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Global mRNA selection mechanisms for translation initiation.

Costello J, Castelli LM, Rowe W, Kershaw CJ, Talavera D, Mohammad-Qureshi SS, Sims PF, Grant CM, Pavitt GD, Hubbard SJ, Ashe MP - Genome Biol. (2015)

Bottom Line: Components of the closed loop complex are highly relevant for many mRNAs, but some heavily translated mRNAs interact poorly with this machinery.Therefore, alternative, possibly Pab1p-dependent mechanisms likely exist to load ribosomes effectively onto mRNAs.Finally, these studies identify and characterize a complex self-regulatory circuit for the yeast 4E-BPs.

View Article: PubMed Central - PubMed

ABSTRACT

Background: The selection and regulation of individual mRNAs for translation initiation from a competing pool of mRNA are poorly understood processes. The closed loop complex, comprising eIF4E, eIF4G and PABP, and its regulation by 4E-BPs are perceived to be key players. Using RIP-seq, we aimed to evaluate the role in gene regulation of the closed loop complex and 4E-BP regulation across the entire yeast transcriptome.

Results: We find that there are distinct populations of mRNAs with coherent properties: one mRNA pool contains many ribosomal protein mRNAs and is enriched specifically with all of the closed loop translation initiation components. This class likely represents mRNAs that rely heavily on the closed loop complex for protein synthesis. Other heavily translated mRNAs are apparently under-represented with most closed loop components except Pab1p. Combined with data showing a close correlation between Pab1p interaction and levels of translation, these data suggest that Pab1p is important for the translation of these mRNAs in a closed loop independent manner. We also identify a translational regulatory mechanism for the 4E-BPs; these appear to self-regulate by inhibiting translation initiation of their own mRNAs.

Conclusions: Overall, we show that mRNA selection for translation initiation is not as uniformly regimented as previously anticipated. Components of the closed loop complex are highly relevant for many mRNAs, but some heavily translated mRNAs interact poorly with this machinery. Therefore, alternative, possibly Pab1p-dependent mechanisms likely exist to load ribosomes effectively onto mRNAs. Finally, these studies identify and characterize a complex self-regulatory circuit for the yeast 4E-BPs.

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The closed loop complex and 4E-BP repression complexes are maintained during purification. (A) A diagram depicting the closed loop complex with eIF4E (4E), eIF4G (4G) and PABP bound to the mRNA; the 4E-BPs Caf20p and Eap1p are also represented competing with the eIF4E-eIF4G interaction. (B) Western blots probed with a protein A peroxidase (PAP) conjugate which detects the TAP-tagged proteins labeled above the blots. Input, flowthrough and eluates are presented on the top, middle and bottom blots respectively. The vast majority of TAP-tagged proteins purified appear in the eluates. (C) Western blots probed with PAP or the antibodies depicted on the right, which detect the components of the closed loop complex (either TAP-tagged or not) depicted on the left. Samples are eluates from TAP-affinity chromatography using strains bearing the TAP-tagged protein depicted above each lane. (D) As for (C), except the components of the 4E-BP complexes were assessed by TAP affinity chromatography.
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Fig1: The closed loop complex and 4E-BP repression complexes are maintained during purification. (A) A diagram depicting the closed loop complex with eIF4E (4E), eIF4G (4G) and PABP bound to the mRNA; the 4E-BPs Caf20p and Eap1p are also represented competing with the eIF4E-eIF4G interaction. (B) Western blots probed with a protein A peroxidase (PAP) conjugate which detects the TAP-tagged proteins labeled above the blots. Input, flowthrough and eluates are presented on the top, middle and bottom blots respectively. The vast majority of TAP-tagged proteins purified appear in the eluates. (C) Western blots probed with PAP or the antibodies depicted on the right, which detect the components of the closed loop complex (either TAP-tagged or not) depicted on the left. Samples are eluates from TAP-affinity chromatography using strains bearing the TAP-tagged protein depicted above each lane. (D) As for (C), except the components of the 4E-BP complexes were assessed by TAP affinity chromatography.

Mentions: Of these mechanisms, the closed loop model (Figure 1A), where a series of protein-RNA and protein-protein interactions bridge a molecular connection between the two ends of the mRNA, has received by far the most attention. The realization that both the 5’ cap and the 3’ poly(A) tail contributed to the translational efficiency of specific reporter mRNAs led to the first suggestions of a closed loop [24,25]. A critical development supporting such a model was that in electroporation studies and various in vitro translation systems, the presence of both a 5’ cap and a 3’ poly(A) tail on a mRNA resulted in a synergistic increase in translation initiation relative to that observed for mRNAs with only a single modification [26–28]. With increased biochemical understanding of the protein components and interactions involved in mRNA recognition came refinements to the model: where eIF4E interacts with the mRNA cap, PABP interacts with the poly(A) tail and eIF4G bridges the two ends of the mRNA leading to the formation of a closed loop [18]. Such a closed loop was observed with atomic force microscopy using purified components [29], and Pab1p has been shown to enhance the interaction of the eIF4G-eIF4E complex with the mRNA [30], with more recent data pointing to a dynamic interaction model where RNA structural alterations also impact upon the efficiency and lifespan of the interactions [31]. Further support for the closed loop model comes from yeast genetics. Mutations that affect interactions between the closed loop components inhibit translation initiation and prevent the cap-poly(A) synergy observed in translation extracts [32,33]. In addition, mutations affecting the eIF4E-eIF4G interaction are synthetic lethal in combination with mutations impacting upon the eIF4G-Pab1p and eIF4G-RNA interactions [33,34].Figure 1


Global mRNA selection mechanisms for translation initiation.

