- Email: [email protected]
Translational halt during elongation caused by G-quadruplex formed by mRNA
Methods 64 (2013) 73–78
Contents lists available at SciVerse ScienceDirect
Methods journal homepage: www.elsevier.com/locate/ymeth
Translational halt during elongation caused by G-quadruplex formed by mRNA Tamaki Endoh a, Yu Kawasaki b, Naoki Sugimoto a,b,⇑ a b
Frontier Institute for Biomolecular Engineering Research (FIBER), Konan University, Kobe, Japan Faculty of Frontiers of Innovative Research in Science and Technology (FIRST), Konan University, Kobe, Japan
a r t i c l e
i n f o
Article history: Available online 7 June 2013 Keywords: Synchronized translation Translation elongation mRNA structure G-quadruplex Kinetic analysis
a b s t r a c t mRNA forms various secondary and tertiary structures that affect gene expression. Although structures formed in the untranslated regions (UTRs) of mRNAs that inhibit translation have been characterized, stable mRNA structures in open reading frames (ORFs) may also cause translational halt or slow translation elongation. We previously established a method, termed a synchronized translation assay, that enables time course analysis of single turnover translation elongation. In this method, translation initiation, which is a rate determining step of the translation procedure, can be ignored because all ribosomes are synchronized on a specific position of mRNA before translation elongation is restarted from this position. In this paper, we used the synchronized translation assay to evaluate the effects of a G-quadruplex structure located at various positions within the mRNA ORF on translational halt. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction During translation, the ribosome, a large RNA–protein complex, polymerizes amino acids based on the sequence encoded in the open reading frame (ORF) of an mRNA to produce functional proteins. mRNAs are able to form various intramolecular secondary and tertiary structures that contribute to the regulation of the translation process. For example, internal ribosomal entry sites (IRESs) allow translation initiation in the absence of initiation factors and riboswitches in 50 untranslated regions (UTRs) of mRNAs regulate translation through RNA conformational transitions in response to metabolites [1–3]. In addition to these functional mRNA structures that control translation through interaction with small molecules or proteins, some RNA structures with high thermodynamic stability, which are either naturally formed or artificially inserted into UTRs or ORFs, suppress ribosome progression [4–6]. Inhibition of the ribosome in its scanning phase by stable RNA structures in 50 UTRs may decrease protein expression, whereas stable structure in the ORF may contribute various biological processes such as ribosomal frameshift [7], no-go mRNA decay [8,9], which is one of the recently discovered mRNA surveillance mechanisms, and co-translational folding of nascent proteins [10–12]. Thus, analyses of mRNA structures that suppress the translation elongation will further expand our understanding of the contributions of mRNA structures to the translation reaction. ⇑ Corresponding author at: Frontier Institute for Biomolecular Engineering Research (FIBER), Konan University, Kobe, Japan. E-mail address: [email protected] (N. Sugimoto). 1046-2023/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ymeth.2013.05.026
The translation reaction can be divided into three processes: initiation, elongation, and termination. It has been suggested that the rate determining step of the translation reaction is initiation, which requires formation of initiation complex formed by several protein factors [13,14]. Thus, if an in vitro translation reaction is stopped at a certain reaction time, the position of ribosomes on the mRNA is heterogeneous depending on the time when the ribosome initiated the translation reaction. To analyze the elongation procedure in detail, the translation reaction must be synchronized to enable time course analysis of translation elongation. We previously reported a procedure for the synchronized translation in a single turnover reaction [15]. Ribosomes were artificially stalled at a specific position of mRNA after initiation of translation reaction, and translation elongation was subsequently restarted. This should be appropriate method for evaluating effects of RNA structures in mRNA ORF on translation elongation. A G-quadruplex is one of the non-canonical structures of nucleic acids formed by guanine rich (G-rich) regions of RNA or DNA with at least four consecutive stretches of more than two guanines [16,17]. Recent research works have been focused on those formed within non-coding regions that impact various biological procedures, such as transcription, mRNA splicing, and translation initiation [18–21]. Especially, G-quadruplexes in ORFs of oncogene mRNAs suppress protein expression from the mRNAs depending on the stability of the quadruplexes by inhibition of the scanning of the ribosome [22]. There is a little information about effect of G-quadruplexes within mRNA ORFs on translation reaction in despite of the G-rich sequences of both non-coding and coding regions of mRNAs would form the G-quadruplex structure. Here,
74
T. Endoh et al. / Methods 64 (2013) 73–78
we evaluated the effects of a G-quadruplex structure on translational halt by using the synchronized translation assay. 2. mRNA design for the synchronized translation To enable the analysis of translation elongation over a time course in a single turnover reaction the mRNA shown in Fig. 1A was designed. This mRNA has three important features: (i) A T7-tag, MASMTGGQQMG, is encoded at N-terminus of the amino acid sequence (Fig. 1A, indicated in blue). This enables purification of translated products that begin from the correct start codon using anti-T7 tag agarose (MBL). Mass spectroscopy can then be used to determine lengths of the protein products to infer where the translation elongation was stalled (Fig. 1B, mass diagram). (ii) A UAG amber codon was inserted after the nucleotide sequence encoding the T7-tag (Fig. 1A, indicated in green). Although the amber codon normally encodes termination of translation, we can incorporate non-natural amino acids into the protein by using amber suppressor tRNA aminoacylated with a non-natural amino acid [23]. Incorporation of the non-natural fluorescent amino acid (CR110-X-AF) using the amber suppressor tRNA (CR110-X-AF-tRNAamber; Protein Express) enables site specific labeling of the translated product (Fig. 1B, green frame) and time-course analysis of translation elongation after separation on SDS–PAGE. (iii) The stop codon at 30 terminus was removed from the mRNA. This prevents turnover of ribosome and ensures a singleturnover reaction. 3. Two-step synchronized translation reaction In the synchronized translation assay, two-step reaction procedure using in vitro translation based on protein synthesis using recombinant elements (PURE) system [24,25], which enables omission of specific protein components required for translation from reaction mixture, was designed to synchronize the ribosomes. The procedure is shown schematically in Scheme 1.
