Characterization of the multimeric structure of poly(A)-binding protein on a poly(A) tail

Eukaryotic mature mRNAs possess a poly adenylate tail (poly(A)), to which multiple molecules of poly(A)-binding protein C1 (PABPC1) bind. PABPC1 regulates translation and mRNA metabolism by binding to regulatory proteins. To understand functional mechanism of the regulatory proteins, it is necessary to reveal how multiple molecules of PABPC1 exist on poly(A). Here, we characterize the structure of the multiple molecules of PABPC1 on poly(A), by using transmission electron microscopy (TEM), chemical cross-linking, and NMR spectroscopy. The TEM images and chemical cross-linking results indicate that multiple PABPC1 molecules form a wormlike structure in the PABPC1-poly(A) complex, in which the PABPC1 molecules are linearly arrayed. NMR and cross-linking analyses indicate that PABPC1 forms a multimer by binding to the neighbouring PABPC1 molecules via interactions between the RNA recognition motif (RRM) 2 in one molecule and the middle portion of the linker region of another molecule. A PABPC1 mutant lacking the interaction site in the linker, which possesses an impaired ability to form the multimer, reduced the in vitro translation activity, suggesting the importance of PABPC1 multimer formation in the translation process. We therefore propose a model of the PABPC1 multimer that provides clues to comprehensively understand the regulation mechanism of mRNA translation.

consideration of the multimerized PABPC1 structure. Whereas a number of the regulatory proteins simultaneously associate with multimeric PABPC1 on poly(A), it remains elusive which PABPC1 molecules interact with each of the regulatory proteins and whether the interactions are cooperative, competitive, or independent. Therefore, to elucidate the mechanisms of translational regulation associated with these PABPC1-binding proteins, it is necessary to know the structure of the PABPC1 multimerized on poly(A), which has remained unsolved.
In the present study, we physicochemically characterized the multimerized structure of the poly(A)-bound PABPC1 by using transmission electron microscopy (TEM), chemical cross-linking, and nuclear magnetic resonance (NMR) spectroscopy. The molecular basis of the multimer formation of the poly(A)-bound PABPC1 multimer revealed here contributes to an understanding how multiple and various PABPC1-binding proteins bind to either of the PABPC1 molecules on poly(A), as this is the state in which the subsequent functions are realized.

Results
Transmission electron microscopy of (multiple) PABPC1-poly(A) complex. Human PABPC1 was expressed in E. coli, and purified to homogeneity. The purified protein exhibited poly(A)-binding affinity with a dissociation constant of 6.9 × 10 −10 M (Supplementary Figure 1a, and Table 1), which is consistent with a previous report 11 .
To characterize how the PABPC1 molecules associate with poly(A), we observed negatively stained poly(A)-bound PABPC1 using a transmission electron microscope (TEM). Figure 2a,b clearly show segmented, wormlike structures. In the enlarged image in Fig. 2c, a number of particles is linearly arrayed. The two-dimensional class averaged image of PABPC1-poly(A) complex resulted in a clearer image shown in Fig. 2d,
Chemical cross-linking of the poly(A)-bound PABPC1 molecules. We further characterized the PABPC1 molecules on poly(A) by chemical cross-linking experiments. As one PABPC1 molecule reportedly covers 23-27 bases of poly(A) 6,11 , we prepared poly(A) RNAs comprising A 24 , A 48 , and A 72 , corresponding to the lengths that are covered by one, two, and three PABPC1 molecules. In addition, a long poly(A), in which the length of 70% of the poly(A) ranges from 150 to 500 bases, was prepared. The concentration corresponding to a 24-base unit of poly(A), c(A 24 ), was adjusted to 1.0 μM in all the samples, which is half of the PABPC1 concentration of 2.0 μM. We used a cross-linker, BS(PEG) 9 , which cross-links two amino groups of the lysine sidechains. As shown in Fig. 3a, the proteins were observed as a smear in the presence of BS(PEG) 9 , probably owing to heterogeneous chemical modification. The smear probably occurred because different numbers of the lysine residues on the molecular surface of PABPC1 were modified by BS(PEG) 9 . The molecular masses of the smears were 70-90, 140-200, and 210-260 kDa, in the presence of A 24 , A 48 , and A 72 , respectively, and there was a much larger mass for the long poly(A). This correlation between the size of the cross-linked molecules and the length of the poly(A) strongly suggests that multiple PABPC1 molecules on the same poly(A) chain form a one-dimensional multimer, which is consistent with the TEM images shown in Fig. 2.

