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RNA Biol. 2011 Sep-Oct; 8(5): 883–892.
Published online Sep 1, 2011. doi:  10.4161/rna.8.5.16022
PMCID: PMC3256357

Interplay between Y-box-binding protein 1 (YB-1) and poly(A) binding protein (PABP) in specific regulation of YB-1 mRNA translation

Abstract

YB-1 is a DNA- and RNA-binding protein that regulates expression of many important genes. Its deficiency or excess may pose threats, including malignant cellular transformation and metastasis, which explains the necessity of strict control over its amount at every level. As we showed previously, the 3′ untranslated region (UTR) of YB-1 mRNA contains a regulatory element specifically binding to YB-1 and PABP (PABPC1). Also, we showed that YB-1 selectively inhibits YB-1 mRNA translation, while PABP stimulates it in a poly(A) tail-independent manner. It was suggested that regulation of YB-1 mRNA translation involves competition between PABP and YB-1 for binding to the regulatory element. Here we offer cogent evidence for this model and add novel details to the mechanism of regulation of YB-1 synthesis. In experiments on regulatory element deletion we showed that it is this element that is responsible for a specific effect of YB-1 and PABP on YB-1 mRNA translation. Mutations eliminating only specific YB-1 affinity for this element suppressed the inhibitory effect of YB-1 and concurrently dramatically decreased the PABP stimulating effect. Mutations reducing only specific PABP affinity for this element, as well as spatial separation of the YB-1- and PABP binding sites, did not affect the YB-1 inhibitory action but completely abolished the positive PABP effect. Together, these results unambiguously prove direct inhibitory action of YB-1 on its mRNA translation, while the positive effect of PABP is realized through displacing YB-1 from the regulatory element.

Key words: YB-1, PABP, translation, regulation, mRNA, 3′ UTR

Introduction

YB-1 is a 36 kDa protein with the extremely conserved cold shock domain. It shows specific and non-specific affinity for DNA and RNA, displays nucleic acid chaperone activities and interacts with many cellular proteins (reviewed in refs. 13). YB-1 participates in almost all events of storing, reproduction and expression of genetic information. In the cell nucleus, it is involved in DNA reparation, replication and possibly recombination,4,5 as well as in mRNA transcription47 and its alternative splicing.8,9 In the cytoplasm, the bulk of YB-1 is associated with translated and untranslated mRNAs, thereby determining their functional activity and stability.1014 At low YB-1/mRNA ratios, YB-1 activates the overall protein synthesis at the stage of initiation, while mRNA saturation with YB-1 is accompanied by complete cessation of the synthesis at the same stage.11,1416 In addition, YB-1 contributes to localization of translationally active mRNAs on actin microfilaments,17 can stimulate tubulin polymerization and may promote association of translationally inactive YB-1-saturated mRNAs with microtubules.18,19 On the cell level, YB-1 contributes to cell resistance against ionizing radiation and DNA-damaging xenobiotics,20 and its nuclear localization is considered to be an early marker of multidrug resistance of cancer cells.21,22 Besides, YB-1 overexpression can induce an epithelial-mesenchymal transition of the cells, and hence, enhance the tumor metastatic ability.23 Since some tumors contain an elevated amount of YB-1 (reviewed in ref. 2), this protein is among the top dozen of prominent cancer markers.22,24

The effect of YB-1 on cellular events is dependent on its amount and nucleocytoplasmic distribution, as well as on its covalent modifications, e.g., phosphorylation.2529 The YB-1 amount in the cell depends on the rates of its synthesis and decay. In its turn, the synthesis rate can be regulated at the level of both transcription30,31 and translation.3235 Earlier, we found that a ~80 nt sequence (regulatory element) within the YB-1 mRNA 3′ UTR specifically interacts with two major proteins of cytoplasmic mRNPs, i.e., with YB-1 itself and PABP.35 Also, it was shown that PABP stimulates YB-1 mRNA translation even in the absence of the 3′-terminal poly(A) tail,35 while YB-1 selectively inhibits YB-1 mRNA translation at its relatively low concentrations optimal for translation of other cellular mRNAs.33,34 Both the positive PABP effect and the negative YB-1 effect are realized solely at the stage of translation initiation and even at the same step of this process, namely, the step of mRNA interaction with the 43S pre-initiation complex (Lyabin DN et al., unpublished data).33 To understand how these two proteins interplay in specific regulation of YB-1 mRNA translation, the role of each of them must be elucidated. Taking into account that YB-1 and PABP compete for binding to the regulatory element within YB-1 mRNA 3′ UTR and that they both are involved in the same step of translation initiation, it could be believed that (1) their action on YB-1 mRNA translation is mediated by their interaction with the regulatory element, and that (2) the effect of one of them is direct, while the role of the other may be reduced to simple displacement of the competitor from the regulatory element.

