 tRNAs through the three binding sites [36e39]. Elongation involves codon is concomitant with the 75% of ribosomes that underwent rotation of the body and platform domains of the small ribosomal frameshifting. These data are interpreted to indicate the initial ki- subunit (30S), swiveling of the 30S head domain, closing of the 30S netic branch point determining the 1 or 0 reading frame is set shoulder and movement of the L1 protein on the large ribosomal before pausing, and is controlled by either formation of the up- subunit (50S) from an open to a closed state [37,40] (Fig. 2A). These stream ShineeDalgarno- antiShineeDalgarno-like interaction or the movements all contribute to at least four degrees of freedom that conformation of the downstream RNA stem loop. the ribosome samples, with each state being populated to varying Analysis of the paused, rotated state revealed that EF-G binds degrees [41]. Previously it was shown that when ribosomes and dissociates multiple times in competition with EF-Tu-Lys-

Fig. 3. Models for 1 programmed frameshifting. It has been proposed that frameshifting occurs A) during accommodation of aminoacyl tRNA on the third codon of the slippery sequence (‘9Å’ model); B) after tRNA accommodation but before peptide bond formation (‘simultaneous slippage’ model); C) while tRNAs are in the hybrid state, which coincides with rotation of the small subunit (‘dynamic’ model); D) during eEF2 catalyzed formation of the post translocation state (‘mechanical’ model); E) during tRNA accommodation but with E and P-site tRNAs in a noncanonical post translocation state. The organization of the ﬁgure is adapted from Brierley et al. [22] with the large subunit shown in grey, the small subunit in blue and the slippery mRNA sequence in red.

tRNALys-GTP ternary complex sampling the AAG (Lys8) codon ribosomes revealed L1 was in an open position. The L1 region was (Fig. 1C). Because the efficiency of frameshifting is dependent on previously shown to be important in mRNA frame maintenance and the identity of the position 8 codon [46], the authors conclude that an open L1 may explain the unexpected observation that E-site this continual A-site sampling by tRNALys defines the reading frame. tRNA dissociates during the paused rotated state observed by Chen In summary, these data support either the ‘9Å’ or ‘three-tRNA’ et al. [46,48,49] or upon the ribosome encountering a base-paired models by which tRNA sampling promotes a shift in the mRNA mRNA element at the mRNA entrance tunnel [42]. frame at the third slippery codon. An extension of this model is that FRET probes positioned on tRNAs and L1 were used to follow the this sampling is a consequence of tRNALys being unable to recognize amount and inter-conversion rates of conformational states of the a rotated 30S stuck in a paused state, consistent with incomplete ribosome in the presence of the dnaX stem loop [44]. When the swiveling of the 30S head domain. ribosome was bound to the dnaX stem loop but in the absence of Using complementary smFRET analyses with a defined pro- EF-G, destabilization of the rotated state occurred (defined as a high grammed ribosome, two additional studies investigated how the L1-tRNA FRET signal) while increasing the population of the non- dnaX mRNA stem loop affects the ribosome's conformational states rotated state (low L1-tRNA FRET signal). This interpretation is [44,45]. Qin et al. used smFRET donoreacceptor pairs that tracked opposite to that of the previous two studies but may be reconciled if ribosomal protein S6 movement relative to protein L9 to define the an open L1 conformation coexists with a rotated 30S subunit in the amount of ribosomes in rotated or non-rotated states (Fig. 2A and presence of the dnaX mRNA as seen by Qin et al. One possible D). When ribosomes are poised for mRNA-tRNA translocation, they reason for these discrepancies may be that L1 stalk dynamics are can sample a rotated (hybrid) state and a non-rotated state. While it being monitored which may not directly correlate to subunit was established that multiple states of 30S subunit rotation are rotation. Upon addition of EF-G, similar ribosome behavior is populated by the ribosome [36,39,47], Qin et al. found that the dnaX observed as in the other smFRET study. Namely, dwell times in the mRNA induces a previously unseen 30S conformation, termed the hybrid state were increased with mRNA-tRNA translocation and ‘hyper-rotated’ state, where the 30S is rotated by 22 from the non- adoption of a post-translocation state accumulates slowly. The rotated state [45]. The hyper-rotated state is dependent on both the authors speculate the dnaX stem loop promotes frameshifting by presence of the stem loop and the nucleotide spacing between it slowing translocation to allow more time to sample upstream and the A-site