In the first reaction, a specific aminoacyl-tRNA synthetase (aaRS) is missing from the reaction mixture. The lack of this aaRS, which results in omission of a specific aminoacyl-tRNA, artificially stalls all ribosomes at a specific position of mRNA (Fig. 1B, first reaction). Release factor (RF) 1 and RF2 are also missing from the first reaction mixture. Amber suppressor tRNA competes with RF1 during normal translation reactions. Thus, lack of RF1 and RF2 enables efficient incorporation of the non-natural amino acid and prevents undesired termination of translation. In the second reaction, the reaction mixture containing all requisite components except ribosome is added to the first reaction to restart the translation elongation (Fig. 1B, second reaction). In order to ensure that any initiation of translation that may occur during the second reaction by backward ribosomes does not mask detection of single-turnover elongation, RF1 is added to the second reaction mix. Addition of RF1 ensures that translation initiated during the second reaction is terminated at the amber codon inserted after the T7-tag (Fig. 1A).
4. Time course analysis of elongation of 12 amino acids Our previous study indicated that the presence of rare codons in the mRNA temporarily halt translation elongation [15]. To analyze the translational halt caused by the mRNA structures, an mRNA must be designed without rare codons prior to the mRNA structure of interest. The mRNA sequence in Fig. 2A was designed as a basal mRNA to study the effect of mRNA structure on the translation elongation. In the previous study, isoleucyl-tRNA synthetase (IleRS) was removed from the reaction mixture to artificially stall the ribosome before an isoleucine codon at the first reaction. Since a portion of ribosomes seemed to cause a + 1 frameshift during the stalling at the first reaction [15], here we removed tyrosyl-tRNA synthetase (TyrRS) from the reaction mixture instead of the IleRS to artificially stall the ribosome before 30th codon encoding tyrosine (Fig. 2A). Addition of reaction mixture containing TyrRS restarts the translation elongation from the tyrosine codon, and the elongation finishes in single turnover at the 30 end of the mRNA following elongation of 12 amino acids (Fig. 2A). In addition to the change in the removed aaRS, PUREfrexÒ (GeneFrontier; http://
Fig. 1. Schematic of synchronized translation assay. (A) General design of mRNA for the synchronized translation. Amino acid sequence of T7-tag for purification of translated product and UAG amber codon for incorporation of non-natural fluorescent amino acid are indicated in blue and green, respectively. (B) In the first reaction, all ribosomes incorporate CR110-X-AF (a non-natural fluorescent amino acid) and stall at a specific codon due to the omission of release factor (RF) and a specific aminoacyl-tRNA synthetase (aaRS). In the second reaction, upon addition of the missing aaRS, all ribosomes restart translation elongation from the stalled position. Translated products are subsequently detected based on the fluorescence signal of CR110-X-AF by SDS–PAGE or by mass spectrometry.
75
T. Endoh et al. / Methods 64 (2013) 73–78
Scheme 1. General experimental flow of the synchronized translation assay.
www.genefrontier.com/english/index.html), a reconstructed in vitro translation system derived from improved purification process of requisite tRNA, ribosome and proteins, was utilized. The customized reaction mixture, in which the TyrRS, RF1, and RF2 were removed, was provided from GeneFrontier Corp. The first reaction was performed in the absence of TyrRS, RF1, and RF2 with various concentrations of tRNA, in which the absorbance at 260 nm of tRNA mix (A260/mL tRNAmix) ranged from 0– to 20. The translation reaction was stopped after a 10-min incubation at 37 °C by addition of 2 M urea, and the sample was treated with RNase A. The translated products were separated on 16% Tris–tricine SDS–PAGE, and the fluorescence signal of CR110-XAF incorporated into the product was imaged using a fluorescence image scanner (Typhoon FLA-7000; GE Life sciences) with 473 nm excitation and 520 nm emission (Fig. 2B). Although a previous study showed production of full-length protein even in the absence of IleRS when A260/mL tRNAmix was higher than 5 [15], a clear main signal was observed at the position slightly higher than the 3.5-kDa marker protein at all the tRNA concentrations used here. Either the change from omission of IleRS to omission of TyrRS or the change from the original PURE system [24] to PUREfrexÒ, or both, resulted in lack of read-through at high tRNA concentrations. The translated product at A260/mL tRNAmix of 20, the tRNA concentration specified by the manufacturer, was purified using an anti-T7-tag antibody. Fig. 2C shows a mass spectrum of the translated product. A single mass signal was observed at 4021.9. This signal corresponds to the expected mass of a 29-residue peptide containing an adenosine moiety produced by stalling of translation elongation before the tyrosine codon (4022.3 with protonation). The adenosine remains after digestion of peptidyl-tRNA, which was retaining in the stalling ribosome, by RNase A. Since the ribosomes were synchronized at a specific position on the mRNA during the first reaction, the second reaction was started by addition of equal volume issue of second reaction mixture containing all requisite components, except the ribosome, into the first reaction. The second reaction mixture was pre-incubated at 37 °C for 10 min, and all experimental procedures were performed in
temperature-controlled room at 37 °C. To stop the translation elongation, the sample was quickly frozen in liquid nitrogen at the reaction time indicated, and urea at 4 °C was added to a final concentration of 2 M. After treatment with RNase A, samples were separated on 16% Tris–tricine SDS–PAGE (Fig. 3A). The signal at the starting position (Fig. 3A, indicated by S) gradually decreased and that at final position (Fig. 3A, indicated by F) increased as a function of time. Although the signal at the starting position did not completely disappear, likely due to inactivation of the ribosome during the first reaction, only the starting and final products were observed. Thus, we assumed a two-stage consecutive elongation reaction: kapp
S!F where S is product of the ribosome stalled before the tyrosine codon, F is the fully elongated product due to ribosomes that reached the 30 end of the mRNA, and kapp is apparent rate constant of translation elongation for the 12 amino acids (Fig. 2A). Degree of translation elongation was evaluated from fluorescence ratio, which is represented as:
ratio ¼ IF =ðIS þ IF Þ where IS and IF are fluorescence intensities at starting (S) and final (F) positions, respectively. The ratio values were plotted versus the second reaction time (Fig. 3B). kapp was calculated using a single exponential equation:
R ¼ A B ekapp
t
where R is fluorescence ratio at reaction time t, A is the fluorescence ratio when the reaction time is infinite, and B is amplitude of the ratio change. The average kapp value calculated from triplicate experiments was 0.20 ± 0.06 s1. From this kapp, t1/2 of the elongation of 12 amino acids was calculated as 3.42 s, indicating that the elongation velocity was 3.5 amino acids s1.
