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Poly(A) binding protein 1 enhances cap-independent translation initiation of neurovirulence factor from avian herpesvirus.

Tahiri-Alaoui A, Zhao Y, Sadigh Y, Popplestone J, Kgosana L, Smith LP, Nair V - PLoS ONE (2014)

Bottom Line: We report a novel viral translational control strategy involving the recruitment of PABP1 to the 5' leader internal ribosome entry site (5L IRES) of an immediate-early (IE) bicistronic mRNA that encodes the neurovirulence protein (pp14) from the avian herpesvirus Marek's disease virus serotype 1 (MDV1).We provide evidence for the interaction between an internal poly(A) sequence within the 5L IRES and PABP1 which may occur concomitantly with the recruitment of PABP1 to the poly(A) tail.RNA interference and reverse genetic mutagenesis results show that a subset of virally encoded-microRNAs (miRNAs) targets the inhibitor of PABP1, known as paip2, and therefore plays an indirect role in PABP1 recruitment strategy by increasing the available pool of active PABP1.

View Article: PubMed Central - PubMed

Affiliation: The Pirbright Institute, Ash Road, Pirbright, Woking, Surrey, United Kingdom.

ABSTRACT
Poly(A) binding protein 1 (PABP1) plays a central role in mRNA translation and stability and is a target by many viruses in diverse manners. We report a novel viral translational control strategy involving the recruitment of PABP1 to the 5' leader internal ribosome entry site (5L IRES) of an immediate-early (IE) bicistronic mRNA that encodes the neurovirulence protein (pp14) from the avian herpesvirus Marek's disease virus serotype 1 (MDV1). We provide evidence for the interaction between an internal poly(A) sequence within the 5L IRES and PABP1 which may occur concomitantly with the recruitment of PABP1 to the poly(A) tail. RNA interference and reverse genetic mutagenesis results show that a subset of virally encoded-microRNAs (miRNAs) targets the inhibitor of PABP1, known as paip2, and therefore plays an indirect role in PABP1 recruitment strategy by increasing the available pool of active PABP1. We propose a model that may offer a mechanistic explanation for the cap-independent enhancement of the activity of the 5L IRES by recruitment of a bona fide initiation protein to the 5' end of the message and that is, from the affinity binding data, still compatible with the formation of 'closed loop' structure of mRNA.

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MDV1-encoded miRNAs target PABP1-interacting protein 2, paip2.(A) The paip2 transcript showing paip2 open reading frame (ORF), the 3' untranslated region (UTR) and the miRNA response elements (MRE). (B) Schematic of the reporter construct containing individual or combined MREs sequences downstream of the simian virus 40 promoter-driven Renilla luciferase cassette from psiCHECK-2 vector. (C) The predicted duplexes between paip2 mRNA and MDV1 miRNAs. The mutated nucleotides within the seed regions of paip2 mRNA are underlined. (D) Luciferase-based miRNA reporter assay. The full length region from the paip2 mRNA that contains all the MREs or the individual MREs and their mutated versions were made as synthetic oligonucleotides and sub-cloned into the sensor plasmid downstream of the Renilla luciferase in psiCHECK-2 vector. The resulting constructs were used to transfect MSB1; an MDV1-transformed CD4+ T-cell line derived from a spleen lymphoma induced by BC-1 strain of MDV1 constitutively expressing viral miRNAs. As positive control for assay validation we have used MRE-M4 that was previously shown to be targeted by MDV1 miRNA-M4. The normalized Renilla luciferase activities from five experiments are shown with the error bars (SEM) relative to that seen for the empty vector psiCHECK-2 which value is set to 1.
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pone-0114466-g005: MDV1-encoded miRNAs target PABP1-interacting protein 2, paip2.(A) The paip2 transcript showing paip2 open reading frame (ORF), the 3' untranslated region (UTR) and the miRNA response elements (MRE). (B) Schematic of the reporter construct containing individual or combined MREs sequences downstream of the simian virus 40 promoter-driven Renilla luciferase cassette from psiCHECK-2 vector. (C) The predicted duplexes between paip2 mRNA and MDV1 miRNAs. The mutated nucleotides within the seed regions of paip2 mRNA are underlined. (D) Luciferase-based miRNA reporter assay. The full length region from the paip2 mRNA that contains all the MREs or the individual MREs and their mutated versions were made as synthetic oligonucleotides and sub-cloned into the sensor plasmid downstream of the Renilla luciferase in psiCHECK-2 vector. The resulting constructs were used to transfect MSB1; an MDV1-transformed CD4+ T-cell line derived from a spleen lymphoma induced by BC-1 strain of MDV1 constitutively expressing viral miRNAs. As positive control for assay validation we have used MRE-M4 that was previously shown to be targeted by MDV1 miRNA-M4. The normalized Renilla luciferase activities from five experiments are shown with the error bars (SEM) relative to that seen for the empty vector psiCHECK-2 which value is set to 1.