codon; however the hyper-rotated state is inde- conformations that allow slipping on the mRNA in the 1 direction. pendent of A-site tRNA occupancy. This study further demonstrated Additional experiments will be needed to understand precisely the hyper-rotated state reduces the dynamic transitions between how ribosome conformations and dynamics control frameshifting the translocation sub-states possibly explaining the long pause but the studies described show smFRET will be a powerful tool in previously seen in the rotated state observed by Chen et al. [46]. unraveling the mechanism of frameshifting. Small angle x-ray scattering (SAXS), a technique that can define the low-resolution structure of a macromolecule, corroborates the 4. Structural studies highlight the important roles tRNAs play formation of the hyper-rotated state deduced from FRET distances. in frameshifting Using FRET probes, the position of the L1 region of the ribosome was also followed (Fig. 2A). While L1 normally adopts a closed The best studied þ1 frameshift examples are promoted by conformation on a rotated ribosome, FRET analysis of hyper-rotated tRNAs that contain nucleotide insertions or deletions, lack

Please cite this article in press as: J.A. Dunkle, C.M. Dunham, Mechanisms of mRNA frame maintenance and its subversion during translation of the genetic code, Biochimie (2015), http://dx.doi.org/10.1016/j.biochi.2015.02.007 J.A. Dunkle, C.M. Dunham / Biochimie xxx (2015) 1e7 5 nucleotide modifications or act under conditions where their region of the ASL, specifically nucleotide 32, with either EF-G cellular concentrations are altered (for review see Ref. [50]). The during translocation [59] or with the ribosome in the P site [60] decoding of a three nucleotide codon by frameshift suppressor suggest that tRNASufA6 may interact with EF-G in a noncanonical tRNAs is not a programmed gene expression event, but rather, this manner, resulting in incomplete or slowed translocation [51] noncanonical decoding is a mechanism by which the wild-type (Fig. 4). An additional possibility is that normal translocation oc- protein amino acid sequence is preserved. Frameshift suppressor curs, but that the 32e38 pairing is not gripped tightly within the P tRNAs have been invaluable tools to understand the general func- site, which may facilitate movement between the anticodon and tion of the ribosome despite the lack of clear molecular basis for codon into a different frame [50,56,61]. their action. Recent structural studies shed light on the molecular Most tRNAs contain post-transcriptional modifications on basis of action of frameshift suppressor tRNAs that contain anti- nucleotide 37, adjacent to the anticodon, which influences frame- Pro codon stem loop (ASL) insertions and also the roles of tRNA mod- shifting [62]. Therefore 70S structures of ASL CGG with and ifications in maintaining the reading frame [51,52]. Although without the m1G37 modification bound to its cognate codon CCG models to explain 1 and þ1 frameshifting have been restricted to were solved to determine the structural basis for this phenomenon. 1 Pro each class [50,53e56], single molecule and structural studies are Surprisingly, the lack of the m G37 modification in ASL CGG also beginning to provide molecular insights that indicate that each may disrupts the 32e38 base pair [51]. These data suggest a common not be as distinct as previously thought [29,44e46,51,52]. mechanism underlying þ1 frameshifting by frameshift-prone SufA6 Pro Frameshift suppressor tRNA , a derivative of tRNA CGG, tRNAs and modification-deficient tRNAs. contains an insertion 30 of the anticodon (after position 37). The absence of an N1-methyl (m1) base modification at G37 in tRNAP- 5. Discovery of programmed frameshifting mediated by ro CGG is known to cause increased levels of þ1 frameshifting [57,58]. trans-acting factors tRNASufA6 stimulates frameshifting on sites with the codon signa- ture CCC-N (where “N” denotes any nucleotide and is read as a Recent identification of protein and microRNA-induced frame- proline CCC codon) (Fig. 1D), and multiple structures of the 70S shifting events indicate the possibility that there are many un- ribosome were solved with the ASLs of tRNASufA6 decoding the known factors that redirect the mRNA reading frame [63e65]. This following proline codons (CCC) containing an additional nucleotide is not surprising given that 1 frameshifting sites were predicted to decoded as part of the codon: CCC-U, CCC-G and CCC-A [51]. In each be widespread in the genomes of eukaryotes [66]. However, vali- case, two Watson-Crick base pairs form between the codon and dation of these studies reveals a new dimension in gene regulation. anticodon, a non Watson-Crick CeC base pair forms at the third (or The reasons viruses utilize frameshifting is clear: frameshifting