Selective Binding to mRNA Duplex Regions by Chemically Modified

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Selective Binding to mRNA Duplex Regions by Chemically Modified PNAs Stimulates Ribosomal Frameshifting Ru Ying Puah, Huan Jia, Manikantha Maraswami, Desiree-Faye Kaixin Toh, Rya Ero, Lixia Yang, Kiran M. Patil, Alan Ann Lerk Ong, Manchugondanahalli S. Krishna, Ruimin Sun, Cailing Tong, Mei Huang, Xin Chen, Teck-Peng Loh, Yonggui Gao, Ding Xiang Liu, and Gang Chen Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00744 • Publication Date (Web): 08 Nov 2017 Downloaded from http://pubs.acs.org on November 9, 2017

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Selective Binding to mRNA Duplex Regions by Chemically Modified PNAs Stimulates Ribosomal Frameshifting

Ru Ying Puah,†,1 Huan Jia,†,1 Manikantha Maraswami,†,1 Desiree-Faye Kaixin Toh,†,1 Rya Ero,‡,1 Lixia Yang,† Kiran M. Patil,† Alan Ann Lerk Ong,† Manchugondanahalli S. Krishna,† Ruimin Sun,⊥ Cailing Tong,† Mei Huang,‡ Xin Chen,⊥ Teck Peng Loh,† Yong-Gui Gao,‡ Ding Xiang Liu,‡, #,* and Gang Chen†,*

†

Division of Chemistry and Biological Chemistry, School of Physical and Mathematical

Sciences, Nanyang Technological University, 21 Nanyang Link, Singapore 637371 ‡

School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive,

Singapore 637551 ⊥

Division of Mathematical Sciences, School of Physical and Mathematical Sciences,

Nanyang Technological University, 21 Nanyang Link, Singapore 637371 #

South China Agricultural University, Guangdong Province Key Laboratory Microbial

Signals & Disease Co, and Integrative Microbiology Research Centre, Guangzhou 510642, Guangdong, People’s Republic of China.

Corresponding Authors: Emails: [email protected] or [email protected] 1

These authors contributed equally to this work. 1 ACS Paragon Plus Environment

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Abstract Minus-one programmed ribosomal frameshifting (–1 PRF) allows the precise maintenance of the ratio between viral proteins, and is involved in the regulation of the half-lives of cellular mRNAs. Minus-one ribosomal frameshifting (–1 RF) is activated by several stimulatory elements such as a heptameric slippery sequence (X XXY YYZ) and an mRNA secondary structure (hairpin or pseudoknot) that is positioned 2-8 nucleotides downstream from the slippery site. Upon –1 RF, the ribosomal reading frame is shifted from the normal zero frame to the –1 frame with the heptameric slippery sequence decoded as XXX YYY Z instead of X XXY YYZ. Our research group has developed chemically-modified Peptide Nucleic Acid (PNA) L and Q monomers to recognize G-C and C-G Watson–Crick base pairs, respectively, through major-groove parallel PNA·RNA-RNA triplex formation. L- and Q-incorporated PNAs show selective binding to double-stranded RNAs (dsRNAs) over single-stranded RNAs (ssRNAs). The sequence specificity and structural selectivity of L- and Q-modified PNAs may allow the precise targeting of desired viral and cellular RNA structures, and thus may serve as valuable biological tools for mechanistic studies and potential therapeutics for fighting diseases. Here, for the first time, we demonstrate by cell-free in vitro translation assays using rabbit reticulocyte lysate that the dsRNA-specific chemically-modified PNAs targeting model mRNA hairpins stimulate –1 RF (from 2% to 32%). An unmodified control PNA, however, shows nonspecific inhibition of translation. Our results suggest that the modified dsRNA-binding PNAs may be advantageous for targeting structured RNAs.