Stabilization of the PABPC1 multimer by the intermolecular interactions between the linker-PABC and the RRMs.
Whereas the RRM region of PABPC1 is responsible for the poly(A) binding 11 , the intermolecular interactions forming the PABPC1 multimer on poly(A) reportedly involve the linker-PABC region 9,11 . Here, we compared the ability to form a multimer on poly(A) between full length PABPC1 and RRM1/2/3/4, which lacks the linker-PABC region, by chemical cross-linking. It should be noted that the RRM1/2/3/4 prepared here exhibited a binding affinity for poly(A) that is comparable to that of the full length PABPC1 (Table 1 and Supplementary Figure 1b).
We prepared several solutions containing 2.0 μM PABPC1 or RRM1/2/3/4 in the presence of increasing amounts of the long poly(A) wherein the molar ratios of a 24-base unit in the long poly(A) over PABPC1 or RRM1/2/3/4 were 1:1, 2:1, 3:1, 4:1, 8:1, and 16:1. As all the PABPC1 or RRM1/2/3/4 molecules were associated with poly(A) at these concentrations, the increase in the molar ratio of [PABPC1 or RRM1/2/3/4]/[A 24 unit of poly(A)] leads to a "one-dimensional dilution" of PABPC1 or RRM1/2/3/4 on poly(A). Figure 3b and d show the SDS-PAGE results after cross-linker treatment, which indicate that RRM1/2/3/4 provides an apparently stronger monomer band as compared to full length PABPC1. The populations of the monomer and the multimer at each poly(A) concentration are plotted in Fig. 3c and e. Whereas approximately 70% of the PABPC1 molecules form a multimer at 16-fold dilution (Fig. 3c), only 63% of the RRM1/2/3/4 molecules form a multimer even at 1-fold dilution, with 8-fold dilution mostly precluding the intermolecular cross-linking of RRM1/2/3/4 (Fig. 3e). These results indicate that the linker and/or PABC regions are crucial for the PABPC1-PABPC1 interactions that are responsible for multimer formation.
As shown in Fig. 3a, however, only poly(A)-bound PABPC1 molecules were cross-linked, whereas no cross-linking was observed in the absence of poly(A), strongly suggesting that multimerization is not due to the mutual intermolecular interaction of the linker-PABC regions that are not involved in the poly(A) binding. Therefore, the direct interactions crucial for the multimer formation are considered to occur between the poly(A)-bound RRM1/2/3/4 region of one PABPC1 molecule and the linker-PABC region of another.
A middle portion of the linker region (residues 428-442) is responsible for the interaction with poly(A)-bound RRM1/2/3/4. To identify which portion of the linker-PABC region is responsible for the intermolecular interactions with poly(A)-bound RRM1/2/3/4, we observed NMR spectra of a number of 15 N-labelled peptide fragments of PABPC1 containing a portion of the linker and PABC (residues 371-430, referred to as Lt1; residues 421-470, Lt2; residues 461-510, Lt3; residues 501-543, Lt4; residues 541-636, PABC) (Fig. 4a), in the absence or presence of unlabeled RRM1/2/3/4 in complex with long poly(A) RNA with a length of 300 to 5,000 bases. Owing to the large size of the multiple molecules of RRM1/2/3/4 bound to the long poly(A), severe line-broadening of the NMR signals of the 1 H-15 N NSQC spectrum was expected upon association of the 15 N-labelled linker or PABC with the huge complex of RRM1/2/3/4-poly(A). Figure 4 and Supplementary Figure 2 show the superposition of the spectra of each PABPC1 region in the absence (black) and presence (red) of RRM1/2/3/4-poly(A). In Fig. 4b, 33 NMR signals for Lt2 exhibited significant line broadening upon addition of RRM1/2/3/4-poly(A), whereas only 9 NMR signals remained observed (Fig. 4b), clearly indicating that Lt2 binds to the poly(A)-bound RRM1/2/3/4. In contrast, the NMR signals for Lt1, Lt3, Lt4, or PABC showed no or subtle spectral changes upon addition of RRM1/2/3/4-poly(A) (Fig. 4c, and Supplementary Figure 2). This indicates that Lt2 contains a region that associates with poly(A)-bound RRM1/2/3/4. NMR resonance assignments of the Lt2 signals (Supplementary Table 1) revealed that the signals for most Lt2 