76
T. Endoh et al. / Methods 64 (2013) 73–78
Fig. 3. Time course of translation elongation of 12 amino acids. (A) Samples taken from the second reaction at indicated reaction times were separated on a 16% Tris– tricine SDS–PAGE. Signals due to the product from the first reaction (S) and final (F) product are indicated. (B) Fluorescence ratio of the signal at the final position related to the sum of fluorescence signals at the starting and the final positions were plotted vs. reaction time. Fit to the equation for two-stage consecutive reaction is shown in gray. Error bars are standard deviation from triplicate experiments.
Fig. 2. Artificial stalling of ribosome before tyrosine codon in the absence of TyrRS. (A) Sequence of basal mRNA. Underlined region is T7-tag sequence. Italicized sequence and asterisk indicate amber codon. In the first reaction, ribosomes are artificially stalled before the 30th codon encoding tyrosine (framed by dashed line) due to omission of tyrosyl-tRNA from the reaction mix. During the second reaction, 12 amino acids are elongated (arrowed line). (B) The first reaction products obtained in the presence of various tRNA concentrations. Translated products were separated on a 16% Tris–tricine SDS–PAGE, and fluorescence signals were imaged using FLA5100 fluorescence image scanner (Fuji Film) with 532 nm excitation and 575 nm emission. (C) Mass spectrometry of the first reaction product. Observed mass of the main signal is indicated. Sequence and calculated mass (mcalc) of the 29residue peptide with N-terminal formyl methionine (fMet), CR110-X-AF, and Cterminal adenosine are indicated.
5. Translational halt caused by G-quadruplex G-quadruplex provides unique structural property, which is stabilized by stacking a number of G-quartets consisting of four guanine bases that interact through Hoogsteen-type hydrogen bonds, and noteworthy stability in the presence of physiological salts concentrations [17]. In addition, we previously reported that the Gquadruplexes are further stabilized under cell-like molecular crowding and encapsulated conditions [26–28]. Thus, the G-quadruplexes on ORFs may suppress translation elongation mediated by their high thermodynamic stability and affect protein expression. We recently showed that gene expression from a reporter construct was suppressed in cells by the presence of a G-quadruplex in an ORF [29]. In addition, the protein expression pattern from human estrogen receptor a (hERa) mRNA was altered depending on the stability of a G-quadruplex formed within the ORF of the hERa mRNA [30]. Here, we evaluated translational halt caused by the G-quadruplex by analyzing the time course of translation elongation through regions with the potential to form the G-quadruplex structure with the synchronized translation assay (Fig. 4A). Reporter mRNAs termed + 0-mRNA to + 6-mRNA were prepared to evaluate effect of G-quadruplex position on the translational halt (Fig. 4B).
These mRNAs contain a G-rich sequence derived from the Escherichia coli eutE gene in various reading frames altered by insertion of one to six adenine nucleotides. Synchronized translation assay was performed using the reporter mRNAs. Clear fluorescence signals were observed from all reporters upon initiation of translation from the synchronized ribosomes. The signals observed at 20 s after restart were located at a slightly higher position in the gel (Fig. 4C, indicated by I) relative to the signal due to the peptide synthesized in the first step of the reaction (Fig. 4C, indicated by S). For certain reporters, fluorescence signal corresponding to translation of the entire message (Fig. 4C indicated by F) was observed at 5 min after restart. The fluorescence signals indicated by I are due to translational halt caused by RNA G-quadruplex formed by the G-rich sequence on the reporter mRNAs. These intermediate products were purified using anti-T7-tag antibody and analyzed by mass spectroscopy (Fig. 4D). In the case of all mRNAs, two main mass signals were observed in the purified products. One is the product corresponding to a 29-residue peptide, which shows a mass signal at 4022; this signal is due to inactivation of ribosome during the first reaction. The other mass signal depended on the position of the G-rich sequence: For the + 0-mRNA and + 1 mRNA the mass signal was observed at 5592; for the + 2-mRNA, +3-mRNA, and + 4-mRNA the signal was observed at 5720; and for the + 5-mRNA and + 6mRNA the signal was observed at 5847. These signals correspond to 42-, 43-, and 44-residues peptides containing an adenosine moiety, respectively, which have calculated mass of 5592.2, 5720.4, and 5848.5, respectively, with protonation. The positions of translational halts were all immediately before the G-rich sequence, at the codon 5–7 nucleotides before the first guanosine of the G-rich sequence (Fig. 4B).
6. Discussion and conclusion Synchronized translation assay was performed using a basal reporter mRNA to evaluate the rate of ribosomal progression in the elongation phase, and mRNAs containing a G-rich sequence were used to investigate the effect of RNA G-quadruplex formation on translation elongation. The rate of translation elongation calcu-
T. Endoh et al. / Methods 64 (2013) 73–78
77
Fig. 4. Translational halt caused by G-quadruplex formation in mRNA. (A) Schematic of synchronized translation assay using mRNA containing a G-rich sequence near the 30 end of the reporter mRNA. (B) Sequence design of +0- to +6-mRNAs containing the G-rich sequence in various reading frames. The G-rich sequence derived from Escherichia coli eutE gene is underlined. Inserted adenines are indicated in italic characters. (C) Synchronized translation products taken from the second reaction at indicated reaction times were separated on a 16% Tris–tricine SDS–PAGE. Signals at starting (S), intermediate (I), and final (F) product positions are indicated. (D) Products isolated from 20-s translation elongation reactions were analyzed by mass spectroscopy. Observed mass of the main signal and position of translational halt are indicated.