2 ACS Paragon Plus Environment

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Introduction Minus-one programmed ribosomal frameshifting (–1 PRF) is a translational recoding mechanism that is employed by many viruses and organisms ranging from prokaryotes to higher eukaryotes.1-3 Minus-one ribosomal frameshifting (–1 RF) is activated by a heptameric slippery sequence (X XXY YYZ) and an mRNA secondary structure that is positioned 2-8 nucleotides (nt) downstream from the slippery site. In viruses and eukaryotes, X, Y, and Z are usually any bases, A or U, and non-G bases, respectively.1 The ribosomal reading frame is shifted, upon –1 RF, from a normal zero frame to a –1 frame with the two codons at the slippery site decoded as XXX and YYY, respectively, instead of XXY and YYZ (Figure 1a). Previous research suggests that –1 RF at a slippery site may be stimulated by a downstream hairpin, pseudoknot, G-quadruplex, or a duplex formed between an antisense oligonucleotide and mRNA.4-17 A downstream mRNA secondary structure causes blockage at the entrance of the mRNA tunnel, hampering the rotation of the ribosomal subunits with respect to each other and therefore, promotes the backward movement of the ribosome by one nucleotide at the slippery site.2, 3, 18-24 Minus-one PRF is often highly regulated with a narrow window of recoding efficiency for a specific virus (e.g., 5% for HIV-1), which allows the precise maintenance of the ratio between viral proteins.2, 3, 25 Either decreasing or increasing the –1 RF may affect viral replication. For example, HIV-1 mutants with an enhanced –1 RFstimulatory mRNA hairpin local stability and thus –1 RF efficiencies are significantly less infectious.26 In addition to being essential for the replication of many viruses, –1 RF is also involved in the regulation of the half-lives of cellular mRNAs.2, 27

The cis-acting –1 RF-stimulatory viral mRNA hairpin or pseudoknot structures are often highly conserved, and thus, are potential target sites for the regulation of –1 RF.28-33 The 3 ACS Paragon Plus Environment

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structural stability of the mRNA secondary structures may be critical for stimulating –1 RF.4, 26, 34-36

Thus, RNA structure-binding ligands may be developed to modulate the structural

stability and –1 RF efficiency.7, 10, 17, 29, 30, 32, 33, 37, 38 In addition, development of site-specific RNA-binding ligands may facilitate a deeper understanding of the complex –1 RF processes. For example, viral mRNA pseudoknots found in beet western yellows virus (BWYV) and murine leukaemia virus (MLV) show protonation dependent pseudoknot structure stabilization, with the fraction of a protonated C or A residue (with near-neutral pKa values and corresponding binding affinities of µM) correlates with the frequency of the translational recoding events.38 Small molecule ligand binding (with binding affinity on the order of nM to µM) to riboswitch aptamer structure also stimulates –1 RF, presumably through the stabilization of the riboswitch aptamer structures.7, 17

Peptide Nucleic Acid (PNA) is a type of artificial nucleic acid, which consists of an uncharged 2-aminoethyl glycine unit instead of the phosphodiester backbone, and a methylene carbonyl linker instead of a cyclic sugar moiety (Figure 2).39-46 PNA’s non-ionic and flexible backbone allows it to form Watson–Crick duplexes with a complementary RNA or DNA in both parallel and antiparallel manners, with significantly enhanced stability compared to DNA/RNA duplexes. Furthermore, PNA has a lower sensitivity towards ionic strength changes, and is resistant to nucleases and proteases.45 Thus, there is a great potential in developing PNAs as chemical probes and potential therapeutic agents.


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Figure 1. Scheme of p2luc dual-luciferase system and PNAs for in vitro translation assays. (a) Experimental mRNA constructs with the insertion between the RLuc and FLuc genes of three model sequences: RNA hairpin 1 (rHP1), ssRNA, and rHP2. The slippery sequence (U UUU UUA) is the same as that found at the HIV-1 frameshift site and is underlined. The 0 frame stop codon (UAG) is shown in orange. Minus-one RF results in the production of both RLuc (0 frame) and FLuc (–1 frame) proteins. The PNA sequences aligned with the targeted regions of the mRNAs are shown. For free rHP1 and rHP2, the hairpin stem is located three nucleotides downstream of the slippery sequence. The PNA·RNA-RNA triplexes, however, are located eight nucleotides downstream of the slippery sequence. An 8-nt spacer is found at the HIV-1 frameshift site. (b) mRNA constructs serving as positive controls for 100% –1 RF efficiency. The control mRNA constructs contain the same hairpins (rHP1 and rHP2) or ssRNA as indicated. The slippery sequences were mutated (indicated by green color) so as to have non-slippery sequences and to have FLuc translated in 0 frame.