To verify these suggestions, we used YB-1 mRNAs either without the regulatory element or with a mutated regulatory element that ensured binding to only one of the major proteins in question. Our in vitro experiments showed that the effect of YB-1 and PABP on YB-1 mRNA translation is conferred by the regulatory element, and that only the negative effect of YB-1 is direct, while the positive effect of PABP is explained by YB-1 displacement from the regulatory element.

Results

To elucidate the relationship of the two major mRNP proteins, YB-1 and PABP, in specific regulation of YB-1 mRNA translation, we studied the effect of each of them on translation of YB-1 mRNAs with mutations preventing their specific interactions with the regulatory element.

Earlier, we showed that two regions in the initial part of the YB-1 mRNA regulatory element are protected by YB-1 from chemical cleavage (Fig. 1A).33 The protected regions are analogous seven-nucleotide sequences UCC AAC A and UCC AGC A that can be considered as members of the UCC AA/GCA motif. We suggested that these sequences are responsible for specific YB-1 interaction with the regulatory element and ensure binding of two YB-1 molecules.33 If so, nucleotide substitutions in these YB-1 mRNA sequences must result in losing their specific affinity for YB-1. In preliminary Gel-shift experiments (Fig. 2) [32P]-labeled 42-nucleotide fragment with the YB-1-protected sequence 1 (UCC AAC A) inserted into a random nucleotide sequence (Fig. 2A) was incubated with YB-1 in the presence of unlabeled RNA fragments of the same length with either the original YB-1-protected sequences (1 or 2) or with altered sequences (as indicated in Fig. 2A) as competitors. The samples were subjected to electrophoresis in non-denaturing PAAG followed by autoradiography. Figure 2B (lane 1) shows the position of the free RNA fragment after electrophoresis. In the presence of YB-1, a complex of lower mobility was formed (Fig. 2B, lane 2). Upon addition of increasing amounts of unlabeled RNA fragments with the original YB-1-protected sequences (UCC AAC A or UCC AGC A) as a competitor, the amount of the radioactive complex dramatically decreased (Fig. 2B, lanes 6–8 and 12–14). On the other hand, unlabeled RNA fragments with a mutated YB-1-protected sequence (UGG AAG A or UCC GGC G) could not displace YB-1 from its complex with the labeled RNA fragment (Fig. 2B, lanes 3–5 and 9–11). This experiment demonstrates specific affinity of YB-1 for the fragments with YB-1-protected sequences, while its affinity for mutated RNA fragments at positions 2, 3, 6 or 4, 5 within the YB-1-protected sites was decreased dramatically. Thus, the YB-1-protected sequences were responsible and sufficient for specific affinity of YB-1 for YB-1 mRNA.

Figure 1
Scheme of YB-1 mRNA, its mutant forms and the corresponding YB-1 mRNA fragments. (A) YB-1 mRNA contains a regulatory element (nt 1,127–1,204). Two specific YB-1-protected sites (nt 1,137–1,144 and 1,164–1,171) and an A-rich specific ...
Figure 2
Specificity of YB-1 interaction with presumable binding sites. (A) 42-nucleotide RNA fragments containing 7-nucleotide YB-1-protected sequences or sequences with indicated nucleotide substitutions were synthesized by in vitro transcription. (B) The [ ...

Accordingly, we synthesized capped poly(A) (C+A) YB-1 mRNA WT (Fig. 1B, i) and YB-1 mRNA mutYB-1 (Fig. 1B, ii), where UUUAUUA was substituted for UCCAACA and UCCAGCA.

It was also shown previously that PABP protected an A-rich sequence (50% A) (nt 1,149–1,204) of the YB-1 mRNA regulatory element against cleavage with RNAse T1 (Fig. 1A).33 Then it was suggested that this sequence ensured specific binding of two PABP molecules to the regulatory element. To verify this suggestion, we synthesized C+A YB-1 mRNA ΔPABP, where we made changes in the PABP-protected A-rich sequence, while the specific YB-1-protected site was preserved intact (Fig. 1B, iii).

Besides, we synthesized C+A YB-1 mRNA ΔRE where a nonspecific sequence of approximately the same length (Fig. 1B, v) was substituted for the entire regulatory element.