lated using the basal reporter mRNA was 3.5 amino acids s1 (Fig. 3B). Since this value corresponds well to the velocity previously estimated using an in vitro translation reaction [11], the ribosomes that restart translation elongation maintain expected properties. In the basal reporter mRNA, the 12 codons elongated at the second reaction do not contain rare codons. By changing these codons to the rare codons, it would be possible to quantitatively evaluate the effect of rare codons on the translation elongation rate. Time course analyses of translation elongation using mRNAs designed to have the G-rich sequence in various reading frames indicated that G-quadruplex formation halted the ribosome at the codon having 5, 6, or 7 spacer nucleotides between the first guanosine of the G-rich sequence (Fig. 4). This positional regularity is likely caused by ribosome dynamics during the translation elongation that the ribosome progresses three nucleotides at a time in every translocation reaction. The 5–7 spacer nucleotides correspond to the number of nucleotides between A-site codon and mRNA entry site of ribosome [31,32]. Highly thermodynamic stability of G-quadruplex was considered to resist helicase activity of ribosome and halts the translation elongation by blocking the mRNA entry into the ribosome. The synchronized translation assay would be an appropriate method for evaluating effects of other RNA structures, such as duplexes and pseudoknots, on the translation elongation.
Foundation at Private Universities (2009–2014), Japan, Nagase Science and Technology Foundation, and the Hirao Taro Foundation of the Konan University Association for Academic Research.
Acknowledgements
[21]
This work was supported in part by Grants-in-Aid for Scientific Research, MEXT-Supported Program for the Strategic Research
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20]
[22] [23]
M. Stoneley, A.E. Willis, Oncogene 23 (2004) 3200–3207. A. Serganov, D.J. Patel, Curr. Opin. Struct. Biol. 22 (2012) 279–286. J.E. Barrick, R.R. Breaker, Genome Biol. 8 (2007) R239. M. Kozak, Proc. Natl. Acad. Sci. USA 83 (1986) 2850–2854. M. Kozak, Mol. Cell. Biol. 9 (1989) 5134–5142. G. Kudla, A.W. Murray, D. Tollervey, J.B. Plotkin, Science 324 (2009) 255–258. D.P. Giedroc, P.V. Cornish, Virus Res. 139 (2009) 193–208. M.K. Doma, R. Parker, Nature 440 (2006) 561–564. Y. Harigaya, R. Parker, RNA 1 (2010) 132–141. A.A. Komar, Trends Biochem. Sci. 34 (2009) 16–24. G. Zhang, M. Hubalewska, Z. Ignatova, Nat. Struct. Mol. Biol. 16 (2009) 274– 280. E.P. O’Brien, M. Vendruscolo, C.M. Dobson, Nat. Commun. 3 (2012) 868. B.S. Laursen, H.P. Sorensen, K.K. Mortensen, H.U. Sperling-Petersen, Microbiol. Mol. Biol. Rev. 69 (2005) 101–123. S. Takahashi, M. Iida, H. Furusawa, Y. Shimizu, T. Ueda, Y. Okahata, J. Am. Chem. Soc. 131 (2009) 9326–9332. T. Endoh, Y. Kawasaki, N. Sugimoto, Anal. Chem. 84 (2012) 857–861. J.R. Williamson, Annu. Rev. Biophys. Biomol. Struct. 23 (1994) 703–730. G.W. Collie, G.N. Parkinson, Chem. Soc. Rev. 40 (2011) 5867–5892. S. Millevoi, H. Moine, S. Vagner, RNA 3 (2012) 495–507. D. Gomez, A. Guedin, J.L. Mergny, B. Salles, J.F. Riou, M.P. Teulade-Fichou, P. Calsou, Nucleic Acids Res. 38 (2010) 7187–7198. V. Marcel, P.L. Tran, C. Sagne, G. Martel-Planche, L. Vaslin, M.P. Teulade-Fichou, J. Hall, J.L. Mergny, P. Hainaut, E. Van Dyck, Carcinogenesis 32 (2010) 271–278. P.H. Wanrooij, J.P. Uhler, T. Simonsson, M. Falkenberg, C.M. Gustafsson, Proc. Natl. Acad. Sci. USA 107 (2010) 16072–16077. A. Bugaut, S. Balasubramanian, Nucleic Acids Res. 40 (2012) 4727–4741. C.J. Noren, S.J. Anthony-Cahill, M.C. Griffith, P.G. Schultz, Science 244 (1989) 182–188.
78
T. Endoh et al. / Methods 64 (2013) 73–78
[24] Y. Shimizu, A. Inoue, Y. Tomari, T. Suzuki, T. Yokogawa, K. Nishikawa, T. Ueda, Nat. Biotechnol. 19 (2001) 751–755. [25] Y. Shimizu, T. Kanamori, T. Ueda, Methods 36 (2005) 299–304. [26] D. Miyoshi, H. Karimata, N. Sugimoto, J. Am. Chem. Soc. 128 (2006) 7957– 7963. [27] S. Pramanik, S. Nagatoishi, N. Sugimoto, Chem. Commun. 48 (2012) 4815– 4817. [28] D. Miyoshi, T. Fujimoto, N. Sugimoto, Top. Curr. Chem. 330 (2012) 87–110.
[29] T. Endoh, Y. Kawasaki, N. Sugimoto, Angew. Chem. Int. Ed. 52 (2013) 5522– 5526. [30] T. Endoh, Y. Kawasaki, N. Sugimoto, Nucleic Acids Res. First published online April 24, 2013, http://dx.doi.org/10.1093/nar/gkt286. [31] L.B. Jenner, N. Demeshkina, G. Yusupova, M. Yusupov, Nat. Struct. Mol. Biol. 17 (2010) 555–560. [32] L. Jenner, P. Romby, B. Rees, C. Schulze-Briese, M. Springer, C. Ehresmann, B. Ehresmann, D. Moras, G. Yusupova, M. Yusupov, Science 308 (2005) 120–123.