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Figure 2. Chemical structures of stable PNA·RNA-RNA base triples and unstable PNA-RNA base pairs. The letter “R” indicates the sugar-phosphate backbone of RNA. (a-d) Chemical structures of triples C+·G-C, T·A-U, L·G-C, and Q·C-G. (e,f) Unstable Watson–Crick-like base pairs of L-G and Q-G.


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Table 1. Sequences of synthesized PNAs and the previously reported47, 48 RNA-binding properties.

PNA Sequence

Tm (°C) (parallel duplex)a

Tm (°C) (antiparallel duplex)a

KD (µM) binding to rHP1b

KD (µM) binding to rHP2b

P3

NH2-Lys-TLTQTTTL-CONH2

10 µM, we observed non-specific inhibition of translation (see Figure S2).


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Figure 4. Effects of PNAs on the expression of FLuc and RLuc. The expression levels are normalized. (a) PNA P3 selectively enhances the expression of FLuc of the experimental mRNA rHP2 construct. (b) PNA P5 selectively enhances the expression of FLuc of the experimental mRNA rHP1 construct. (c) Unmodified PNA P1 shows non-selective inhibition of the expression of both FLuc and RLuc. (d,e) Plots of normalized FLuc and RLuc expression levels versus fractions of mRNA hairpin bound by dsRNA-binding PNA. The fraction of mRNA hairpin bound by PNA is calculated assuming the binding reaches an equilibrium.


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We tested if pre-incubation of PNA with mRNA would affect the efficacy of –1 RF stimulation. We observed that even without the 20-min pre-incubation, P3 and P5 are still able to induce –1 RF of rHP2 and rHP1, respectively (Figure S3). The results are consistent with the fact that PNA·RNA-RNA triplex formation is relatively fast,59,

60

and the two

modified PNAs (P3 and P5) activate –1 RF during the multiple turnovers of translation elongation. We also tested if the PNAs affect the activities of the luciferases by adding the PNAs after the translation reactions were completed. For all the mRNAs, upon the posttranslational addition of the PNAs (modified PNAs P3 and P5 and unmodified PNA P1), we observed no significant changes in the –1 RF efficiency (Figure S4), and the expression levels of RLuc and FLuc (Figure S5). Thus, the unmodified and modified PNAs show no direct effect on the luciferase activities. Taken together, our results verify that the changes in –1 RF efficiency (Figure 3) result from the specific interactions between the mRNA hairpins and the dsRNA-binding modified PNAs (Figure 1).

Unmodified PNAs are known to be able to bind to complementary ssRNAs and partially structured RNAs through strand invasion.61 Thus, compared to our chemically-modified PNAs, unmodified PNAs may show less selectivity and specificity in binding to structured RNAs. For example, the unmodified PNA P1 (Figure 1) can bind to rHP1 (Figure 2a,b, Table 1).47,

48

In addition, PNA P1 can form parallel and antiparallel duplexes with 5ʹ-

AGAGAAAG-3ʹ and 3ʹ-AGAGAAAG-5ʹ, respectively (Table 1). PNA P1, however, shows no binding to rHP2 (Table 1). We tested the effect of PNA P1 on –1 RF. As illustrated in Figure 4c, for all mRNAs (ssRNA, rHP1, and rHP2), with increasing concentration of unmodified PNA P1 added, the expression levels of both FLuc and RLuc drop significantly to baseline level. It is possible that PNA P1 inhibits translation by binding to rRNAs and/or coding regions of Renilla/firefly luciferase genes. By examining the sequences of Homo 16 ACS Paragon Plus Environment

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sapiens ribosomal RNAs (rRNAs) (similar to rabbit rRNAs)62 and p2luc vector, several possible off-target sites containing sequences similar to 5ʹ-AGAGAAAG-3ʹ and 3ʹAGAGAAAG-5ʹ were identified (Figure 5 and Appendix in Supporting Information). We used the secondary structures of human rRNAs because (i) the secondary structure of the large subunit rabbit rRNA is not available,62 (ii) human rRNAs are very similar to rabbit rRNAs, and (iii) the near-atomic resolution structure of the human ribosome has recently become available.63, 64

Out of the three predicted binding regions in 18S rRNA (Figure 5), two (HS1 and HS3) are buried within the 40S subunit and likely not accessible to P1 PNA (Figure S6). In contrast, HS2 region is located at the back of 40S neck and almost entirely exposed to solvent (Figure 6a and Figure S6) making it a likely binding site for PNA P1. Given that the dynamics (rolling and swiveling of 40S head) of the neck region is highly important for the efficiency and fidelity of translocation, P1 PNA interaction with HS2 region could explain the nonspecific inhibition of translation.

