InteractiveFly: GeneBrief

Pabp2: Biological Overview | References

Translational control of maternal mRNA through regulation of poly(A) tail length is crucial during early development. The nuclear poly(A) binding protein, PABP2, was identified biochemically from its role in nuclear polyadenylation. This study analyzed the in vivo function of PABP2 in Drosophila. PABP2 is required in vivo for polyadenylation, and Pabp2 function, including poly(A) polymerase stimulation, is essential for viability. An unanticipated cytoplasmic function is demonstrated for PABP2 during early development. In contrast to its role in nuclear polyadenylation, cytoplasmic PABP2 acts to shorten the poly(A) tails of specific mRNAs. PABP2, together with the deadenylase CCR4, regulates the poly(A) tails of oskar and cyclin B mRNAs, both of which are also controlled by cytoplasmic polyadenylation. Both Cyclin B protein levels and embryonic development depend upon this regulation. These results identify a regulator of maternal mRNA poly(A) tail length and highlight the importance of this mode of translational control (Benoit, 2005).

During early development in most species, regulation of gene expression is strictly posttranscriptional. One major posttranscriptional regulatory mechanism involves variations in poly(A) tail length, which regulate mRNA expression by affecting both mRNA stability and translation. Cytoplasmic changes in mRNA poly(A) tail length by deadenylation and polyadenylation play, thus, an essential role in controlling the production of key proteins during early development. While cytoplasmic polyadenylation has been studied extensively, the mechanisms underlying the control of poly(A) tail length in the cytoplasm are unknown (Benoit, 2005).

In Xenopus oocytes, cytoplasmic poly(A) tail elongation requires cis elements, including the cytoplasmic polyadenylation element (CPE) located in the 3' UTR of mRNAs and the nuclear polyadenylation signal, AAUAAA. CPEs are bound by the CPE binding protein (CPEB; see Drosophila CPEB, Orb), a primary factor in cytoplasmic polyadenylation, which also requires a poly(A) polymerase (PAP) and a complex that binds the AAUAAA element, called the Cleavage and Polyadenylation Specificity Factor (CPSF) (Mendez, 2000). Cytoplasmic elongation of the poly(A) tail leads to translational activation by remodeling the mRNP: in the repressed state, a translational repressor called Maskin binds to CPEB and eIF4E, the cap binding initiation factor, and precludes the eIF4E-eIF4G interaction that is required for translation initiation. When polyadenylation occurs, the elongated poly(A) tail is bound by the cytoplasmic poly(A) binding protein, PABP, which then interacts with eIF4G. This promotes the association between eIF4E and eIF4G (Cao, 2002), thereby allowing translation initiation (Benoit, 2005).

The role of the poly(A) tail in translational control in Drosophila is more controversial. In early embryos, a long poly(A) tail is both necessary and sufficient to induce translation of bicoid and Toll mRNAs, which encode the anterior morphogen and a determinant of dorsoventral polarity, respectively (Salles, 1994; Schisa, 1998). Translation of the posterior determinant oskar (osk) mRNA, is also highly regulated during oogenesis. Translation is repressed until mid-oogenesis, and subsequently in the oocyte, as osk mRNA is being transported to the posterior pole. During this transport, a major translational repressor is Bruno, whose mechanism of action was found to be independent of the 5' cap and the poly(A) tail in vitro. A recent study, however, has identified a new translational repressor of osk mRNA, called Cup, which interacts with both Bruno and eIF4E. This strongly suggests that Cup/Bruno-mediated translational repression is cap-dependent, acting to prevent the eIF4E-eIF4G interaction in a manner similar to Maskin. While the mechanism underlying the release of Bruno repression at the posterior pole is unknown, accumulation of Osk protein at the posterior of the oocyte requires osk mRNA cytoplasmic polyadenylation involving Orb, the Drosophila homolog of CPEB and Drosophila PAP (Benoit, 2005 and references therein).

