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Regulation of Programmed Ribosomal Frameshifting by Co-Translational Refolding RNA Hairpins

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Regulation of Programmed Ribosomal Frameshifting by Co-Translational Refolding RNA Hairpins Regulation of Programmed Ribosomal Frameshifting by Co-Translational Refolding RNA Hairpins Che-Pei Cho, Szu-Chieh Lin, Ming-Yuan Chou, Hsiu-Ting Hsu, Kung-Yao Chang* Institute of Biochemistry, National Chung-Hsing University, Taichung, Taiwan, Republic of China Abstract RNA structures are unwound for decoding. In the process, they can pause the elongating ribosome for regulation. An example is the stimulation of -1 programmed ribosomal frameshifting, leading to 39 direction slippage of the reading-frame during elongation, by specific pseudoknot stimulators downstream of the frameshifting site. By investigating a recently identified regulatory element upstream of the SARS coronavirus (SARS-CoV) 21 frameshifting site, it is shown that a minimal functional element with hairpin forming potential is sufficient to down-regulate21 frameshifting activity. Mutagenesis to disrupt or restore base pairs in the potential hairpin stem reveals that base-pair formation is required for21 frameshifting attenuation in vitro and in 293T cells. The attenuation efficiency of a hairpin is determined by its stability and proximity to the frameshifting site; however, it is insensitive to E site sequence variation. Additionally, using a dual luciferase assay, it can be shown that a hairpin stimulated +1 frameshifting when placed upstream of a +1 shifty site in yeast. The investigations indicate that the hairpin is indeed a cis-acting programmed reading-frame switch modulator. This result provides insight into mechanisms governing21 frameshifting stimulation and attenuation. Since the upstream hairpin is unwound (by a marching ribosome) before the downstream stimulator, this study’s findings suggest a new mode of translational regulation that is mediated by the reformed stem of a ribosomal unwound RNA hairpin during elongation. Citation: Cho C-P, Lin S-C, Chou M-Y, Hsu H-T, Chang K-Y (2013) Regulation of Programmed Ribosomal Frameshifting by Co-Translational Refolding RNA Hairpins. PLoS ONE 8(4): e62283. doi:10.1371/journal.pone.0062283 Editor: Volker Thiel, Kantonal Hospital St. Gallen, Switzerland Received December 20, 2012; Accepted March 19, 2013; Published April 29, 2013 Copyright: ß 2013 Cho et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by Grants NSC 99-2311-B-005-004-MY3, NSC 99-2627-M-005-002 and NSC 101-2627-M-005-007 from the National Science Council of Taiwan (to K.-Y. Chang). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction or actively by creating tension/strain to communicate with transfer RNA (tRNA)-mRNA linkages to destabilize the P site Sequence complementarities and three-nucleotide based genetic codon-anticodon helix in the 0-frame (XXY). It also eventually codes in messenger RNA (mRNA) imbue interesting features for facilitates re-pairing of tRNA with the21 frame mRNA (XXX) translation. These include a) an intra-molecular duplex (formed [2,3,8,9,10]. via sequence complementarities) having to be unwound for Other factors have been suggested in the modulation of decoding, and b) one of three potential reading-frames having to frameshifting efficiency [5]. In particular, an optimally placed be maintained for faithful protein synthesis. The ribosome internal Shine-Dalgarno (SD) sequence in prokaryotic mRNA may possesses helicase activity that allows for the unwinding of RNA serve as a 21 PRF stimulator by pairing with the anti-SD element duplexes [1,2] while reading-frame maintenance is closely coupled in 16S ribosomal RNA (rRNA) of the 70S ribosome [11]. In with translocation [3]. However, unwinding of specific RNA addition, 21 frameshifting efficiency can be affected positively or structures can pause or stall the ribosome for further elongation negatively by flanking sequences upstream of a slippery site regulation [4]. In particular, specific mRNA signals can program a [12,13]. Previously, we identified a 170-nucleotide RNA element ribosome to switch reading-frames during elongation, with the (ATT), upstream of the21 PRF slippery site of SARS-CoV ribosome slipping backward (toward the 59-direction) or forward mRNA, capable of down-regulating viral21 PRF [14]. Recently, (toward the 39-direction) by a single nucleotide. It then continues ATT was shown to optimize viral replication and was suggested to translation in the new21or+1 reading-frame. Such21or+1 act by causing a fraction of the elongating ribosome to fall-off in programmed ribosomal frameshifting (PRF) has been character- front