The potential P1 PNA binding sites (six altogether) of 28S rRNA (Figure 5) exhibit varied structural contexts in ribosome (Figure S6). While the HL1 region is not far from GTPase activation site of the ribosome, it is buried within 60S subunit. In contrast, HL2 region is solvent accessible yet far away from ribosomal regions directly linked to translation. Another potential P1 binding site, HL5, while surrounded by 60S structure, is located close to 40S in a region likely involved in inter-subunit communication during translation.


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Figure 5. Potential off-target binding sites of unmodified PNA P1 in homo sapiens rRNAs. The identified sequences are similar to RNA 5ʹ-AGAGAAAG-3ʹ (shown in red) or RNA 3ʹAGAGAAAG-5ʹ (shown in blue) and may form parallel or antiparallel PNA-RNA duplexes with unmodified PNA P1, respectively.


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Curiously, three regions (HL3, HL4, and HL6) are located in the vicinity of the nascent peptide exit tunnel and are at least partially accessible to PNA P1 (Figure 6b,c and Figure S6). HL3 region is located adjacent to the tunnel exit on solvent side of 60S, and PNA P1 binding could affect trans factors involved in nascent protein folding or transport. HL4 aligns the inside of the tunnel, with the tunnel diameter of 15-20 Å. Thus, even with PNA bound, there is likely enough room for peptide chain to pass. However the dynamic interactions with the tunnel are believed to facilitate protein folding, so the effect could be more indirect. HL6 is situated close to the tunnel entrance as well as the peptidyl transferase center at the subunit interface side (Figure 6b,c). The diameter of the ribosome exit tunnel in the vicinity of HL6 is about 10 Å. Thus, PNA binding to HL6 could physically hinder nascent chain proceeding through the tunnel, as this part is the narrowest region of the tunnel and even small molecules like antibiotics are known to affect nascent chain tunnel interactions. Furthermore, HL6 region is likely involved in the stabilization of 28S rRNA U4452 residue (Figure 6b) that has been proposed63 to function as a gatekeeper to correctly orient the early growing nascent peptide.

Taken together, it is highly probable that the nonspecific inhibition of translation by P1 is due to duplex formation between P1 and the identified rRNA regions, resulting in the obstruction of ribosomal functions.65, 66 In addition, binding of P1 to the coding regions of RLuc gene (see Appendix in Supporting Information) may affect the ribosomal translocation on mRNA. Thus, for the PNAs tested here, the chemically-modified dsRNA-binding PNAs show more specific binding with reduced off-target binding.


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Figure 6. Potential binding sites of unmodified PNA P1 in human ribosome 3D structure. Predicted P1 PNA binding sites were mapped onto H. sapiens 80S ribosome structure solved by cryo-electron microscopy (PDB ID: 5T2C).64 Figures were generated using PyMol Molecular Graphics System, Schrödinger, LLC. (a) Close-up view (from the 40S solvent side) of the 18S rRNA HS2 sequence predicted to interact with P1 PNA. HS2 sequence is shown in red sticks. 40S proteins in the vicinity of HS2 are indicated. SA stands for 40S ribosomal protein SA. (b,c) Close-up views of the 28S rRNA HL6, HL4, and HL3 sequences (shown in red sticks) predicted to interact with P1 PNA from the 60S intersubunit and solvent side, respectively. 28S rRNA U4452 residue located at the entrance of the nascent peptide exit tunnel is shown in magenta sticks. 5.8S rRNA is shown in yellow cartoon. 60S proteins in the vicinity of predicted P1 binding rRNA sequences are also indicated. Asterisk marks the nascent peptide exit tunnel through the 60S subunit.

The identified potential binding sites in rRNAs for the unmodified PNA P1 are essentially single-stranded, and may not be recognized by chemically-modified PNAs P3 and P5 because P3 and P5 show selective binding towards dsRNAs over ssRNAs (Table 1).47 Consistently, a less prominent reduction was observed in the RLuc readings upon the addition of P3 and P5 (Figure 4). Our data indicate that chemically-modified PNAs P3 and P5 site-specifically bind to rHP2 and rHP1, respectively, with reduced off-target binding to ribosome or other regions of mRNA.