Since, hypothetically, YB-1 and PABP influence YB-1 mRNA translation through their competition for binding to the regulatory element, our next step was to learn the effect of each of them on translation of YB-1 mRNA with separated YB-1- and PABP-protected sites (to exclude their competition). For this purpose, we synthesized C+A YB-1 mRNA with separated YB-1- and PABP-protected sites (YB-1 mRNA SPS). This mRNA contained two YB-1-protected sites with a mutated spacer between them, a 50 nt insert, and the PABP-protected site with a mutated YB-1-protected site inside it (Fig. 1B, v).

YB-1 and PABP affinity for the original and mutated YB-1 mRNA regulatory element.

Before studying the effect of YB-1 and PABP on YB-1 mRNA translation, we analyzed interactions of YB-1 and PABP with YB-1 mRNA fragments (nt 1,070–1,240) containing either the wild type or a mutant regulatory element in rabbit reticulocyte lysate to check specific affinity of these fragments for YB-1 and PABP. For this purpose, the fragments with either the full-length regulatory element (nt 1,127–1,204, WT) or with a substitution (Fig. 1B, i–v), as well as the nonspecific control RNA sample (see Materials and Methods), were biotin-modified at the 3′ end, immobilized on Streptavidin Sepharose, and used to adsorb rabbit reticulocyte lysate proteins. YB-1 and PABP were detected by appropriate antibodies. Figure 3A shows that PABP and YB-1 binding to the WT fragment was more efficient than their binding to the fragment of nonspecific RNA (control) (cf. lanes 2 and 1). The interaction between the mutYB-1 RNA fragment and YB-1 went down to the level of control (lane 4, bottom part). In contrast, the level of specific PABP binding remained unchanged (cf. lanes 2 and 4, upper part). The ΔPABP fragment kept binding to YB-1 efficiently, while its binding to PABP was strongly decreased (lane 5). There was no specific YB-1 and PABP binding to the ΔRE fragment (lane 3). Lastly, the fragment with separated YB-1- and PABP-protected sites kept binding specifically both to YB-1 and PABP (lane 6).

Figure 3
Specificity of YB-1 and PABP interactions with presumable binding sites in the YB-1 mRNA regulatory element. (A) The proteins were adsorbed from rabbit reticulocyte lysate (300 µl) on Streptavidin Sepharose with 60 pmol of either biotinylated ...

The filter binding assay was used to measure apparent dissociation constants of the YB-1- and PABP complexes with YB-1 mRNA fragments (nt 1,070–1,240) containing the WT regulatory element or mutYB-1, or ΔRE, or ΔPABP, or SPS.

As seen from Figure 3B and Table 1, KDapp of the YB-1 and PABP complexes with the original YB-1 mRNA fragment are very close (about 3.9 ± 0.2 nM and 3.8 ± 0.2 nM, respectively). Presumably, this provides optimal conditions for competition between the two proteins whose observed concentrations in the cell are close to each other.

Table 1
Apparent dissociation constants (KDapp) of the YB-1 or PABP complexes with YB-1 mRNA fragments containing wt or mutated regulatory element

Unlike KDapp of the YB-1 complex with the original YB-1 mRNA fragment, KDapp of the YB-1 complex with the mutYB-1 fragment is about 12 ± 1.9 nM (p < 0.01, Student t-test), i.e., YB-1 affinity for the mutYB-1 fragment is three times lower. Approximately the same has been revealed as to YB-1 affinity for the ΔRE fragment (KDapp = 10 ± 0.1 nM; p < 0.001, Student t-test). It is of importance that PABP affinity for the mutYB-1 fragment remained unchanged, as compared with that for the WT fragment (Fig. 3C and Table 1, KDapp = 3.9 ± 0.4 nM and 3.8 ± 0.2 nM, respectively).

Thus, mutations in the YB-1-protected sites, as well as substitution of a non-specific sequence for the regulatory element, decrease YB-1 affinity for RNA down to the nonspecific level, while mutations in the YB-1-protected sites produce no effect on specific binding of PABP.

Also, as follows from Figure 3C and Table 1, the absence of the PABP-protected site from the regulatory element (ΔPABP fragment) reduced affinity of the latter for PABP 5-fold, as compared with its affinity for the WT fragment (KDapp = 17.7 ± 0.6 nM and 3.8 ± 0.2 nM, respectively; p < 0.001, Student's t test), while specific YB-1 affinity showed no difference from the WT fragment (Fig. 3B and Table 1, KDapp = 3.9 ± 0.2 nM and 3.9 ± 0.2 nM, respectively). In other words, substitution of a nonspecific sequence for the PABP-protected site within the regulatory element produced no effect on specificity of YB-1 binding.