Before cytoplasmic regulation can occur, poly(A) tails are added to mRNAs in the nucleus in a cotranscriptional reaction involving endonucleolytic cleavage followed by polyadenylation (Wahle, 1999). In mammals, the reaction involves two signals flanking the cleavage site, the upstream polyadenylation signal, AAUAAA, and a downstream GU-rich element. Cleavage requires several cleavage factors, including CPSF, and polyadenylation of cleaved RNAs can be recapitulated in vitro with CPSF, PAP, and the nuclear poly(A) binding protein, PABP2 (PABPN1 in mammals) (Bienroth, 1993). While PAP has a very low affinity for, and binds aspecifically to, RNA, specificity is achieved through the recognition of the AAUAAA element by CPSF, which then tethers PAP to the RNA by direct protein-protein interaction. While CPSF thus stimulates PAP, complete stimulation occurs only in the additional presence of PABP2, which binds the poly(A) tail when it has reached ten residues in size. At this point, the reaction becomes processive, and a complete poly(A) tail is synthesized very rapidly and without dissociation of PAP from the RNA. PABP2 has a second function in nuclear polyadenylation, namely, to control poly(A) tail length: once the poly(A) tail has reached full length (250 residues in mammalian cells), the reaction becomes slow and distributive (Wahle, 1995). These two functions of PABP2 in nuclear polyadenylation are carried out by different domains of the protein, and they can be uncoupled by point mutations (Kerwitz, 2003). These data have led to a model in which multiple PABP2 proteins coat the growing poly(A) tail, with only one of them directly interacting with PAP (Benoit, 2005).

Although PABP2 is nuclear at steady-state levels in somatic cells, it shuttles from nuclear to cytoplasmic compartments (Calado, 2000). While a possible role for PABP2 in mRNA export has not been investigated, PABP2 has been found to be associated with an mRNA during its docking at the nuclear pore, and it was present on the cytoplasmic side of the nuclear envelope (Bear, 2003). This suggests that the exchange between nuclear PABP2 and cytoplasmic PABP on poly(A) tails occurs in the cytoplasm (Benoit, 2005).

This study used Pabp2 mutants to address the in vivo role of PABP2 in Drosophila.PABP2 has a role in poly(A) tail lengthening in somatic tissues, and that this function is essential for viability. The cytoplasmic presence of PABP2 is described in oocytes and early embryos (Benoit, 1999; full text of article), and it is shown that cytoplasmic PABP2 binds to poly(A) tails at these stages and shortens poly(A) tails of specific mRNAs, in conjunction with the deadenylase CCR4. Cytoplasmic poly(A) tail length control by PABP2 is essential for development, as embryos depleted of PABP2 show early developmental arrest, with elongated poly(A) tails of key maternal mRNAs (Benoit, 2005).

An important set of biochemical data has led to a precise description of PABP2 function in nuclear polyadenylation (Kühn, 2004). In in vitro polyadenylation assays, PABP2 has two distinct roles: it stimulates PAP to make polyadenylation processive, and it controls poly(A) tail length, with polyadenylation becoming distributive once the tail has reached 250 nucleotides. This length control involves a measurement of the poly(A) tail by PABP2 (Wahle, 1995). Drosophila PABP2 tested in mammalian polyadenylation-reconstituted assays also shows these two functions (Benoit, 1999). Using a null Pabp2 allele, this study shows that poly(A) tails measured either on total or on individual mRNAs are shorter in Pabp2 mutants than they are in wild-type, consistent with a role for PABP2 in poly(A) tail elongation. The short poly(A) tails in mutant embryos appear to result from deadenylation of existing mRNAs and progressive reduction of new mRNA synthesis as the PABP2 level decreases. Poly(A) tails of newly synthesized Hsp70 mRNA were of similar size in the Pabp2⁵⁵ mutant and in wild-type, although they were present in a small amount in the mutant and were thought to be synthesized with the remaining maternal PABP2. The finding that newly synthesized mRNAs with short poly(A) tails do not accumulate when PABP2 is limiting suggests that PABP2 is absolutely required for polyadenylation and that PAP is unable to produce stable poly(A) tails in the absence of PABP2. In agreement with this, it was found that PABP2 is essential for viability, and specifically for cell viability, since Pabp2⁵⁵ mutant somatic or germline clones were found to not survive. Moreover, lethality in the absence of PAPB2 may be caused by a lack of PAP stimulation, since a Pabp2 transgene bearing a point mutation that prevents PAP stimulation is unable to rescue the lethality of the null allele Pabp2⁵⁵. Taken together, these results strongly suggest that the function of PABP2 in mRNA polyadenylation is essential, and that PAP in the absence of PABP2 is incapable of producing stable polyadenylated mRNAs (Benoit, 2005).