residues (residues 427 to 470) except for the N and C terminal 9 residues were broadened ( Fig. 4b), presumably owing to the increase in the rotational correlation time upon binding to the huge RRM1/2/3/4-poly(A) complex. To identify interacting Lt2 residues while avoiding severe line-broadening, we performed NMR titration experiments using a RRM1/2/3/4-A 24 complex, which possesses a much smaller molecular weight than the long poly(A)-bound multiple RRM1/2/3/4. Figure 4d shows an overlay of a series of 1 H-15 N HSQC spectra of 15 N-labelled Lt2 upon the sequential addition of RRM1/2/3/4-A 24 . Curve fitting of the chemical shift changes for the signals from W437 and T438 using a 1:1 binding model resulted in the K d values ranging from 10 to 54 μM (Fig. 4e). Although the NMR spectral change did not saturate during the titration, the chemical shift differences between free Lt2 and Lt2 (100 μM) in the presence of RRM1/2/3/4-A 24 (150 μM) plotted in Fig. 4f indicate that the signal for W437 exhibited the largest chemical shift change, and that the 15-residue portion from I428 to A442 exhibited significant chemical shift changes. These data strongly suggest that the a 15-residue portion containing W437 is responsible for binding to RRM1/2/3/4 in the poly(A)-bound PABPC1. It should be noted that no typical secondary structure was predicted for Lt2 by its chemical shift values [27][28][29][30]  We then tried to identify which of RRM1 or RRM2 acts as the linker binding domain. As the binding affinity of RRM1 or RRM2 for poly(A) is not sufficiently high to form a stable complex with poly(A), we evaluated the intermolecular paramagnetic relaxation enhancement (PRE) effects on Lt2 of a spin label introduced to RRM1 or RRM2 in RRM1/2/3/4-poly(A). When some of the residues of 15 N-labelled Lt2 approaches within 20 Å of the spin-labelled residue on RRM1 or RRM2 in A 24 -bound RRM1/2/3/4, NMR signals of the Lt2 residues are broadened and reduce their intensities by the PRE effect, leading to the identification which of RRM1 or RRM2 bind to Lt2 (Supplementary Figure 4a). It should be noted that the residues to which the spin labels (MTSL) are chemically attached (E29C in RRM1 or D117C in RRM2) exist at the corresponding positions in each RRM (Supplementary Figure 4b).
We observed HSQC spectra of 15 N-labelled Lt2 in the presence of the A 24 -bound, spin-labelled RRM1/2/3/4, in both the radical (paramagnetic) form and reduced (diamagnetic) form of MTSL. The PRE effects were evaluated by the signal intensity ratios in the presence of the radical (paramagnetic) form and the reduced (diamagnetic) form, by assuming constant intrinsic R 2 values among all the Lt2 residues. If the spin label of the radical form of MTSL lay within approximately 20 Å from the amide group of Lt2, the amide NMR signal of Lt2 would be broadened by the PRE effect, thus reducing signal intensity. Figure 5c shows that almost no intensity reduction by PRE was observed following use of the spin-labelled RRM1. Conversely, significant intensity reductions (<0.5) were observed for the signals of A429, S434, R436, W437, A439, and R443 in the sample containing spin-labelled RRM2, representing the PRE effects on these residues (Fig. 5d). Based on these results, we concluded that the linker residues in the Lt2 region bind to RRM2.
The Lt2 region plays an important role in PABPC1 multimerization on poly(A). We next investigated the role of the interactions of the linker region in the multimerization of PABPC1. Chemical cross-linking experiments were performed with a deletion mutant of PABPC1 that lacks the Lt2 region (PABPC1ΔLt2) as well as for another mutant that lacks the Lt4 region (PABPC1ΔLt4) as a control, in the same manner as shown in Fig. 3b- where   (Fig. 6b). These values are lower than those for PABPC1 (97, 86, and 69%, respectively, Fig. 3c), but markedly higher than those of RRM1/2/3/4 (63, 28, and 7%, respectively, Fig. 3e). Conversely, the value for PABPC1ΔLt4 were 83, 74, and 67%, respectively (Fig. 6d), which are comparable to those for PABPC1.