The replacement of the entire regulatory element (ΔRE fragment) resulted in a still lower affinity of the YB-1 mRNA fragment for PABP as compared with its affinity for the WT fragment (Fig. 3C and Table 1, KDapp = 45.3 ± 1.5 nM; p < 0.01, Student's t test). Lastly, separation of the specific YB-1- and PABP-protected sites within the regulatory element had no effect on YB-1 and PABP affinity for the YB-1 mRNA fragment (Fig. 3C and Table 1).

Taken together, these results indicate that YB-1- and PABP-protected sequences, detected previously in reference 33, really provide specific binding of YB-1 and PABP to the regulatory element. Substitutions in the YB-1-protected sequences eliminated specific YB-1 binding, leaving affinity for PABP unchanged. Deletion of the PABP-protected site from the YB-1 mRNA regulatory element resulted in a dramatic decrease of its affinity for PABP and had no effect on YB-1 binding. Separation of these sites, that excluded competition between YB-1 and PABP, left affinity of the fragment for the both proteins unchanged.

The effects of YB-1 and PABP on translation of YB-1 mRNA and its mutant forms.

To elucidate the effect of the above mutations in the regulatory element on translation, we used C+A YB-1 mRNAs (WT and mutated) and Luciferase mRNA as a control shown in Figure 1B. All these mRNA samples were translated in a rabbit reticulocyte cell-free translation system to study the effect of YB-1 or PABP on their translation.

Figure 4A and B illustrate the effect of YB-1 on translation of the above mRNA forms. As seen, YB-1 inhibited translation of YB-1 mRNA mutYB-1 and YB-1 mRNA ΔRE much weaker than translation of YB-1 mRNA WT, but somewhat stronger than that of control Luciferase mRNA. Translation of YB-1 mRNA ΔPABP and YB-1 mRNA SPS was still dramatically suppressed by YB-1. As appears, the presence of intact specific YB-1-protected sites within the regulatory element is crucial for the YB-1 inhibitory effect on its own synthesis. Deletion of the PABP-protected site, and hence, a decreased level of PABP binding, or separation of the YB-1- and PABP-protected sites did not decrease the capability of YB-1 to inhibit its own synthesis in a rabbit reticulocyte cell-free translation system. Thus, YB-1 has a direct inhibitory effect on YB-1 mRNA translation that is independent of specific interaction of PABP with the regulatory element.

Figure 4
YB-1 and PABP effects on translation of YB-1 mRNA and its mutant forms. (A and C) 0.1 pmol of C+A wild type, mutated YB-1 mRNAs, and Luciferase mRNA, as internal control, were translated in a rabbit reticulocyte cell-free system in the presence ...

Figure 4C and D demonstrate the PABP effect on translation of the same mRNA samples. PABP stimulates translation of YB-1 mRNA WT stronger than that of all YB-1 mRNA mutant forms and of Luciferase mRNA. Thus, in the absence of specific YB-1 binding to the regulatory element of YB-1 mRNA mutYB-1 and YB-1 mRNA ΔRE or in the absence of overlapping of the YB-1- and PABP-protected sites, the PABP effect on their translation is mild and analogous to that on Luciferase mRNA translation (control). Thus, the stimulating effect of PABP depends on specific binding of YB-1 to the regulatory element.

Taken together, these results are evidence for the direct inhibitory effect of YB-1 on translation of its own mRNA. This inhibition is explained by YB-1 binding to specific sites within the YB-1 mRNA regulatory element. The presence of the PABP-protected site is required only to exert displacing of YB-1 by PABP, and thereby to suppress the YB-1 inhibitory effect.

Discussion

Since YB-1 performs many functions in the cell and participates in its crucial events, synthesis of this protein must be under strict control. There are reports that the following transcription factors are involved in regulation of transcription of the YB-1 gene: Twist,36 GATA,37 Math2 (Neurod6),38 p73, c-Myc и Max.39

A few studies have been reported on regulation of YB-1 synthesis at the posttranscriptional level. Specifically, Fukuda and colleagues32 have demonstrated that in human cells KB3-1 and H1299 YB-1 selectively inhibits its own synthesis through specific binding to YB-1 mRNA 5′ UTR. As reported, the studied YB-1 mRNA 5′ UTR is 200 nt longer than human YB-1 mRNA 5′ UTR and those from mouse, rat and rabbit, as given in PubMed Data Base (e.g., NM_004559.3 for human YB-1 mRNA), as well as rabbit YB-1 mRNA used in our experiments. It is this additional 200 nt sequence localized in the initial part of the 5′ UTR that specifically interacts with YB-1. The YB-1 mRNAs that differ from each other in length of 5′ UTR could have resulted from YB-1 transcription from different starting points, depending of cell and tissue types. Regardless of the mechanism providing these two types of YB-1 mRNAs, it is obvious that the described translational autoregulation of YB-1 synthesis is restricted only to mRNAs with a more extended 5′ UTR.