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To rule out the possibility that mRNAs degrade upon the addition of PNAs, we carried out agarose gel electrophoresis of the model mRNAs in the absence and presence of PNAs. The gel results reveal no mRNA degradation upon the incubation with the PNAs (P3, P5, and P1) (Figure S1). Taken together, our results suggest that, compared to unmodified PNAs, the Qand L-modified dsRNA-binding PNAs are superior in sequence-specifically and selectively targeting the dsRNA regions of structured RNAs.

Conclusions In summary, our results suggest that relatively short chemically-modified dsRNA-binding PNAs may be used to probe the complex ribosomal translation processes. Formation of a major-groove parallel PNA·RNA-RNA triplex may stimulate –1 RF due to the stabilization of mRNA duplex structures, and/or the interference of the direct interactions of mRNA duplex at the entrance tunnel with the ribosome at the slippery site. The chemically-modified PNAs platform may be applied in future to the targeting of viral RNA structures to uncover the roles of cis-acting mRNA structures in stimulating –1 PRF. To allow the production of a protein in the downstream –1 frame, the PNA·RNA-RNA triplex needs to be unwound for continued translation in –1 frame. It is known that an overly stable mRNA structure may block translation instead of stimulate –1 RF.67 Thus, we expect that the stimulation of –1 RF and other translational regulations will be highly dependent on the sequence, length, and binding position of the dsRNA-binding PNAs. Future structural and mechanistic studies will help develop dsRNA-binding PNAs with further improved efficacy and selectivity in stimulating –1 RF. Our work also provides insights into future development of highly selective and potent PNAs targeting rRNA structures. With the advances in PNA delivery technologies,47, 68-79 our work on the model mRNAs may also serve as a basis for the future 21 ACS Paragon Plus Environment

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development of antivirals targeting viral mRNA structures that stimulate –1 PRF, and therapeutics targeting many other biomedically-important RNAs.

Associated Content Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Detailed experimental protocols and list of DNA oligonucleotides used. Agarose gel data (Figure S1), Nonspecific inhibition of translation observed for PNAs P3 and P5 at > 10 µM (Figure S2), frameshifting assay data without mRNA-PNA pre-incubation (Figure S3), minus-one RF efficiency data with PNAs added after the in vitro translation reactions (Figure S4), relative RLuc and FLuc activities with the PNAs added after the in vitro translation reactions (Figure S5), overview of the distribution and location of the putative P1 PNA binding sites in the ribosome (Figure S6), detailed sequences of the plasmids containing segments similar to 5ʹ-AGAGAAAG-3ʹ and 3ʹ-AGAGAAAG-5ʹ, and secondary structures of rRNAs.

Author Information Corresponding Authors [email protected] and [email protected] ORCID: Gang Chen: 0000-0002-8772-9755 Notes 22 ACS Paragon Plus Environment

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The authors declare no competing financial interest.

Acknowledgement This work was supported by the grants from Singapore Ministry of Education (MOE) Tier 1 (RGT3/13 and RG42/15 to G.C.) and MOE Tier 2 (MOE2013-T2-2-024 and MOE2015-T21-028 to G.C., and MOE2014-T2-1-083 to Y.-G.G.). We thank Prof Sam Butcher for sharing the experimental protocols, and for providing the p2luc plasmids which was originally constructed by Profs John Atkins and Raymond Gesteland, Prof Robin Gutell for sharing the secondary structures of rRNAs, and Prof Rene Olsthoorn for critically reading the manuscript.

References

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For Table of Contents Use Only


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Figure 1 190x160mm (300 x 300 DPI)

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Figure 2 190x220mm (300 x 300 DPI)


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Figure 3 190x145mm (300 x 300 DPI)


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Figure 4 190x189mm (300 x 300 DPI)


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Figure 5 190x219mm (300 x 300 DPI)


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(b)


(c)

60S

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1 +d ACS Plus Environment sRN bi Paragon 3 Zero frame nding PNAMinus-one frame A CC UUU UUU AGG G 4CCU UUU UUA GGG

2 40S

Selective Binding to mRNA Duplex Regions by Chemically Modified

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