These results indicate that the Lt2 region largely contributes to the formation of the PABPC1 multimer on poly(A). As deletion of the Lt4 region, which is the same length as Lt2, showed much less effect on the

PABPC1 multimerization is important for cap/poly(A)-dependent translation. It has been
reported that PABPC1 on the poly(A) tail of mRNA enhances translation in a cap/poly(A)-dependent manner 31 . Here, we investigated the effects of PABPC1 multimerization on cap/poly(A)-dependent translation by using PABPC1ΔLt2 as a multimerization-deficient mutant in a cell-free translation system with a nuclease-treated rabbit reticulocyte lysate (RRL).
The translation system established here exhibited significant enhancement of translation in the presence of both cap and poly(A), whereas almost no enhancement was observed in the absence of a 5′-cap (Fig. 7a). Next, we depleted endogenous PABPC1 by using GST-Paip2 (Fig. 7b) 31 , and then added PABPC1ΔLt2, which possesses impaired multimerization activity. Addition of full length PABPC1 or PABPC1ΔLt4 rather than PABPC1ΔLt2 was also performed as control experiments.
Depletion of endogenous PABPC1 reduced the translation of mRNA with a cap/poly(A) to 1/3 of the rate before the depletion. Conversely, the addition of full-length PABPC1 enhanced the translation by 2.5 fold, representing 80% of that before the depletion, suggesting that the incomplete recovery of translation activity reflects the difference between endogenous rabbit PABPC1 and the recombinant human PABPC1 that was added to the system. Although the addition of PABPC1ΔLt4 exhibited essentially the same enhancement of translation as that from the full-length PABPC1, addition of PABPC1ΔLt2 showed only 1.7-fold enhancement, which is significantly smaller than that obtained with the full-length PABPC1 or PABPC1ΔLt4 (Fig. 7).
In contrast, the translation of mRNA without poly(A), which occurs at approximately 40% the rate of mRNA possessing a poly(A) (Fig. 7c), was reduced to 75% of that following endogenous PABPC1 depletion (Fig. 7d). This reduced translation activity was unchanged upon addition of full-length PABPC1, PABPC1ΔLt2, or PABPC1ΔLt4.
These results indicate that cap/poly(A)-dependent translation is suppressed by deletion of the Lt2 region of poly(A)-bound PABPC1, which is a critical region for PABPC1 multimerization on poly(A). Therefore, the PABPC1 multimer on poly(A) is suggested to play important roles in translation.

Discussion
To date, the structure of multiple PABPC1 molecules bound to a long poly(A) chain has remained unsolved, owing in part to its huge molecular size and the heterogeneity in poly(A) length and thus in the number of PABPC1 molecules bound to the poly(A). Here, we first visualized the PABPC1-poly(A) complex as a TEM image, in which several PABPC1 molecules are linearly arrayed, forming a wormlike structure (Fig. 2). This one-dimensional array is consistent with the chemical cross-linking results showing a graded increase in the size of the cross-linked PABPC1 molecules in response to the increase in the number of the A 24 units in poly(A), which is the length of poly(A) recognized by a single PABPC1 molecule 6,11 (Fig. 3a). Furthermore, the chemical cross-linking experiments with the one-dimensional dilution of PABPC1 on poly(A) clearly indicated that the poly(A)-bound PABPC1 molecule interacts with the PABPC1 molecules on the same poly(A) chain (Fig. 3b).
The analyses for the NMR spectral changes revealed that the intermolecular interactions responsible for PABPC1 multimerization occur between RRM2 of one molecule and a 15-residue portion of the linker region containing W437 (residues 428-442) of another PABPC1 molecule on poly(A) (Figs 4 and 5). The largest chemical shift change was observed for W437, and the largest intensity reduction by PRE from the spin label on D117 in RRM2 was observed for the sidechain of W437. Furthermore, the Lt2 region possesses no typical secondary structure in the free state, as predicted by chemical shift values (Supplementary Figure 3). Therefore, it is strongly suggested that W437 plays the most important role in the multimerization of PABPC1 on poly(A). The PABPC1 mutant lacking the Lt2 region (PABPC1ΔLt2) exhibited markedly less ability to form a multimer (Fig. 6), confirming that the Lt2 region contains a portion contributing to multimer formation. To date, several crystal and NMR structures of RRM from other proteins in complex with a Trp-containing peptide have been reported, where a Trp sidechain is inserted into a cleft formed by two helices of RRM on the surface of RRM that is opposite to the RNA-binding surface [32][33][34][35][36] (Supplementary Figure 5a). In the PABPC1 linker region, W437 is the only Trp residue, which seems to be why the W437-containing Lt2 region is critical for the multimer formation of PABPC1 on poly(A). It should be noted that the Trp-binding site of the RRM from other proteins correspond to the eIF4G binding site on RRM2 of PABPC1 (Supplementary Figure 5b), which is proximal to D117 that is spin-labelled in the PRE experiment.