Recently, Kato and colleagues40 reported that overnight stimulation of mesangial cells with transforming growth factor TGFβ resulted in a dramatic decrease of both YB-1 and YB-1 mRNA. In parallel, they observed an increase of the level of microRNA-216a (miR-216a) having a potential binding site in the YB-1 mRNA 3′ UTR. Experiments on reporter Luciferase mRNA containing either YB-1 mRNA 3′ UTR or that with mutated miR-216a binding site confirmed that this microRNA is involved in negative regulation of YB-1 mRNA stability.

Previously, we showed that the YB-1 mRNA 3′ UTR fragment is able to inhibit translation of various mRNAs at the stage of initiation.35 We proposed that this fragment contains a nucleotide sequence specifically interacting with proteins that participate in translation initiation. This sequence was named a regulatory element. Next, we demonstrated that the regulatory element specifically binds to two major mRNP proteins, YB-1 and PABP.35 These proteins interact with overlapping binding sites of this element in a competitive manner. YB-1 selectively inhibited its own synthesis at its concentrations optimal for translation of other mRNAs, while PABP stimulated translation of YB-1 mRNA in a poly(A) tail-independent manner.33,35 Then, we proposed that the negative effect of YB-1 and the positive effect of PABP resulted from their specific interaction with the regulatory element.

To verify this suggestion, in this study we substituted a random nucleotide sequence of about the same length for the regulatory element and showed that such a substitution prevents both selective autoregulation of YB-1 mRNA translation and stimulation of translation of capped poly(A)YB-1 mRNA caused by PABP.

It also remained unclear what role each of the two proteins (YB-1 and PABP) plays in translational control of YB-1 mRNA expression. Inasmuch as these proteins competed for the regulatory element and the effect of each of them became apparent upon initiation of translation and, what is more, at the same step of this process, three proposals can be made relative to their interplay during translational control of YB-1 synthesis.

It may be believed that regulation of YB-1 mRNA translation initiation by the two major mRNP proteins is explained by the direct stimulating effect of PABP. This effect may be realized through the translation stimulation mechanism similar to the mechanism typical of most poly(A)+ mRNAs, which involves PABP interaction with the poly(A) tail and eIF4G.41 In this case, the inhibitory role of YB-1 would be reduced to blocking the positive action of PABP through its displacing from the YB-1 mRNA regulatory element.

On the other hand, it may be suggested that YB-1 bound to the regulatory element of its own mRNA directly inhibits initiation of its translation by a so far unknown mechanism, while the stimulating effect of PABP is simply a result of YB-1 displacement from the regulatory element and thus blocking its inhibitory action.

Lastly, it could be proposed that each of the two proteins has its own direct mechanism of regulation of translation initiation, and that their competition for binding to the regulatory element of YB-1 mRNA only enhances the positive or negative effect of each of them on translation.

To choose among the above alternatives of the YB-1 mRNA translational control, we performed mutagenesis in the regulatory element of this mRNA or in its fragment containing the regulatory element and studied the influence of these mutations on affinity of the two major proteins for RNA fragments, as well as the influence of YB-1 and PABP on translation of the mutant mRNAs in rabbit reticulocyte lysate.

Our experiments on binding of the YB-1 mRNA 3′ UTR fragment containing the regulatory element with two native seven-nucleotide motifs to rabbit reticulocyte lysate proteins or to isolated YB-1 confirmed the elevated affinity of YB-1 for this element and demonstrated the loss of specific affinity upon replacement of nucleotides in the both sequences protected by YB-1. At the same time, the mutant fragment retained completely its high affinity for PABP.

The experiments on binding of the ΔPABP fragment to rabbit reticulocyte lysate proteins confirmed a loss of the fragment affinity for PABP with a complete retention of its specific affinity for YB-1. The experiments on binding of isolated PABP to the YB-1 mRNA 3′ UTR fragment with a deleted A-rich region revealed that affinity of this fragment for PABP was partially lost. The binding curve of the ΔPABP fragment somewhat differed from those of ΔRE- and non-specific fragments. This difference could be explained by the presence of a retained YB-1-protected sequence within the PABP-protected site, provided that PABP had an elevated affinity for this sequence.