Contents lists available at SciVerse ScienceDirect
Methods journal homepage: www.elsevier.com/locate/ymeth
Translational halt during elongation caused by G-quadruplex formed by mRNA Tamaki Endoh a, Yu Kawasaki b, Naoki Sugimoto a,b,⇑ a b
Frontier Institute for Biomolecular Engineering Research (FIBER), Konan University, Kobe, Japan Faculty of Frontiers of Innovative Research in Science and Technology (FIRST), Konan University, Kobe, Japan
a r t i c l e
i n f o
Article history: Available online 7 June 2013 Keywords: Synchronized translation Translation elongation mRNA structure G-quadruplex Kinetic analysis
a b s t r a c t mRNA forms various secondary and tertiary structures that affect gene expression. Although structures formed in the untranslated regions (UTRs) of mRNAs that inhibit translation have been characterized, stable mRNA structures in open reading frames (ORFs) may also cause translational halt or slow translation elongation. We previously established a method, termed a synchronized translation assay, that enables time course analysis of single turnover translation elongation. In this method, translation initiation, which is a rate determining step of the translation procedure, can be ignored because all ribosomes are synchronized on a specific position of mRNA before translation elongation is restarted from this position. In this paper, we used the synchronized translation assay to evaluate the effects of a G-quadruplex structure located at various positions within the mRNA ORF on translational halt. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction During translation, the ribosome, a large RNA–protein complex, polymerizes amino acids based on the sequence encoded in the open reading frame (ORF) of an mRNA to produce functional proteins. mRNAs are able to form various intramolecular secondary and tertiary structures that contribute to the regulation of the translation process. For example, internal ribosomal entry sites (IRESs) allow translation initiation in the absence of initiation factors and riboswitches in 50 untranslated regions (UTRs) of mRNAs regulate translation through RNA conformational transitions in response to metabolites [1–3]. In addition to these functional mRNA structures that control translation through interaction with small molecules or proteins, some RNA structures with high thermodynamic stability, which are either naturally formed or artificially inserted into UTRs or ORFs, suppress ribosome progression [4–6]. Inhibition of the ribosome in its scanning phase by stable RNA structures in 50 UTRs may decrease protein expression, whereas stable structure in the ORF may contribute various biological processes such as ribosomal frameshift [7], no-go mRNA decay [8,9], which is one of the recently discovered mRNA surveillance mechanisms, and co-translational folding of nascent proteins [10–12]. Thus, analyses of mRNA structures that suppress the translation elongation will further expand our understanding of the contributions of mRNA structures to the translation reaction. ⇑ Corresponding author at: Frontier Institute for Biomolecular Engineering Research (FIBER), Konan University, Kobe, Japan. E-mail address: [email protected] (N. Sugimoto). 1046-2023/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ymeth.2013.05.026
The translation reaction can be divided into three processes: initiation, elongation, and termination. It has been suggested that the rate determining step of the translation reaction is initiation, which requires formation of initiation complex formed by several protein factors [13,14]. Thus, if an in vitro translation reaction is stopped at a certain reaction time, the position of ribosomes on the mRNA is heterogeneous depending on the time when the ribosome initiated the translation reaction. To analyze the elongation procedure in detail, the translation reaction must be synchronized to enable time course analysis of translation elongation. We previously reported a procedure for the synchronized translation in a single turnover reaction [15]. Ribosomes were artificially stalled at a specific position of mRNA after initiation of translation reaction, and translation elongation was subsequently restarted. This should be appropriate method for evaluating effects of RNA structures in mRNA ORF on translation elongation. A G-quadruplex is one of the non-canonical structures of nucleic acids formed by guanine rich (G-rich) regions of RNA or DNA with at least four consecutive stretches of more than two guanines [16,17]. Recent research works have been focused on those formed within non-coding regions that impact various biological procedures, such as transcription, mRNA splicing, and translation initiation [18–21]. Especially, G-quadruplexes in ORFs of oncogene mRNAs suppress protein expression from the mRNAs depending on the stability of the quadruplexes by inhibition of the scanning of the ribosome [22]. There is a little information about effect of G-quadruplexes within mRNA ORFs on translation reaction in despite of the G-rich sequences of both non-coding and coding regions of mRNAs would form the G-quadruplex structure. Here,
74
T. Endoh et al. / Methods 64 (2013) 73–78
we evaluated the effects of a G-quadruplex structure on translational halt by using the synchronized translation assay. 2. mRNA design for the synchronized translation To enable the analysis of translation elongation over a time course in a single turnover reaction the mRNA shown in Fig. 1A was designed. This mRNA has three important features: (i) A T7-tag, MASMTGGQQMG, is encoded at N-terminus of the amino acid sequence (Fig. 1A, indicated in blue). This enables purification of translated products that begin from the correct start codon using anti-T7 tag agarose (MBL). Mass spectroscopy can then be used to determine lengths of the protein products to infer where the translation elongation was stalled (Fig. 1B, mass diagram). (ii) A UAG amber codon was inserted after the nucleotide sequence encoding the T7-tag (Fig. 1A, indicated in green). Although the amber codon normally encodes termination of translation, we can incorporate non-natural amino acids into the protein by using amber suppressor tRNA aminoacylated with a non-natural amino acid [23]. Incorporation of the non-natural fluorescent amino acid (CR110-X-AF) using the amber suppressor tRNA (CR110-X-AF-tRNAamber; Protein Express) enables site specific labeling of the translated product (Fig. 1B, green frame) and time-course analysis of translation elongation after separation on SDS–PAGE. (iii) The stop codon at 30 terminus was removed from the mRNA. This prevents turnover of ribosome and ensures a singleturnover reaction. 3. Two-step synchronized translation reaction In the synchronized translation assay, two-step reaction procedure using in vitro translation based on protein synthesis using recombinant elements (PURE) system [24,25], which enables omission of specific protein components required for translation from reaction mixture, was designed to synchronize the ribosomes. The procedure is shown schematically in Scheme 1.