One important conclusion presented in this paper is the identification of an unexpected function for PABP2 in regulating poly(A) tail length of cytoplasmic mRNAs during early development. Using two hypomorphic Pabp2 alleles, it was found that a reduced amount of PABP2 leads to elongated poly(A) tails in two mRNAs regulated by cytoplasmic polyadenylation. Three sets of data indicate that this function of PABP2 is cytoplasmic: (1) the poly(A) tail elongation phenotype on the involved mRNAs is the opposite of the Pabp2 mutant phenotype on total mRNAs, also visible in the same RNA preparations on the control sop mRNA; (2) a reduced level of PABP2 restores longer poly(A) tails on osk and cyc B mRNAs, but not on sop mRNA, in orb^mel ovaries in which cytoplasmic polyadenylation is impaired; (3) PABP2 is cytoplasmic in oocytes, and in early embryos prior to the onset of zygotic transcription, it binds poly(A) tails of mRNAs that are also bound by the cytoplasmic proteins Orb and PABP, and it is recruited into cytoplasmic cyc B mRNA particles (Benoit, 2005).

This cytoplasmic function of PABP2 is essential for early development. PABP2 is required to shorten poly(A) tails of, at least, osk and cyc B mRNAs, and in Pabp2 mutant germline clones the lengthening of cyc B poly(A) tails correlates with higher levels of Cyc B protein and with embryonic phenotypes similar to those produced by a high dosage of maternal Cyc B. Misregulation of other maternal mRNAs could also contribute to the lethality of embryos from these germline clones, since cytoplasmic PABP2 probably regulates several of them. The maternal-effect embryonic lethality of Pabp2⁶ is strongly rescued by the Pabp2-I61S transgene, which lacks the nuclear function of PAP stimulation; this suggests that this lethality results from a defect in the cytoplasmic function of PABP2. In addition, the synergistic effect of the simultaneous decrease in PABP2 and CCR4 amounts in the female germline, which leads to important embryonic lethality and elongated poly(A) tails of osk and cyc B mRNAs, also indicates an essential function of cytoplasmic PABP2 in shortening poly(A) tails at these stages. Finally, consistent with PABP2 playing a major role in the cytoplasm during early development is the recent identification (Good, 2004) of a cytoplasmic PABP2 specific to embryos in Xenopus and mouse (Benoit, 2005).

Several lines of evidence suggest that PABP2 regulates poly(A) tail length in the cytoplasm by using a different mechanism than that used during nuclear polyadenylation. Termination of poly(A) tail elongation during nuclear polyadenylation is thought to result from a PABP2-dependent remodeling of the polyadenylation complex that blocks PAP stimulation. This remodeling depends on the complete coating of the poly(A) tail by PABP2 (Kerwitz, 2003). In sharp contrast, studies of cytoplasmic polyadenylation in Drosophila embryos suggest that the reaction is not processive and does not involve PAP stimulation by PABP2. Cytoplasmic polyadenylation of bicoid mRNA in embryos is slow, with poly(A) tail elongation depending on the level of PAP (Juge, 2002). Very long poly(A) tails are produced by overexpression of PAP, without poly(A) tail length control. Consistent with this, it was found that cytoplasmic PABP and PABP2 are present on the same mRNA poly(A) tails in ovary and early embryo extracts, thereby precluding complete coating of the poly(A) tail by PABP2 (Benoit, 2005).

In yeast, poly(A) tail length control involves deadenylation by the PAN (Pan2/Pan3) deadenylase, which is activated by poly(A) tail bound PABP (Brown, 1998). It is proposed that, similarly, during early Drosophila development, cytoplasmic PABP2 controls the poly(A) tail length of key mRNAs whose turnover and translatability are specifically regulated, by modulating the activity of a deadenylase. Poly(A) tail length control of these mRNAs would thus be achieved by the balance between cytoplasmic polyadenylation and deadenylation. A major deadenylation complex in Drosophila is the CCR4/NOT complex, in which CCR4 is the deadenylase (Temme, 2004). ccr4 function is essential in the female germline, where it regulates poly(A) tail lengths of cyc B mRNA and other cell cycle regulators (Morris, 2005). This study found that Pabp2 and ccr4 act in conjunction in shortening poly(A) tails of specific mRNAs, consistent with a possible role of PABP2 in stimulation of CCR4 activity. In yeast, deadenylation by the CCR4/NOT complex is inhibited in vitro by PABP (Tucker, 2002). If this regulation is conserved in metazoans, the presence of PABP2 on poly(A) tails could modulate this effect of PABP (Benoit, 2005).