Then cap+ and poly(A) YB-1 mRNA with mutations in YB-1- and PABP binding sites were translated in a rabbit reticulocyte cell-free translation system in the presence of increasing YB-1 and PABP amounts. We have shown that YB-1 capability to inhibit its own synthesis is explained by the existence of a specific YB-1 binding site within the regulatory sequence of its own mRNA. Mutations in the YB-1 binding sites result in elimination of the stimulating effect of PABP. More importantly, spatial separation of YB-1 and PABP binding sites also leads to loss of the PABP stimulating effect. Besides, YB-1 preserves its ability to selectively inhibit translation of YB-1 mRNA in case of deletion of the PABP binding site from this mRNA or separating the YB-1 and PABP binding sites. These results are in agreement with the supposition of the direct negative effect of YB-1 on initiation of YB-1 mRNA translation and indirect PABP-induced stimulation of YB-1 mRNA translation, mostly through YB-1 displacement from the regulatory element of YB-1 mRNA. These results also bring solely YB-1 in the focus of our further studies.

It still remains unclear how 3′ UTR-associated YB-1 can suppress initiation of YB-1 mRNA translation that occurs at the 5′ end of the molecule. Perhaps this happens through bringing the 3′ and 5′ ends together due to certain structural peculiarities of YB-1 mRNA itself. Alternatively, these ends might approach each other as a result of interaction between proteins associated with the 3′ UTR (including YB-1) and proteins specifically bound to the 5′ UTR. Also, it is possible that the elevated affinity of the regulatory element for YB-1 promotes YB-1 mRNA saturation and packaging at a lower YB-1/mRNA ratio than that typical of ordinary mRNAs, which results in YB-1 mRNA transition into an untranslated state with retention of active translation of the other mRNAs.

Materials and Methods

Plasmid construction.

The pBluescript II SK YB-1 WT construct containing rabbit YB-1 cDNA and the pT3-Luc construct encoding Luciferase (Luc) were described earlier in references 35 and 42.

The pBluescript II SK YB-1 mutYB-1 construct was obtained using site-specific mutagenesis by overlap extension. The overlapping primers 5′-AAG AAA TGA ATA TGA AAT TTT ATT AAT AAG AAA TGA ACA AAA GAT TGG A-3′ and 5′-CTT ATT AAT AAA ATT TCA TAT TCA TTT CTT CTT ATT AAA TGA CTA AAC CGG A-3′ contained a mutation leading to substitution of UUUAUUA for the both WT YB-1-protected sites (UCC AAC A and UCC AGC A) within YB-1 mRNA.

A portion of YB-1 cDNA encoding the PABP-protected site (nucleotides from 1,174 to 1,204) was deleted by PCR amplification of pBluescript II SK YB-1 WT, in which primers were designed in the inverted tail-to-tail direction. The forward primer was 5′-TTA AGT GCT TGC TTT TTG CCC-3′, and the reverse primer was 5′-CAG TCA GTC AGT CAG TCA GTC AGT CAG TCA GTC AGT CAG TCA GTC AGT ATT GCT GGA Ata aCA TAa TCA aaT Caa CTT GTT GGA TGA C-3′. Substitutions in the sequence encoding the PABP-protected site overlapping with the YB-1-protected site are shown by lower case characters; the insert replacing the deleted part of the PABP-protected site is bold. The gap between 5′ ends of the primers corresponded to the deleted region. After PCR, the amplified linear DNA was self-ligated. The obtained construct was named pBluescript II SK YB-1 ΔPABP.

The YB-1 cDNA sequence encoding the regulatory element (nucleotides from 1,127 to 1,204) was deleted by PCR amplification of pBluescript II SK YB-1 WT, in which primers were designed in the inverted tail-to-tail direction. The forward primer was 5′-TTA AGT GCT TGC TTT TTG CCC-3′, and the reverse primer was 5′-CGG ATG ATG GTA GAG ATG TTA AGC-3′. The gap between 5′ ends of the primers corresponded to the deleted region. After PCR, the amplified linear DNA was ligated with a 66 bp blunt-ended fragment of the pBluescript II SK plasmid. The obtained construct was named pBluescript II SK YB-1 ΔRE (Δ Regulatory Element).