Based on the results of cross-linking showing one-dimensional array, and NMR results demonstrating that the intermolecular interactions between RRM2 and the Lt2 region, particularly, residues 428-442 that contribute to the multimer formation, a schematic of the repeating nature of the PABPC1 multimer is shown in Fig. 8. The direction of RRM1/2/3/4 to poly(A) is given according to that of RRMs 1 and 2 to poly(A) in the crystal structure 37 , where RRMs 1 and 2 bind to the 3′ and 5′ sides of poly(A), respectively. Although RRM1/2/3/4 might adopt certain structure in complex with poly(A), it is depicted in a linear fashion, as the structure of RRMs 3 and 4 in the poly(A)-bound state remains unknown.
The K d value for binding of Lt2 to RRM1/2/3/4-A 24 was estimated to be less than 100 μM, where a protein concentration as high as 1 mM is necessary to saturate the binding. However, as the Lt2 is tethered to the C-terminus of RRM4, the apparent Lt2 concentration is markedly high in the periphery of the RRM4. Considering the 70-residue long unstructured region tethering the Lt2 region to the C-terminus of RRM4, with a reported separation of 7.0 nm 38 , the apparent concentration of the Lt2 existing within a sphere with a radius of 7.0 nm can be estimated as 1.2 mM, based on the following formula: This apparent high concentration of the Lt2 region of poly(A)-bound PABPC1 enables the Lt2 region to bind to the RRM2 in the neighbouring PABPC1 bound to the same poly(A) chain, and thus to form a multimer.
When the wild-type PABPC1 was substituted in the RRL by PABPC1ΔLt2, which possesses an impaired ability to form a multimer on poly(A), translation was effectively suppressed (Fig. 7). This result suggests that PABPC1 multimerization is important in the translation system. It has been reported that translation is further enhanced as the length of the poly(A) increases 39 , which appears to be related to the increase in the number of PABPC1 molecules in the multimer bound to the poly(A) chain.
PABPC1 is known to bind a wide range of proteins such as the translation initiation factor eIF4G 14 , translation termination factor eRF3 15 , PABP-interacting protein 1 (Paip1) 16 and 2 (Paip2) 17 that enhance and suppress translation, subunits in the deadenylase complex Tob-Caf1 19 , and Pan2-Pan3 20 . The respective binding sites have also been identified: RRM2 for eIF4G, PABC for eRF3 and deadenylase complexes, and RRMs and PABC for Paip1 and Paip2. It should be noted that to our best knowledge, no factors have yet been reported to bind to the Lt2 region.
Although it remains unknown how PABPC1 multimerization enhances translation, it is likely to increase the binding affinity for PABPC1-binding proteins that affect the translation activity. In the multimer, the multiple binding sites of a particular target protein are exposed on the multimer surface at locally high concentrations, which enhances the rebinding of proteins that dissociate from one of the PABPC1 molecules. This effect decreases the apparent k off value, through the rebinding of the dissociated molecules to the neighbouring sites, resulting in an increase in binding affinity. Accordingly, in our previous report, we showed that the binding affinity of a PABC-binding peptide derived from eRF3 for PABC of the multiple PABPC1 molecules bound to a long poly(A) chain was 50-fold higher than that for a monomeric PABC 26 .
Notably, proteases from polio virus 40,41 , Coxsackie virus 42 , and HIV 43 digest the linker region of PABPC1, resulting in the removal of PABC, which is the binding site for translational factors such as eRF3. Our results suggest that the digestion of the linker by the viral proteases significantly impairs the capacity for multimerization, which appears to represent another mechanism underlying the translation suppression mediated by these viruses.
In summary, we have physicochemically characterized the structure of multiple PABPC1 molecules bound to a long poly(A) chain as constituting a one-dimensional wormlike form. We have identified the interactions responsible for PABPC1 multimerization, which occur at residues in the linker region and the RRM2, and shown that such interaction is important for translational activity. The molecular basis of the interactions related to the poly(A)-bound PABPC1 multimer revealed here contributes to an understanding how the various PABPC1-binding proteins regulate translation and mRNA degradation on the poly(A)-bound PABPC1.