wobble) position, and no interaction occurs with the downstream allows for an important regulatory switch independent of the nucleotide U/G/A that is part of the four nucleotide codon. These expression of additional factors, while also increasing viral genome results establish a þ1 slip of the reading frame likely occurs after density to encode multiple polypeptides per open reading frame. completion of tRNA selection in the A site, either during mRNA- However, a high-density genome is not required for eukaryotic cells, tRNA translocation to the P site, or within the P site itself. These therefore it is unclear why frameshifting would exist in eukaryotes. structures also revealed that the U32-A38 base pair, which sits at This point is further underscored by that fact that most computa- the top of the anticodon loop, is disrupted in the context of struc- tionally predicted 1 frameshift signals direct ribosomes to tures containing a þ1 frameshift-prone tRNA and corresponding encounter stop codons that truncate the protein shortly after the shift four nucleotide codons (Fig. 4). Interestingly, the 32e38 interaction [65]. However, trans-activation of a 1 frameshifting signal would is restored when a þ1 frameshift-prone tRNA interacts with a offer an additional layer of regulatory complexity of translational cognate codon that supports 0 frame decoding, signifying both the control. The recent identification of a microRNA (miRNA)- frameshift suppressor tRNA and a specific four nucleotide codon activated 1 frameshift site in the human CCR5 gene provides the first are required for the frameshift event [51]. Interactions between this example of frameshifting by a trans-activating RNA molecule [65]. Putative miRNA sites within the CCR5 1 frameshift signal suggested that the signal may be regulated in an RNA-mediated manner [65]. The putative CCR5 1 frameshift signal is similar to viral frameshift signals: a slippery sequence and a downstream pseudoknot located adjacent to the mRNA entrance tunnel. This study demonstrated that miR-1224 promoted 1 frameshifting by direct interaction with the CCR5 1 frameshift signal. The authors also concluded that frameshifting is, in this case, not a mechanism to express a different protein, but rather, a mechanism by which the ribosome may be directed to an adjacent stop codon, stimulating the nonsense mediated decay of the CCR5 transcript. It is still unclear why mRNA degradation via 1 frameshifting would be initi- ated as a mechanism to control protein expression, but the authors propose a number of scenarios related to circumventing viral in- vasion as possibilities. Another example of trans-activation of frameshifting was recently identified in which a viral protein redirects the mRNA frame. Again, prediction of a possible frameshifting site in the Fig. 4. Base pairing at 32e38 of the ASL is important for frame maintenance. Base Porcine Reproductive and Respiratory Syndrome Virus (PRRV) was pairing within the anticodon stem loop of tRNA (stem, purple; anticodon loop, yellow) the starting point for these experiments [63]. The first indication is important for decoding of the mRNA codon (green). X-ray crystal structures of þ1 that this gene may undergo potentially novel regulation was the frameshift-prone tRNAs bound to the 70S ribosome reveal disruption of the 32e38 extremely high synonymous-site conservation in the 30 region of pair. Cryo-electron microscopy particle reconstructions have shown domain IV of EF-G (red) contacts the ASL in the vicinity of the 32e38 base pair before translocation in the the viral nsp2 coding region, and the absence of out-of-frame stop A site, suggesting how the 32e38 base pair may be linked to frameshifting [59]. codons, typically representative of alternative, overlapping protein

Please cite this article in press as: J.A. Dunkle, C.M. Dunham, Mechanisms of mRNA frame maintenance and its subversion during translation of the genetic code, Biochimie (2015), http://dx.doi.org/10.1016/j.biochi.2015.02.007 6 J.A. Dunkle, C.M. Dunham / Biochimie xxx (2015) 1e7 coding regions. The second indication was that the 30 region of nsp2 Acknowledgements revealed additional similarities to viral 1 frameshifting sites with a slippery site followed by a conserved sequence spaced 10 nucle- Research is supported by the National Institute of General otides downstream. However, other features were uncharacteristic Medical Sciences of the NIH under award number R01GM093278 of typical 1 viral frameshift signals; for example, ribosomes read and the Pew Scholar in the Biomedical Sciences Program (CMD). a þ1 mRNA reading frame and there was no predicted structured We thank Graeme L. Conn for critical reading of the manuscript. RNA region in the conserved downstream element [63]. 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