Mentions: The internal poly(A) has the potential to interact with the PABP1 and therefore modulates the activity of the 5L IRES. To show that the internal poly(A) has indeed the ability to interact with PABP1 we performed electrophoretic mobility shift assay (EMSA) and demonstrated the occurrence of 5Lwt IRES/PABP1 complex that is seen in all combinations except with 5Lmt2&3 and 5Lmt2 that display poor 5L reporter activity (Fig. 1D and 1E). Our mutation analyses and EMSA are in agreement with previous findings [24] that showed A11 and A12 are capable of competing effectively with A25 for PABP1, whereas A9 and A10 are not. To further demonstrate the specificity of the interaction between internal poly(A) and PABP1, we performed affinity binding assays (Fig. 2A). The recombinant PABP1 binds tightly to the 5Lwt and to all mutants except when the length the internal poly(A) is reduced to less than 10-nucleotides, see for example 5Lmt2 and 5Lmt2&3 (Fig. 2A), and the binding affinity does not significantly change when the internal poly(A) length increases beyond A11, compare for example 5Lwt and 5Lmt1 (Fig. 2A). There is a good correlation between the binding affinity of PABP1 to the internal poly(A) and the activity of the 5L reporter (Fig. 2B). The importance of PABP1 for the activity of the 5L reporter was further demonstrated by siRNA-mediated PABP1 depletion. DF-1 cells were co-transfected with siRNA that targets PABP1 or control siRNA and with the reporter construct depicted in (Fig. 3A). Following 48 hour incubation the activity of the 5L reporter was assessed by measuring the luciferase activities from R-Luc and F-Luc. There is 80% decrease in the activity of the 5L reporter in the siPABP1 as compared to the control siRNA (Fig. 3A). When we used a reporter construct that lacks the 5L IRES and in which the R-Luc is under canonical cap-dependent translation and the F-Luc under ICR IRES control (S1A Figure, pR/ICR-F reporter), we found that siRNA-mediated PABP1 depletion caused only about 40% reduction in R-Luc activity with no apparent effect on the activity of F-Luc which is now controlled by the ICR IRES (S1A Figure), indicating the specific effect of PABP1 depletion on the activity of the 5L reporter. Northern blotting analysis shows that the decrease in the activity of the 5L reporter is not due to the stability or abundance of the reporter mRNAs (Fig. 3A and S1A Figure). Immunoblotting analysis reveals ∼75% decrease in the level of PABP1 in cells transfected with siPABP1, and as reported by another study [25] we also observed a concomitant decrease in the level of paip2, whereas the level of other translation factors such as eIF4E and eIF4A appeared unchanged (Fig. 3B). The above data indicate that PABP1 is involved in the regulation of the activity of the 5L IRES within the mRNA reporter, most likely via its interaction with the internal 5' poly(A). To gain further insights on how the PABP1 may mediate the regulation of the 5L IRES we investigated the interplay between the 3' end poly(A) tail, the internal poly(A) and the 5' cap structure using in vitro engineered mRNA reporters depicted in (Fig. 3C). The rationale behind this is that the activity of some cellular IRESes has been shown to be enhanced by poly(A) tail in the absence of PABP1 [26], [27]. Furthermore, we have previously shown that the 5L IRES efficiently initiates translation when cap-dependent translation initiation is inhibited [22]. Using RNA transfection experiments we show here that when the cap structure (7mGpppG) is replaced by the cap analogue (ApppG) the activity of the 5Lwt reporter increases by at least 5-fold in the presence of the poly(A) tail and only by ∼2-fold in the absence of the poly(A) tail (Fig. 3C). However, in the presence of the cap structure the absence of the poly(A) tail does not significantly alter the activity of the 5Lwt reporter (Fig. 3C), suggesting the possibility that the internal poly(A) within the 5L IRES may assume some of the functions of the poly(A) tail such as the direct recruitment of PABP1 to the 5L IRES located at the 5' end of the bicistronic mRNA reporter. Mutations within the internal poly(A), for example 5Lmt2&3, that simultaneously reduce the activity of the 5L reporter and the binding of the PABP1 also make the activity of the 5L reporter insensitive to the nature of the 5' and 3' ends of the bicistronic mRNA (Fig. 3C). In all the combinations tested the activity of the 5Lmt2&3 reporter is reduced by more than 70% (Fig. 3C, 5Lmt2&3-R/ICR-F). The importance of the interplay between the 3' poly(A) tail, the internal poly(A) within the 5L IRES for the activity of the 5L reporter was further demonstrated by using reporter mRNA that lacks the 5L IRES and in which the R-Luc is under canonical cap-dependent translation and the F-Luc under ICR IRES control (S1B Figure, R/ICR-F mRNA reporter). The data show that the activity of the ICR IRES is slightly enhanced in the absence of both the cap-structure and the 3' poly(A) tail and as expected the R-Luc activity which is now under canonical cap-dependent translation initiation was severely impaired in the absence the cap structure and the 3' poly(A) tail (S1B Figure). Northern blotting shows that variations in the activity of the 5L reporter are not due to the stability or the abundance of the reporter mRNAs (Fig. 3D and S1B Figure). These results indicate that the internal poly(A) and the poly(A) tail may work in synergy to enhance the activity of the 5L IRES within the mRNA reporter possibly by bridging the ends of the message in a cap-independent manner.