Costello J, Castelli LM, Rowe W, Kershaw CJ, Talavera D, Mohammad-Qureshi SS, Sims PF, Grant CM, Pavitt GD, Hubbard SJ, Ashe MP - Genome Biol. (2015)

The closed loop complex and 4E-BP repression complexes are maintained during purification. (A) A diagram depicting the closed loop complex with eIF4E (4E), eIF4G (4G) and PABP bound to the mRNA; the 4E-BPs Caf20p and Eap1p are also represented competing with the eIF4E-eIF4G interaction. (B) Western blots probed with a protein A peroxidase (PAP) conjugate which detects the TAP-tagged proteins labeled above the blots. Input, flowthrough and eluates are presented on the top, middle and bottom blots respectively. The vast majority of TAP-tagged proteins purified appear in the eluates. (C) Western blots probed with PAP or the antibodies depicted on the right, which detect the components of the closed loop complex (either TAP-tagged or not) depicted on the left. Samples are eluates from TAP-affinity chromatography using strains bearing the TAP-tagged protein depicted above each lane. (D) As for (C), except the components of the 4E-BP complexes were assessed by TAP affinity chromatography.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig1: The closed loop complex and 4E-BP repression complexes are maintained during purification. (A) A diagram depicting the closed loop complex with eIF4E (4E), eIF4G (4G) and PABP bound to the mRNA; the 4E-BPs Caf20p and Eap1p are also represented competing with the eIF4E-eIF4G interaction. (B) Western blots probed with a protein A peroxidase (PAP) conjugate which detects the TAP-tagged proteins labeled above the blots. Input, flowthrough and eluates are presented on the top, middle and bottom blots respectively. The vast majority of TAP-tagged proteins purified appear in the eluates. (C) Western blots probed with PAP or the antibodies depicted on the right, which detect the components of the closed loop complex (either TAP-tagged or not) depicted on the left. Samples are eluates from TAP-affinity chromatography using strains bearing the TAP-tagged protein depicted above each lane. (D) As for (C), except the components of the 4E-BP complexes were assessed by TAP affinity chromatography.
Mentions: Of these mechanisms, the closed loop model (Figure 1A), where a series of protein-RNA and protein-protein interactions bridge a molecular connection between the two ends of the mRNA, has received by far the most attention. The realization that both the 5’ cap and the 3’ poly(A) tail contributed to the translational efficiency of specific reporter mRNAs led to the first suggestions of a closed loop [24,25]. A critical development supporting such a model was that in electroporation studies and various in vitro translation systems, the presence of both a 5’ cap and a 3’ poly(A) tail on a mRNA resulted in a synergistic increase in translation initiation relative to that observed for mRNAs with only a single modification [26–28]. With increased biochemical understanding of the protein components and interactions involved in mRNA recognition came refinements to the model: where eIF4E interacts with the mRNA cap, PABP interacts with the poly(A) tail and eIF4G bridges the two ends of the mRNA leading to the formation of a closed loop [18]. Such a closed loop was observed with atomic force microscopy using purified components [29], and Pab1p has been shown to enhance the interaction of the eIF4G-eIF4E complex with the mRNA [30], with more recent data pointing to a dynamic interaction model where RNA structural alterations also impact upon the efficiency and lifespan of the interactions [31]. Further support for the closed loop model comes from yeast genetics. Mutations that affect interactions between the closed loop components inhibit translation initiation and prevent the cap-poly(A) synergy observed in translation extracts [32,33]. In addition, mutations affecting the eIF4E-eIF4G interaction are synthetic lethal in combination with mutations impacting upon the eIF4G-Pab1p and eIF4G-RNA interactions [33,34].Figure 1

Bottom Line: Components of the closed loop complex are highly relevant for many mRNAs, but some heavily translated mRNAs interact poorly with this machinery.Therefore, alternative, possibly Pab1p-dependent mechanisms likely exist to load ribosomes effectively onto mRNAs.Finally, these studies identify and characterize a complex self-regulatory circuit for the yeast 4E-BPs.

View Article: PubMed Central - PubMed

ABSTRACT

Background: The selection and regulation of individual mRNAs for translation initiation from a competing pool of mRNA are poorly understood processes. The closed loop complex, comprising eIF4E, eIF4G and PABP, and its regulation by 4E-BPs are perceived to be key players. Using RIP-seq, we aimed to evaluate the role in gene regulation of the closed loop complex and 4E-BP regulation across the entire yeast transcriptome.

Results: We find that there are distinct populations of mRNAs with coherent properties: one mRNA pool contains many ribosomal protein mRNAs and is enriched specifically with all of the closed loop translation initiation components. This class likely represents mRNAs that rely heavily on the closed loop complex for protein synthesis. Other heavily translated mRNAs are apparently under-represented with most closed loop components except Pab1p. Combined with data showing a close correlation between Pab1p interaction and levels of translation, these data suggest that Pab1p is important for the translation of these mRNAs in a closed loop independent manner. We also identify a translational regulatory mechanism for the 4E-BPs; these appear to self-regulate by inhibiting translation initiation of their own mRNAs.

Conclusions: Overall, we show that mRNA selection for translation initiation is not as uniformly regimented as previously anticipated. Components of the closed loop complex are highly relevant for many mRNAs, but some heavily translated mRNAs interact poorly with this machinery. Therefore, alternative, possibly Pab1p-dependent mechanisms likely exist to load ribosomes effectively onto mRNAs. Finally, these studies identify and characterize a complex self-regulatory circuit for the yeast 4E-BPs.

Show MeSH