of the ATT [15]. Understanding how21 PRF attenuation is ized in prokaryotes and eukaryotes and is usually related to specific achieved would not only shed light on how a 21 PRF stimulator cellular functions [5]. promotes21 frameshifting and provide insight into the mechanism A slippery sequence (XXXYYYZ) and optimally placed governing reading-frame control, but also may have potential for downstream stimulator structures on mRNA are the two in-cis antiviral applications because21 PRF efficiency is crucial for the elements required for efficient eukaryotic21 PRF [6]. However, the precise21 PRF stimulation mechanism remains unclear [7]. replication of several human viral pathogens, including HIV and Most models of21 PRF stimulation propose that a specific SARS coronavirus (SARS-CoV) [15,16]. structural or mechanical feature of the stimulator resists the Here, we identify a minimal element in SARS-CoV ATT as the unwinding activity of ribosomal helicases [2]. This is done either major determinant of21 PRF attenuation function. Additionally, passively by serving as a roadblock to pause ribosomal movement we show that attenuation efficiency is not sensitive to E site PLOS ONE | www.plosone.org 1 April 2013 | Volume 8 | Issue 4 | e62283 Refolding RNA Hairpin Regulates Frameshifting sequence variation, suggesting flanking-sequences effect is not the 0 frame E site of the ribosome when the slippery sequence main cause of attenuation. We further demonstrate that this occupied ribosomal P and A sites. This invasion could interfere minimal element acted through a hairpin form with attenuation with the proposed reading-frame maintenance function of the E efficiency determined by hairpin stability and spacing to the site [18]. However, mutagenesis analysis indicates that in vitro slippery site. Importantly, this potential hairpin also enhanced +1 attenuation activity of the 6BPGC hairpin is not sensitive to base- frameshifting in yeast, indicating that in addition to being a 21 pair formation involving 39- flanking UU sequences (Figs. S1A and frameshifting attenuator, it can serve as a +1 frameshifting S1B). On the other hand, disrupting potential base pairs at 39- stimulator. Together, these results indicate that the upstream flanking CG impaired attenuation activity by a third (Fig. S1B RNA hairpin functions as a cis-acting RNA motif in programmed compares UUCG-6BPGC with GAAA-6BPGC). Especially im- frameshifting regulation. Finally, our findings also indicate base- portant is the disruption of two GC base pairs at the bottom of the pair reformation involving the terminal sequences of the 39-half of hairpin stem (6BPGC12AG). Such a disruption dramatically the hairpin stem as being crucial for attenuation. This implies the reduced attenuation activity. This evidence seems to show that two existence of a refolding hairpin stem in close proximity to the extra GC base pairs involving 39- flanking CG lead to an extended ribosomal E site. stem and contribute to attenuation activity. However, only one potential GC base pair exists in the corresponding region of wild- Results type SARS-CoV viral RNA sequences (Fig. 3A), suggesting that this extra base pair is not essential for attenuation activity by the The Minimal Upstream Attenuation Determinant May Act wild-type SARS-CoV attenuator hairpin. To see if these through a Hairpin Form observations are valid and applicable to a different stimulator in To search for a minimal determinant within ATT, we other biological systems, selected 6BPGC 59-flanking sequence performed sequential 59sequence deletions of ATT and compared mutants were placed upstream of a distinct 21 PRF stimulator, the relative frameshifting activity of the different deletion variants the DU177 pseudoknot [19], and examined for their attenuation (Fig. 1). We found that viral sequences, covering nucleotides 13363 activity in 293T cells. The results (Fig. S1C) indicate that the two to 13387, possessed substantial21 PRF attenuation activity (Fig. 1) potential base pairs involving E-site sequences are not the main and the ability to form a stable stem-loop structure (Fig. 2A). cause of observed attenuation activity in 293T cell cultures. However, the base of the predicted hairpin stem is only 4 nucleotides away from the 59-edge of the slippery site. It is possible Attenuation Efficiency is Positively Correlated with that a hairpin stem cannot be formed when the slippery site Hairpin Stability occupies ribosomal P and A sites. The identified attenuator hairpin contained a single nucleotide In the past, the refolding pathway of unwound RNA structures within ribosome cores has not been comprehensively investigated. G bulge and
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