Poly(A) binding protein 1 enhances cap-independent translation initiation of neurovirulence factor from avian herpesvirus.

Tahiri-Alaoui A, Zhao Y, Sadigh Y, Popplestone J, Kgosana L, Smith LP, Nair V - PLoS ONE (2014)

MDV1-encoded miRNAs target PABP1-interacting protein 2, paip2.(A) The paip2 transcript showing paip2 open reading frame (ORF), the 3' untranslated region (UTR) and the miRNA response elements (MRE). (B) Schematic of the reporter construct containing individual or combined MREs sequences downstream of the simian virus 40 promoter-driven Renilla luciferase cassette from psiCHECK-2 vector. (C) The predicted duplexes between paip2 mRNA and MDV1 miRNAs. The mutated nucleotides within the seed regions of paip2 mRNA are underlined. (D) Luciferase-based miRNA reporter assay. The full length region from the paip2 mRNA that contains all the MREs or the individual MREs and their mutated versions were made as synthetic oligonucleotides and sub-cloned into the sensor plasmid downstream of the Renilla luciferase in psiCHECK-2 vector. The resulting constructs were used to transfect MSB1; an MDV1-transformed CD4+ T-cell line derived from a spleen lymphoma induced by BC-1 strain of MDV1 constitutively expressing viral miRNAs. As positive control for assay validation we have used MRE-M4 that was previously shown to be targeted by MDV1 miRNA-M4. The normalized Renilla luciferase activities from five experiments are shown with the error bars (SEM) relative to that seen for the empty vector psiCHECK-2 which value is set to 1.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4263670&req=5