In the first reaction, a specific aminoacyl-tRNA synthetase (aaRS) is missing from the reaction mixture. The lack of this aaRS, which results in omission of a specific aminoacyl-tRNA, artificially stalls all ribosomes at a specific position of mRNA (Fig. 1B, first reaction). Release factor (RF) 1 and RF2 are also missing from the first reaction mixture. Amber suppressor tRNA competes with RF1 during normal translation reactions. Thus, lack of RF1 and RF2 enables efficient incorporation of the non-natural amino acid and prevents undesired termination of translation. In the second reaction, the reaction mixture containing all requisite components except ribosome is added to the first reaction to restart the translation elongation (Fig. 1B, second reaction). In order to ensure that any initiation of translation that may occur during the second reaction by backward ribosomes does not mask detection of single-turnover elongation, RF1 is added to the second reaction mix. Addition of RF1 ensures that translation initiated during the second reaction is terminated at the amber codon inserted after the T7-tag (Fig. 1A).
4. Time course analysis of elongation of 12 amino acids Our previous study indicated that the presence of rare codons in the mRNA temporarily halt translation elongation [15]. To analyze the translational halt caused by the mRNA structures, an mRNA must be designed without rare codons prior to the mRNA structure of interest. The mRNA sequence in Fig. 2A was designed as a basal mRNA to study the effect of mRNA structure on the translation elongation. In the previous study, isoleucyl-tRNA synthetase (IleRS) was removed from the reaction mixture to artificially stall the ribosome before an isoleucine codon at the first reaction. Since a portion of ribosomes seemed to cause a + 1 frameshift during the stalling at the first reaction [15], here we removed tyrosyl-tRNA synthetase (TyrRS) from the reaction mixture instead of the IleRS to artificially stall the ribosome before 30th codon encoding tyrosine (Fig. 2A). Addition of reaction mixture containing TyrRS restarts the translation elongation from the tyrosine codon, and the elongation finishes in single turnover at the 30 end of the mRNA following elongation of 12 amino acids (Fig. 2A). In addition to the change in the removed aaRS, PUREfrexÒ (GeneFrontier; http://
Fig. 1. Schematic of synchronized translation assay. (A) General design of mRNA for the synchronized translation. Amino acid sequence of T7-tag for purification of translated product and UAG amber codon for incorporation of non-natural fluorescent amino acid are indicated in blue and green, respectively. (B) In the first reaction, all ribosomes incorporate CR110-X-AF (a non-natural fluorescent amino acid) and stall at a specific codon due to the omission of release factor (RF) and a specific aminoacyl-tRNA synthetase (aaRS). In the second reaction, upon addition of the missing aaRS, all ribosomes restart translation elongation from the stalled position. Translated products are subsequently detected based on the fluorescence signal of CR110-X-AF by SDS–PAGE or by mass spectrometry.
75
T. Endoh et al. / Methods 64 (2013) 73–78
Scheme 1. General experimental flow of the synchronized translation assay.
www.genefrontier.com/english/index.html), a reconstructed in vitro translation system derived from improved purification process of requisite tRNA, ribosome and proteins, was utilized. The customized reaction mixture, in which the TyrRS, RF1, and RF2 were removed, was provided from GeneFrontier Corp. The first reaction was performed in the absence of TyrRS, RF1, and RF2 with various concentrations of tRNA, in which the absorbance at 260 nm of tRNA mix (A260/mL tRNAmix) ranged from 0– to 20. The translation reaction was stopped after a 10-min incubation at 37 °C by addition of 2 M urea, and the sample was treated with RNase A. The translated products were separated on 16% Tris–tricine SDS–PAGE, and the fluorescence signal of CR110-XAF incorporated into the product was imaged using a fluorescence image scanner (Typhoon FLA-7000; GE Life sciences) with 473 nm excitation and 520 nm emission (Fig. 2B). Although a previous study showed production of full-length protein even in the absence of IleRS when A260/mL tRNAmix was higher than 5 [15], a clear main signal was observed at the position slightly higher than the 3.5-kDa marker protein at all the tRNA concentrations used here. Either the change from omission of IleRS to omission of TyrRS or the change from the original PURE system [24] to PUREfrexÒ, or both, resulted in lack of read-through at high tRNA concentrations. The translated product at A260/mL tRNAmix of 20, the tRNA concentration specified by the manufacturer, was purified using an anti-T7-tag antibody. Fig. 2C shows a mass spectrum of the translated product. A single mass signal was observed at 4021.9. This signal corresponds to the expected mass of a 29-residue peptide containing an adenosine moiety produced by stalling of translation elongation before the tyrosine codon (4022.3 with protonation). The adenosine remains after digestion of peptidyl-tRNA, which was retaining in the stalling ribosome, by RNase A. Since the ribosomes were synchronized at a specific position on the mRNA during the first reaction, the second reaction was started by addition of equal volume issue of second reaction mixture containing all requisite components, except the ribosome, into the first reaction. The second reaction mixture was pre-incubated at 37 °C for 10 min, and all experimental procedures were performed in
temperature-controlled room at 37 °C. To stop the translation elongation, the sample was quickly frozen in liquid nitrogen at the reaction time indicated, and urea at 4 °C was added to a final concentration of 2 M. After treatment with RNase A, samples were separated on 16% Tris–tricine SDS–PAGE (Fig. 3A). The signal at the starting position (Fig. 3A, indicated by S) gradually decreased and that at final position (Fig. 3A, indicated by F) increased as a function of time. Although the signal at the starting position did not completely disappear, likely due to inactivation of the ribosome during the first reaction, only the starting and final products were observed. Thus, we assumed a two-stage consecutive elongation reaction: kapp
S!F where S is product of the ribosome stalled before the tyrosine codon, F is the fully elongated product due to ribosomes that reached the 30 end of the mRNA, and kapp is apparent rate constant of translation elongation for the 12 amino acids (Fig. 2A). Degree of translation elongation was evaluated from fluorescence ratio, which is represented as:
ratio ¼ IF =ðIS þ IF Þ where IS and IF are fluorescence intensities at starting (S) and final (F) positions, respectively. The ratio values were plotted versus the second reaction time (Fig. 3B). kapp was calculated using a single exponential equation:
R ¼ A B ekapp
t
where R is fluorescence ratio at reaction time t, A is the fluorescence ratio when the reaction time is infinite, and B is amplitude of the ratio change. The average kapp value calculated from triplicate experiments was 0.20 ± 0.06 s1. From this kapp, t1/2 of the elongation of 12 amino acids was calculated as 3.42 s, indicating that the elongation velocity was 3.5 amino acids s1.
