An Efficient System for Cap- and Poly(A)-Dependent Translation In Vitro

Svitkin, Yuri V.; Sonenberg, Nahum

doi:10.1385/1-59259-750-5:155

Cited by 63 publications

(112 citation statements)

References 21 publications

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“…Consequently, we used an alternative approach first noted by Svitkin and Sonenberg (2004), who used poly(A)-binding protein-interacting protein, PAIP, to remove PABP from the poly(A) tail (Khaleghpour et al 2001). In vitro, both Xenopus and human PAIP2 have a strong affinity for Xenopus and human PABP as well as ePAB (Fig.…”

Section: Epab Promotes Long Poly(a) Tailsmentioning

confidence: 99%

RINGO/cdk1 and CPEB mediate poly(A) tail stabilization and translational regulation by ePAB

Kim¹,

Richter²

2007

Genes Dev.

105

106

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One activity that controls mRNA translation in vertebrate oocytes, embryos, and neurons is cytoplasmic polyadenylation. In Xenopus oocytes, where much of the biochemistry of this process has been elucidated, nuclear pre-mRNAs containing a cytoplasmic polyadenylation element (CPE) in their 3 untranslated regions (UTRs) have long poly(A) tails; once the RNAs are spliced and transported to the cytoplasm, the tails are shortened. Following the resumption of meiosis, the poly(A) tails are lengthened and translation ensues. CPEB is a sequence-specific RNA-binding protein that coordinates these events and does so by binding to the CPE as well as several factors including Gld2, a poly(A) polymerase, and PARN [poly(A)-specific ribonuclease], a deadenylase. Here, we show that ePAB, embryonic poly(A)-binding protein, transiently associates with the polyadenylation complex; it initially interacts with CPEB, but after polyadenylation, it binds the poly(A) tail. ePAB dissociation from CPEB is regulated by RINGO (Rapid Inducer of G 2 /M progression in Oocytes), a cyclin B1-like cofactor that activates cdk1, a protein kinase that phosphorylates CPEB. Subsequent ePAB binding to the poly(A) tail is necessary to protect the homopolymer from degradation by deadenylating enzymes. Poly(A)-bound ePAB also interacts with eIF4G, which instigates translation initiation of CPEB-bound mRNAs.[Keywords: RINGO; CPEB; cytoplasmic polyadenylation; poly(A); translation; ePAB] Supplemental material is available at http://www.genesdev.org.

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Section: Epab Promotes Long Poly(a) Tailsmentioning

confidence: 99%

RINGO/cdk1 and CPEB mediate poly(A) tail stabilization and translational regulation by ePAB

Kim¹,

Richter²

2007

Genes Dev.

105

106

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“…Association of human eIF4GI and PABP was detected without addition of poly(A), and treatment of proteins with micrococcal nuclease did not decrease the interaction (19). We showed previously that, in a Krebs-2 cell extract, either PABP depletion or PABP inactivation by Paip2 inhibited the translation of poly(A) mRNA (29,33,38). Consistent with these observations, our current results demonstrate that PABP can mediate in cis translational stimulation of mRNAs that are devoid of a poly(A) tail.…”

Section: Discussionmentioning

confidence: 82%

“…In addition, eIF4GI failed to copurify with PABP that was pulled down with GST-Paip2-coupled beads (lane 5). These results indicate that eIF4GI and Paip2 binding to PABP is (33). Bound proteins were eluted in Laemmli sample buffer, resolved by SDS͞7.5% PAGE, and subjected to Western blotting using anti-eIF4GI and anti-PABP antibodies.…”

Section: Paip2mentioning

confidence: 88%

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A mechanism of translational repression by competition of Paip2 with eIF4G for poly(A) binding protein (PABP) binding