pone-0114466-g005: MDV1-encoded miRNAs target PABP1-interacting protein 2, paip2.(A) The paip2 transcript showing paip2 open reading frame (ORF), the 3' untranslated region (UTR) and the miRNA response elements (MRE). (B) Schematic of the reporter construct containing individual or combined MREs sequences downstream of the simian virus 40 promoter-driven Renilla luciferase cassette from psiCHECK-2 vector. (C) The predicted duplexes between paip2 mRNA and MDV1 miRNAs. The mutated nucleotides within the seed regions of paip2 mRNA are underlined. (D) Luciferase-based miRNA reporter assay. The full length region from the paip2 mRNA that contains all the MREs or the individual MREs and their mutated versions were made as synthetic oligonucleotides and sub-cloned into the sensor plasmid downstream of the Renilla luciferase in psiCHECK-2 vector. The resulting constructs were used to transfect MSB1; an MDV1-transformed CD4+ T-cell line derived from a spleen lymphoma induced by BC-1 strain of MDV1 constitutively expressing viral miRNAs. As positive control for assay validation we have used MRE-M4 that was previously shown to be targeted by MDV1 miRNA-M4. The normalized Renilla luciferase activities from five experiments are shown with the error bars (SEM) relative to that seen for the empty vector psiCHECK-2 which value is set to 1.
Mentions: The internal poly(A) has the potential to interact with the PABP1 and therefore modulates the activity of the 5L IRES. To show that the internal poly(A) has indeed the ability to interact with PABP1 we performed electrophoretic mobility shift assay (EMSA) and demonstrated the occurrence of 5Lwt IRES/PABP1 complex that is seen in all combinations except with 5Lmt2&3 and 5Lmt2 that display poor 5L reporter activity (Fig. 1D and 1E). Our mutation analyses and EMSA are in agreement with previous findings [24] that showed A11 and A12 are capable of competing effectively with A25 for PABP1, whereas A9 and A10 are not. To further demonstrate the specificity of the interaction between internal poly(A) and PABP1, we performed affinity binding assays (Fig. 2A). The recombinant PABP1 binds tightly to the 5Lwt and to all mutants except when the length the internal poly(A) is reduced to less than 10-nucleotides, see for example 5Lmt2 and 5Lmt2&3 (Fig. 2A), and the binding affinity does not significantly change when the internal poly(A) length increases beyond A11, compare for example 5Lwt and 5Lmt1 (Fig. 2A). There is a good correlation between the binding affinity of PABP1 to the internal poly(A) and the activity of the 5L reporter (Fig. 2B). The importance of PABP1 for the activity of the 5L reporter was further demonstrated by siRNA-mediated PABP1 depletion. DF-1 cells were co-transfected with siRNA that targets PABP1 or control siRNA and with the reporter construct depicted in (Fig. 3A). Following 48 hour incubation the activity of the 5L reporter was assessed by measuring the luciferase activities from R-Luc and F-Luc. There is 80% decrease in the activity of the 5L reporter in the siPABP1 as compared to the control siRNA (Fig. 3A). When we used a reporter construct that lacks the 5L IRES and in which the R-Luc is under canonical cap-dependent translation and the F-Luc under ICR IRES control (S1A Figure, pR/ICR-F reporter), we found that siRNA-mediated PABP1 depletion caused only about 40% reduction in R-Luc activity with no apparent effect on the activity of F-Luc which is now controlled by the ICR IRES (S1A Figure), indicating the specific effect of PABP1 depletion on the activity of the 5L reporter. Northern blotting analysis shows that the decrease in the activity of the 5L reporter is not due to the stability or abundance of the reporter mRNAs (Fig. 3A and S1A Figure). Immunoblotting analysis reveals ∼75% decrease in the level of PABP1 in cells transfected with siPABP1, and as reported by another study [25] we also observed a concomitant decrease in the level of paip2, whereas the level of other translation factors such as eIF4E and eIF4A appeared unchanged (Fig. 3B). The above data indicate that PABP1 is involved in the regulation of the activity of the 5L IRES within the mRNA reporter, most likely via its interaction with the internal 5' poly(A). To gain further insights on how the PABP1 may mediate the regulation of the 5L IRES we investigated the interplay between the 3' end poly(A) tail, the internal poly(A) and the 5' cap structure using in vitro engineered mRNA reporters depicted in (Fig. 3C). The rationale behind this is that the activity of some cellular IRESes has been shown to be enhanced by poly(A) tail in the absence of PABP1 [26], [27]. Furthermore, we have previously shown that the 5L IRES efficiently initiates translation when cap-dependent translation initiation is inhibited [22]. Using RNA transfection experiments we show here that when the cap structure (7mGpppG) is replaced by the cap analogue (ApppG) the activity of the 5Lwt reporter increases by at least 5-fold in the presence of the poly(A) tail and only by ∼2-fold in the absence of the poly(A) tail (Fig. 3C). However, in the presence of the cap structure the absence of the poly(A) tail does not significantly alter the activity of the 5Lwt reporter (Fig. 3C), suggesting the possibility that the internal poly(A) within the 5L IRES may assume some of the functions of the poly(A) tail such as the direct recruitment of PABP1 to the 5L IRES located at the 5' end of the bicistronic mRNA reporter. Mutations within the internal poly(A), for example 5Lmt2&3, that simultaneously reduce the activity of the 5L reporter and the binding of the PABP1 also make the activity of the 5L reporter insensitive to the nature of the 5' and 3' ends of the bicistronic mRNA (Fig. 3C). In all the combinations tested the activity of the 5Lmt2&3 reporter is reduced by more than 70% (Fig. 3C, 5Lmt2&3-R/ICR-F). The importance of the interplay between the 3' poly(A) tail, the internal poly(A) within the 5L IRES for the activity of the 5L reporter was further demonstrated by using reporter mRNA that lacks the 5L IRES and in which the R-Luc is under canonical cap-dependent translation and the F-Luc under ICR IRES control (S1B Figure, R/ICR-F mRNA reporter). The data show that the activity of the ICR IRES is slightly enhanced in the absence of both the cap-structure and the 3' poly(A) tail and as expected the R-Luc activity which is now under canonical cap-dependent translation initiation was severely impaired in the absence the cap structure and the 3' poly(A) tail (S1B Figure). Northern blotting shows that variations in the activity of the 5L reporter are not due to the stability or the abundance of the reporter mRNAs (Fig. 3D and S1B Figure). These results indicate that the internal poly(A) and the poly(A) tail may work in synergy to enhance the activity of the 5L IRES within the mRNA reporter possibly by bridging the ends of the message in a cap-independent manner.