Protein purification details are described in the Supplementary methods. Briefly, the GST-fusion proteins were expressed in Escherichia coli cells and purified using a Glutathione-Sepharose 4B column (GE Healthcare), followed by digestion by factor Xa (Novagen) or PreScission Protease (GE Healthcare). The cleaved GST and the non-cleaved fusion proteins were removed by using a Glutathione-Sepharose 4B column. The bound nucleic acids were removed via a cation exchange column (HiTrap SP HP, GE Healthcare), followed by gel filtration using HiLoad 16/600 Superdex 75 pg (GE Healthcare).
Lt1, Lt2, Lt3, and Lt4 were further purified by high-performance liquid chromatography (Shimadzu Corp., Kyoto, Japan) using a reverse-phase C18 column (GL Sciences, Tokyo, Japan). The molecular weights of Lt1, Lt2, Lt3, and Lt4 were confirmed by MALDI-TOF mass spectrometry, using an AXIMA-CFR Plus mass spectrometer (Shimadzu Corp.  The samples were adsorbed onto thin carbon films supported by copper mesh grids, which were rendered hydrophilic in advance by glow discharge under low air pressure. The PABPC1-poly(A) complexes were negatively stained with EM stainer (Nisshin EM, Tokyo, Japan), blotted, and dried in air. Samples were observed using a JEM1230 transmission electron microscope (JEOL, Tokyo, Japan) at an acceleration voltage of 100 kV. Images were recorded using a TVIPS F114T CCD camera (Oslo, Norway).
For 2D classification, a total of 193 boxed images were manually extracted from 39 independently recorded CCD images with 128 by 128 pixel size using BOXER software in EMAN2 44 . We applied eight rounds of reference free 2D-class averaging using EMAN2 and they were classified into six classes. NMR analyses. Data were collected on Bruker Avance 400, 500, or 600 spectrometers using triple-resonance or cryogenic probes. All spectra were processed using Bruker TopSpin 3.5 software, and the data were analysed with Sparky (T. D. Goddard and D. G. Kneller, Sparky 3, University of California, San Francisco, CA). Titrations of 15 N-labelled Lt1, Lt2, Lt3, Lt4, and PABC with unlabelled RRM1/2/3/4, RRM1/2, and RRM3/4 in the presence of long poly(A) or A 24 were monitored by 1 H-15 N heteronuclear single quantum coherence (HSQC) spectra at 303 K in a buffer containing 26 mM NaH 2 PO 4 (pH 6.0), 100 mM NaCl, and 8% 2 H 2 O.
Chemical shift changes were calculated using the following equation 45 : where Δ 1H and Δ 15N are the chemical shift changes in the 1 H and 15 N dimensions, respectively. Sequential assignments of the backbone NMR resonances of Lt2 were achieved by HNCACB, CBCA(CO)NH, C(CO)NH, HN(CA)CO, HNCO, and 15 N-edited TOCSY-HSQC experiments at 303 K in the buffer containing 26 mM NaH 2 PO 4 (pH 6.0), 100 mM NaCl, 0.5 mM ethylenediaminetetraacetic acid (EDTA), and 8% 2 H 2 O.
In vitro translation assay. Luciferase mRNAs with or without an A 72 tail were synthesized with T7 RNA polymerase after linearization of pBK-5F-Luc-pA with BsmBI or XbaI, respectively. pBK-5F-Luc-pA was generated by inserting the following three PCR fragments into the XhoI and ManHI site of pBluescript II SK(-): 5xFlag-tag that was amplified using the primer pair NH733/NH124 and pCMV-5xFlag 46 as a template, luciferase cDNA that was amplified using the primer pair NH363/NH373 and pGL4.16 (Promega) as a template, and β-globin 3'UTR that contains 72 nts of poly(A) tail that was amplified using the primer pair NH734/NH770 and pFlag-CMV/TO-BGG 18 as a template. To synthesize capped mRNAs, 3′-O-Me-m 7 G(5′) pppG (Stratagene, Santa Clara, CA, USA) was used.
One-way analysis of variance (ANOVA) followed by the Tukey test was used to evaluate differences among the four groups. Differences were considered to be significant at P < 0.05. Data availability. All data generated or analysed during this study are included in this published article and its Supplementary Information files.