The pBluescript II SK YB-1 SPS (Separated Protected Sites) construct was obtained using site-specific mutagenesis by overlap extension. Two PCR products were amplified using the pBluescript II SK YB-1 WT plasmid as template and primers 1 and 2 (5′-GGT GGA GTT CCA GTG CAA GG-3′ and 5′-CAG TCA GTC AGT CAG TCA GTC AGT CAG TCA GTC AGT CAG TCA GTC AGT ATT GCT GGA Ata aCA TAa TCA aaT Caa CTT GTT GGA TGA C-3′, respectively) or 3 and 4 (5′ACT GAC TGA CTG ACT GAC TGA CTG ACT GAC TGA CTG ACT GAC TGA CTG ACA AGA AAT GAA TAT GAA ATT ttA ttA ATA AGA AAT GAA C-3′ and 5′-CCG CGG TGG CGG CAG ATC TAG AAC TAG T-3′, respectively). The obtained fragments were combined using the overlapping regions (bold) and the flanking primers 1 and 4. Substitutions in the sequences encoding YB-1- and PABP-protected sites are shown in lower case characters. The resultant PCR product was treated with XagI and BamHI and ligated with the pBluescript II SK YB-1 WT plasmid treated with the same restriction endonucleases. The obtained construct was named pBluescript II SK YB-1 SPS. All primers were synthesized by “Syntol” (Russia).

In vitro transcription.

Luciferase (Luc) cap+poly(A) (C+A) mRNA was transcribed by T3 RNA polymerase from pT3Luc linearized with BamHI. Transcriptions of rabbit YB-1 mRNA and its fragments were carried out by T7 RNA polymerase.

The DNA template for C+A YB-1 mRNA WT was pBluescript II SK YB-1 WT linearized with BamHI; for C+A YB-1 mRNA ΔPABP, it was pBluescript II SK YB-1 ΔPABP linearized with BamHI; for C+A YB-1 mRNA mutYB-1-pBluescript II SK YB-1 mutYB-1 linearized with BamHI; for C+A YB-1 mRNA ΔRE-pBluescript II SK YB-1 ΔRE linearized with BamHI; for C+A YB-1 mRNA SPS-pBluescript II SK YB-1 SPS linearized with BamHI.

Templates for in vitro synthesis of the YB-1 mRNA fragments (nt 1,070–1,240) containing the WT regulatory element or mutated regulatory elements were generated by PCR using pBluescript II SK YB-1 WT, pBluescript II SK YB-1 ΔPABP, pBluescript II SK YB-1 mutYB-1, pBluescript II SK YB-1 ΔRE or pBluescript II SK YB-1 SPS as a template. The forward primer was 5′-TAA TAC GAC TCA CTA TAG GGA ATT CGT CCG CTC TCG AGG CTG-3′ and contained a T7 promoter region. The reverse primer was 5′-GCA GAT AGT TCT AGA AAT CTG GTC AAC GGG CA-3′. A 200 nt nonspecific RNA fragment was transcribed by T7 RNA polymerase from pET28a linearized with XhoI.

The transcription was performed as described previously in reference 43. Capped mRNA transcripts were obtained by a reaction where a mixture of 0.2 mM GTP and 1 mM 3′-0-Me-m7G5′ppp(5′) G (NEB, #S1411L) was used instead of 5 mM GTP. To generate [32P]-labeled RNA fragments, 0.1 µCi/µl [32P]UTP (2,000 Ci/mM; IBCh, Russia) was added to the reaction, and the concentration of unlabeled UTP was reduced to 0.05 mM.

In vitro translation assays.

Translation of exogenous mRNA in a rabbit reticulocyte cell-free system was performed as described elsewhere in reference 44. The incubation mixture (15 µl) contained: reticulocyte lysate (7.5 µl), 10 mM Hepes-KOH, pH 7.6, 100 mM KOAc, 1 mM Mg(OAc)2, 8 mM creatine phosphate, 0.5 mM spermidine, 0.2 mM GTP, 0.8 mM ATP, 1 mM dithiothreitol, 25 µM of each of 20 amino acids except for the labeled one; 10 µM [14C]-Tyr (482 mCi/mmol, GE Healthcare, #CFB74) was added. Appropriate mRNA was added to the mixture as indicated in the figure legends. Translation was carried out at 30°C for 1 h. [14C]-Tyr-incorporating proteins were analyzed by 12% SDS-PAGE followed by autoradiography. The relative amount of radioactivity in the bands was determined using a Packard Cyclone Storage Phosphor System (Packard Instrument Company, Inc.).

PABP and YB-1 purification.

Recombinant PABP with His tag and recombinant YB-1 were expressed and purified as described previously in references 45 and 46.

Isolation of rabbit reticulocyte lysate proteins using biotinylated RNA.