76
T. Endoh et al. / Methods 64 (2013) 73–78
Fig. 3. Time course of translation elongation of 12 amino acids. (A) Samples taken from the second reaction at indicated reaction times were separated on a 16% Tris– tricine SDS–PAGE. Signals due to the product from the first reaction (S) and final (F) product are indicated. (B) Fluorescence ratio of the signal at the final position related to the sum of fluorescence signals at the starting and the final positions were plotted vs. reaction time. Fit to the equation for two-stage consecutive reaction is shown in gray. Error bars are standard deviation from triplicate experiments.
Fig. 2. Artificial stalling of ribosome before tyrosine codon in the absence of TyrRS. (A) Sequence of basal mRNA. Underlined region is T7-tag sequence. Italicized sequence and asterisk indicate amber codon. In the first reaction, ribosomes are artificially stalled before the 30th codon encoding tyrosine (framed by dashed line) due to omission of tyrosyl-tRNA from the reaction mix. During the second reaction, 12 amino acids are elongated (arrowed line). (B) The first reaction products obtained in the presence of various tRNA concentrations. Translated products were separated on a 16% Tris–tricine SDS–PAGE, and fluorescence signals were imaged using FLA5100 fluorescence image scanner (Fuji Film) with 532 nm excitation and 575 nm emission. (C) Mass spectrometry of the first reaction product. Observed mass of the main signal is indicated. Sequence and calculated mass (mcalc) of the 29residue peptide with N-terminal formyl methionine (fMet), CR110-X-AF, and Cterminal adenosine are indicated.
5. Translational halt caused by G-quadruplex G-quadruplex provides unique structural property, which is stabilized by stacking a number of G-quartets consisting of four guanine bases that interact through Hoogsteen-type hydrogen bonds, and noteworthy stability in the presence of physiological salts concentrations [17]. In addition, we previously reported that the Gquadruplexes are further stabilized under cell-like molecular crowding and encapsulated conditions [26–28]. Thus, the G-quadruplexes on ORFs may suppress translation elongation mediated by their high thermodynamic stability and affect protein expression. We recently showed that gene expression from a reporter construct was suppressed in cells by the presence of a G-quadruplex in an ORF [29]. In addition, the protein expression pattern from human estrogen receptor a (hERa) mRNA was altered depending on the stability of a G-quadruplex formed within the ORF of the hERa mRNA [30]. Here, we evaluated translational halt caused by the G-quadruplex by analyzing the time course of translation elongation through regions with the potential to form the G-quadruplex structure with the synchronized translation assay (Fig. 4A). Reporter mRNAs termed + 0-mRNA to + 6-mRNA were prepared to evaluate effect of G-quadruplex position on the translational halt (Fig. 4B).
These mRNAs contain a G-rich sequence derived from the Escherichia coli eutE gene in various reading frames altered by insertion of one to six adenine nucleotides. Synchronized translation assay was performed using the reporter mRNAs. Clear fluorescence signals were observed from all reporters upon initiation of translation from the synchronized ribosomes. The signals observed at 20 s after restart were located at a slightly higher position in the gel (Fig. 4C, indicated by I) relative to the signal due to the peptide synthesized in the first step of the reaction (Fig. 4C, indicated by S). For certain reporters, fluorescence signal corresponding to translation of the entire message (Fig. 4C indicated by F) was observed at 5 min after restart. The fluorescence signals indicated by I are due to translational halt caused by RNA G-quadruplex formed by the G-rich sequence on the reporter mRNAs. These intermediate products were purified using anti-T7-tag antibody and analyzed by mass spectroscopy (Fig. 4D). In the case of all mRNAs, two main mass signals were observed in the purified products. One is the product corresponding to a 29-residue peptide, which shows a mass signal at 4022; this signal is due to inactivation of ribosome during the first reaction. The other mass signal depended on the position of the G-rich sequence: For the + 0-mRNA and + 1 mRNA the mass signal was observed at 5592; for the + 2-mRNA, +3-mRNA, and + 4-mRNA the signal was observed at 5720; and for the + 5-mRNA and + 6mRNA the signal was observed at 5847. These signals correspond to 42-, 43-, and 44-residues peptides containing an adenosine moiety, respectively, which have calculated mass of 5592.2, 5720.4, and 5848.5, respectively, with protonation. The positions of translational halts were all immediately before the G-rich sequence, at the codon 5–7 nucleotides before the first guanosine of the G-rich sequence (Fig. 4B).
6. Discussion and conclusion Synchronized translation assay was performed using a basal reporter mRNA to evaluate the rate of ribosomal progression in the elongation phase, and mRNAs containing a G-rich sequence were used to investigate the effect of RNA G-quadruplex formation on translation elongation. The rate of translation elongation calcu-
T. Endoh et al. / Methods 64 (2013) 73–78
77
Fig. 4. Translational halt caused by G-quadruplex formation in mRNA. (A) Schematic of synchronized translation assay using mRNA containing a G-rich sequence near the 30 end of the reporter mRNA. (B) Sequence design of +0- to +6-mRNAs containing the G-rich sequence in various reading frames. The G-rich sequence derived from Escherichia coli eutE gene is underlined. Inserted adenines are indicated in italic characters. (C) Synchronized translation products taken from the second reaction at indicated reaction times were separated on a 16% Tris–tricine SDS–PAGE. Signals at starting (S), intermediate (I), and final (F) product positions are indicated. (D) Products isolated from 20-s translation elongation reactions were analyzed by mass spectroscopy. Observed mass of the main signal and position of translational halt are indicated.