Karim

Svitkin

Kahvejian

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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The eukaryotic mRNA 3 poly(A) tail and the 5 cap cooperate to synergistically enhance translation. This interaction is mediated by the cap-binding protein eIF4E, the poly(A) binding protein (PABP), and eIF4G, a scaffolding protein that bridges between eIF4E and PABP to bring about the circularization of the mRNA. The translational repressor, Paip2 (PABP-interacting protein 2), inhibits translation by promoting the dissociation of PABP from poly(A). Here we report on the existence of an alternative mechanism by which Paip2 inhibits translation by competing with eIF4G for binding to PABP. We demonstrate that Paip2 can abrogate the translational activity of PABP, which is tethered to the 3 end of the mRNA. Thus, Paip2 can inhibit translation by a previously unrecognized mechanism, which is independent of its ability to disrupt PABP-poly(A) interaction.circularization ͉ translation initiation T ranslational control is an important means by which cells govern gene expression. Initiation, the rate-limiting step of translation, is often the target of translational control (1). This control involves in many circumstances the cap structure at the mRNA 5Ј end and the poly(A) tail at the mRNA 3Ј end. Although both mRNA terminal structures stimulate translation on their own, their combined translational enhancement is synergistic. Such a mechanism was demonstrated in yeast, plant, and mammalian systems (2, 3) and plays a key role during development of Xenopus, Drosophila, and mouse (4). Translational synergy was also recapitulated in vitro (5-9). Hence, the cap-poly(A) tail synergy represents a common paradigm for translational control.The mechanism by which the mRNA 3Ј poly(A) tail synergizes with the 5Ј cap to stimulate translation was first documented in yeast, where PABP was shown to interact directly with the eIF4G subunit of the cap-binding complex eIF4F (see below) (10, 11). In yeast, PABP is an essential protein because deletion of the PABP1 gene is lethal (12). However, because deletion of the eIF4G binding site in PABP causes only a mild effect on yeast cell growth (13), it is not clear that PABP-eIF4G interaction is the only mechanism by which PABP regulates translation and cell growth in yeast. In addition, Searfoss et al. (14) demonstrated that yeast Ski proteins inhibit the activity of eIF5 and eIF4B in 60S ribosomal subunit joining and that this inhibition is reversed by PABP. These results implicate an important role for PABP in 60S ribosomal subunit joining. In contrast to yeast, the expression of an eIF4G mutant that does not interact with PABP in Xenopus oocytes repressed translation of polyadenylated mRNAs and inhibited progesteroneinduced oocyte maturation (15).PABP contains four RNA-recognition motifs (RRMs) and a proline-rich C-terminal region, which is N-terminal to a highly evolutionarily conserved sequence termed PABC (see below) (16,17). eIF4F is composed of the cap-binding subunit eIF4E, an RNA-dependent ATPase͞ATP-dependent RNA helicase, eIF4A, and eIF4G (reviewed in ref. 18). The latter serves...

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“…In vitro translations in rabbit reticulocyte lysates and wheat germ extracts were carried out using 35 S-methionine as instructed by the manufacturer (Promega). Krebs translation extracts were prepared and used for in vitro translation reactions, as previously described [23].…”

Section: In Vitro Transcription/translation Reactionsmentioning

confidence: 99%

The Tie2 5′ untranslated region is inhibitory to 5′ end‐mediated translation initiation

Park

Lee

2006

FEBS Letters

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Tie2 is an endothelium-specific receptor tyrosine kinase required for normal blood vessel maturation, remodeling, and stability. Tie2 expression is also upregulated in various cancers implicating a role in tumor angiogenesis. Its mRNA transcript contains an unusually long (372 nucleotides) 5 0 untranslated region (UTR) with five upstream open reading frames (uORFs) and an internal ribosome entry site (IRES) that allows this mRNA to be translated under hypoxic conditions. This sets up an alternative initiation pathway with the potential to clash with 5 0 end-mediated initiation from the same template. Herein, we define experimental conditions under which the Tie2 IRES is not active, allowing us to assess the contribution of the 5 0 UTR to cap-dependent translation on the Tie2 transcript. We find that the Tie2 5 0 UTR is inhibitory to translation initiation with ribosome flow decreasing following encounters with each uORF. No single uORF was found to harbor significant cisacting inhibitory activity. Our results suggest that the uORFs within the Tie2 5 0 UTR serve to decrease the percent of ribosomes competent for reinitiation as these traverse the mRNA 5 0 UTR, thus minimizing interference with the IRES.

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An Efficient System for Cap- and Poly(A)-Dependent Translation In Vitro

Cited by 63 publications

References 21 publications

RINGO/cdk1 and CPEB mediate poly(A) tail stabilization and translational regulation by ePAB

RINGO/cdk1 and CPEB mediate poly(A) tail stabilization and translational regulation by ePAB

A mechanism of translational repression by competition of Paip2 with eIF4G for poly(A) binding protein (PABP) binding

The Tie2 5′ untranslated region is inhibitory to 5′ end‐mediated translation initiation

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