YB-1 mRNA fragments (WT and mutated), as well as a 200 nt nonspecific RNA fragment, were biotinylated as described in reference 47. Briefly, RNA (60 pmol) was oxidized in 50 µl of 66 mM NaOAc buffer (pH 4.5) containing 5 mM NaIO4 as the oxidizing agent. The oxidation was carried out on ice in the dark for 45 min. The oxidized RNA was subsequently precipitated with isopropanol and dissolved in 50 µl of RNase-free water. Then, 5 µl of 1 M NaOAc (pH 6.1), 5 µl of 10% SDS, and 150 µl of 10 mM biotin hydrazide long arm (Calbiochem, #203110) (freshly dissolved in DMSO) were added to the sample. On the RNA, the oxidized diol groups were biotinylated by 3 h incubation at room temperature (22–26°C) in the dark. Finally, RNA was precipitated with ethanol and dissolved in 70 µl of RNase-free water.

For isolation of rabbit reticulocyte lysate proteins we used Streptavidin Sepharose (GE Healthcare, #17-5113-01) that had been preincubated overnight in Blocking Buffer (10 mM Hepes-KOH, pH 7.6, 100 mM KCl, 1 mg/ml BSA, 0.2 mg/ml glycogen, 0.2 mg/ml E. coli total RNA). Rabbit reticulocyte lysate (400 µl) was first precleared by incubating with 100 µl of 50% slurry preblocked Streptavidin Sepharose for 1 h at 4°C in Binding Buffer 10 mM Hepes-KOH, pH 7.6, 100 mM KCl, 3 mM MgCl2, 2 mM DTT, 0.2 mM vanadyl ribonucleoside complex (Fluka, #94740). Beads were pelleted, and the supernatant was incubated with 60 pmol biotinylated RNA probe for 30 min at room temperature. The mixture was then incubated with 50 µl of 50% slurry preblocked Streptavidin Sepharose in Binding Buffer for 30 min at room temperature. Beads were pelleted and washed 10 times in Binding Buffer and twice in Binding Buffer with 300 mM KCl. Proteins were eluted from the beads by incubating with Sample Buffer for SDS-PAGE and resolved by SDS-PAGE (12%) followed by western blot analysis using specific rat antibodies against YB-1 (IMTEK, Russia) and rat antibodies against PABP (prepared by D. Lyabin).

Filter binding assay.

Increasing amounts of YB-1 or PABP were incubated with the 0.02 pmol in vitro transcribed [32P]-labeled RNA for 15 min at 30°C in 50 µl of binding buffer (20 mM Hepes 7.5, 100 mM KCl, 2 mM MgCl2). The reaction mixtures were filtered through a nitrocellulose (GE Healthcare, RPN3032D) and nylon membrane (GE Healthcare, RPN303B) using a Bio-dot apparatus (Bio-Rad, #170-6545), and then washed twice with 200 µl of ice-cold binding buffer. The [32P] label retained on the nitrocellulose and nylon membrane was quantified by phosphorimaging analysis using a Packard Cyclone Storage Phosphor System (Packard Instrument Company, Inc.). The apparent dissociation constants (KDapp) were the concentration at which 50% of the RNA bound at saturation was retained on the filter and were calculated from graphs of the protein concentration versus the fraction bound (nitrocellulose/nylon + nitrocellulose). No RNA was retained by the nitrocellulose itself.

Electrophoretic mobility shift assay (Gel-shift).

Recombinant YB-1 was incubated with [32P]-labeled RNA (10,000 cpm) in a final volume of 10 µl (10 mM Hepes-KOH, pH 7.6, 100 mM KCl, 0.5 mM dithiothreitol, 1 mg/ml bovine serum albumin) at 30°C for 15 min. After incubation, the protein-RNA complexes were analyzed by 6% PAGE in 0.5x TBE buffer (1x: 89 mM Tris, 89 mM boric acid, 2 mM EDTA) followed by autoradiography.

Data analysis.

Means ± SD are depicted. Student t-test for repeated measurements was used for comparisons between data sets, and p < 0.05 was considered significant.

Acknowledgments

This work was supported by Programs on “Molecular and Cellular Biology” and “Basic Sciences to Medicine” of the Russian Academy of Sciences and Russian Foundation for Basic Research (#11-04-00267). We thank Prof. N. Sonenberg for providing pT3-Luc and pET3B PABP-His, E. Serebrova and T. Kuvshinkina for help in manuscript preparation.

Abbreviation

YB-1
Y-box binding protein 1
PABP
poly(A)-binding protein (PABPC1)
UTR
untranslated region of mRNA

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