lated using the basal reporter mRNA was 3.5 amino acids s1 (Fig. 3B). Since this value corresponds well to the velocity previously estimated using an in vitro translation reaction [11], the ribosomes that restart translation elongation maintain expected properties. In the basal reporter mRNA, the 12 codons elongated at the second reaction do not contain rare codons. By changing these codons to the rare codons, it would be possible to quantitatively evaluate the effect of rare codons on the translation elongation rate. Time course analyses of translation elongation using mRNAs designed to have the G-rich sequence in various reading frames indicated that G-quadruplex formation halted the ribosome at the codon having 5, 6, or 7 spacer nucleotides between the first guanosine of the G-rich sequence (Fig. 4). This positional regularity is likely caused by ribosome dynamics during the translation elongation that the ribosome progresses three nucleotides at a time in every translocation reaction. The 5–7 spacer nucleotides correspond to the number of nucleotides between A-site codon and mRNA entry site of ribosome [31,32]. Highly thermodynamic stability of G-quadruplex was considered to resist helicase activity of ribosome and halts the translation elongation by blocking the mRNA entry into the ribosome. The synchronized translation assay would be an appropriate method for evaluating effects of other RNA structures, such as duplexes and pseudoknots, on the translation elongation.
Foundation at Private Universities (2009–2014), Japan, Nagase Science and Technology Foundation, and the Hirao Taro Foundation of the Konan University Association for Academic Research.
Acknowledgements
[21]
This work was supported in part by Grants-in-Aid for Scientific Research, MEXT-Supported Program for the Strategic Research
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20]
[22] [23]
M. Stoneley, A.E. Willis, Oncogene 23 (2004) 3200–3207. A. Serganov, D.J. Patel, Curr. Opin. Struct. Biol. 22 (2012) 279–286. J.E. Barrick, R.R. Breaker, Genome Biol. 8 (2007) R239. M. Kozak, Proc. Natl. Acad. Sci. USA 83 (1986) 2850–2854. M. Kozak, Mol. Cell. Biol. 9 (1989) 5134–5142. G. Kudla, A.W. Murray, D. Tollervey, J.B. Plotkin, Science 324 (2009) 255–258. D.P. Giedroc, P.V. Cornish, Virus Res. 139 (2009) 193–208. M.K. Doma, R. Parker, Nature 440 (2006) 561–564. Y. Harigaya, R. Parker, RNA 1 (2010) 132–141. A.A. Komar, Trends Biochem. Sci. 34 (2009) 16–24. G. Zhang, M. Hubalewska, Z. Ignatova, Nat. Struct. Mol. Biol. 16 (2009) 274– 280. E.P. O’Brien, M. Vendruscolo, C.M. Dobson, Nat. Commun. 3 (2012) 868. B.S. Laursen, H.P. Sorensen, K.K. Mortensen, H.U. Sperling-Petersen, Microbiol. Mol. Biol. Rev. 69 (2005) 101–123. S. Takahashi, M. Iida, H. Furusawa, Y. Shimizu, T. Ueda, Y. Okahata, J. Am. Chem. Soc. 131 (2009) 9326–9332. T. Endoh, Y. Kawasaki, N. Sugimoto, Anal. Chem. 84 (2012) 857–861. J.R. Williamson, Annu. Rev. Biophys. Biomol. Struct. 23 (1994) 703–730. G.W. Collie, G.N. Parkinson, Chem. Soc. Rev. 40 (2011) 5867–5892. S. Millevoi, H. Moine, S. Vagner, RNA 3 (2012) 495–507. D. Gomez, A. Guedin, J.L. Mergny, B. Salles, J.F. Riou, M.P. Teulade-Fichou, P. Calsou, Nucleic Acids Res. 38 (2010) 7187–7198. V. Marcel, P.L. Tran, C. Sagne, G. Martel-Planche, L. Vaslin, M.P. Teulade-Fichou, J. Hall, J.L. Mergny, P. Hainaut, E. Van Dyck, Carcinogenesis 32 (2010) 271–278. P.H. Wanrooij, J.P. Uhler, T. Simonsson, M. Falkenberg, C.M. Gustafsson, Proc. Natl. Acad. Sci. USA 107 (2010) 16072–16077. A. Bugaut, S. Balasubramanian, Nucleic Acids Res. 40 (2012) 4727–4741. C.J. Noren, S.J. Anthony-Cahill, M.C. Griffith, P.G. Schultz, Science 244 (1989) 182–188.
78
T. Endoh et al. / Methods 64 (2013) 73–78
[24] Y. Shimizu, A. Inoue, Y. Tomari, T. Suzuki, T. Yokogawa, K. Nishikawa, T. Ueda, Nat. Biotechnol. 19 (2001) 751–755. [25] Y. Shimizu, T. Kanamori, T. Ueda, Methods 36 (2005) 299–304. [26] D. Miyoshi, H. Karimata, N. Sugimoto, J. Am. Chem. Soc. 128 (2006) 7957– 7963. [27] S. Pramanik, S. Nagatoishi, N. Sugimoto, Chem. Commun. 48 (2012) 4815– 4817. [28] D. Miyoshi, T. Fujimoto, N. Sugimoto, Top. Curr. Chem. 330 (2012) 87–110.
[29] T. Endoh, Y. Kawasaki, N. Sugimoto, Angew. Chem. Int. Ed. 52 (2013) 5522– 5526. [30] T. Endoh, Y. Kawasaki, N. Sugimoto, Nucleic Acids Res. First published online April 24, 2013, http://dx.doi.org/10.1093/nar/gkt286. [31] L.B. Jenner, N. Demeshkina, G. Yusupova, M. Yusupov, Nat. Struct. Mol. Biol. 17 (2010) 555–560. [32] L. Jenner, P. Romby, B. Rees, C. Schulze-Briese, M. Springer, C. Ehresmann, B. Ehresmann, D. Moras, G. Yusupova, M. Yusupov, Science 308 (2005) 120–123.