Bottom Line: We report a novel viral translational control strategy involving the recruitment of PABP1 to the 5' leader internal ribosome entry site (5L IRES) of an immediate-early (IE) bicistronic mRNA that encodes the neurovirulence protein (pp14) from the avian herpesvirus Marek's disease virus serotype 1 (MDV1).We provide evidence for the interaction between an internal poly(A) sequence within the 5L IRES and PABP1 which may occur concomitantly with the recruitment of PABP1 to the poly(A) tail.RNA interference and reverse genetic mutagenesis results show that a subset of virally encoded-microRNAs (miRNAs) targets the inhibitor of PABP1, known as paip2, and therefore plays an indirect role in PABP1 recruitment strategy by increasing the available pool of active PABP1.

View Article: PubMed Central - PubMed

Affiliation: The Pirbright Institute, Ash Road, Pirbright, Woking, Surrey, United Kingdom.

ABSTRACT
Poly(A) binding protein 1 (PABP1) plays a central role in mRNA translation and stability and is a target by many viruses in diverse manners. We report a novel viral translational control strategy involving the recruitment of PABP1 to the 5' leader internal ribosome entry site (5L IRES) of an immediate-early (IE) bicistronic mRNA that encodes the neurovirulence protein (pp14) from the avian herpesvirus Marek's disease virus serotype 1 (MDV1). We provide evidence for the interaction between an internal poly(A) sequence within the 5L IRES and PABP1 which may occur concomitantly with the recruitment of PABP1 to the poly(A) tail. RNA interference and reverse genetic mutagenesis results show that a subset of virally encoded-microRNAs (miRNAs) targets the inhibitor of PABP1, known as paip2, and therefore plays an indirect role in PABP1 recruitment strategy by increasing the available pool of active PABP1. We propose a model that may offer a mechanistic explanation for the cap-independent enhancement of the activity of the 5L IRES by recruitment of a bona fide initiation protein to the 5' end of the message and that is, from the affinity binding data, still compatible with the formation of 'closed loop' structure of mRNA.

Show MeSH
Related in: MedlinePlus