Search PubMed for articles about Drosophila Pabp2

Bear, D.G., et al. (2003). Nuclear poly(A)-binding protein PABPN1 is associated with RNA polymerase II during transcription and accompanies the released transcript to the nuclear pore. Exp. Cell Res. 286: 332-344. PubMed citation: 12749861

Benoit, B., et al. (1999). The Drosophila poly(A)-binding protein II is ubiquitous throughout Drosophila development and has the same function in mRNA polyadenylation as its bovine homolog in vitro. Nucleic Acids Res. 27(19): 3771-8. Medline abstract: 10481015

Benoit, B., et al. (2005). An essential cytoplasmic function for the nuclear poly(A) binding protein, PABP2, in poly(A) tail length control and early development in Drosophila. Dev. Cell. 9(4): 511-22. Medline abstract: 16198293

Bienroth, S., Keller, W. and Wahle, E. (1993). Assembly of a processive mRNA polyadenylation complex. EMBO J. 12: 585-594. Medline abstract: 8440247

Brown, C. E. and Sachs, A. B. (1998). Poly(A) tail length control in Saccharomyces cerevisiae occurs by message-specific deadenylation. Mol. Cell. Biol. 18: 6548-6559. Medline abstract: 9774670

Calado, A., et al. (2000). Deciphering the cellular pathway for transport of poly(A)-binding protein II. RNA 6: 245-256. Medline abstract: 10688363

Cao, Q. and Richter, J. D. (2002). Dissolution of the maskin-eIF4E complex by cytoplasmic polyadenylation and poly(A)-binding protein controls cyclin B1 mRNA translation and oocyte maturation. EMBO J. 21: 3852-3862. Medline abstract: 12110596

Good, P.J., Abler, L., Herring, D. and Sheets, M. D. (2004). Xenopus embryonic poly(A) binding protein 2 (ePABP2) defines a new family of cytoplasmic poly(A) binding proteins expressed during the early stages of vertebrate development. Genesis 38: 166-175. Medline abstract: 15083517

Juge, F., Zaessinger, S., Temme, C., Wahle, E. and Simonelig, M. (2002). Control of poly(A) polymerase level is essential to cytoplasmic polyadenylation and early development in Drosophila. EMBO J. 21: 6603-6613. Medline abstract: 12456666

Kerwitz, Y., et al. (2003). Stimulation of poly(A) polymerase through a direct interaction with the nuclear poly(A) binding protein allosterically regulated by RNA, EMBO J. 22: 3705-3714. Medline abstract: 12853485

Kühn, U. and Wahle, E. (2004). Structure and function of poly(A) binding proteins. Biochim. Biophys. Acta 1678: 67-84. Medline abstract: 15157733

Mendez, R., et al. (2000). Phosphorylation of CPEB by Eg2 mediates the recruitment of CPSF into an active cytoplasmic polyadenylation complex, Mol. Cell 6: 1253-1259. Medline abstract: 11106762

Morris, Z., Hong, A., Lilly, M. A. and Lehmann, R. (2005). twin, a CCR4 homolog, regulates cyclin poly(A) tail length to permit Drosophila oogenesis. Development 132: 1165-1174. Medline abstract: 15703281

Salles, F. J. et al. (1994). Coordinate initiation of Drosophila development by regulated polyadenylation of maternal messenger RNAs. Science 266: 1996-1999. Medline abstract: 7801127

Schisa, J. A. and Strickland, S. (1998). Cytoplasmic polyadenylation of Toll mRNA is required for dorsal-ventral patterning in Drosophila embryogenesis. Development 125: 2995-3003. Medline abstract: 9655821

Temme, C., et al. (2004). A complex containing the CCR4 and CAF1 proteins is involved in mRNA deadenylation in Drosophila. EMBO J. 23. 2862-2871. Medline abstract: 15215893

Tucker, M., et al. (2002). Ccr4p is the catalytic subunit of a Ccr4p/Pop2p/Notp mRNA deadenylase complex in Saccharomyces cerevisiae, EMBO J. 21: 1427-1436. Medline abstract: 11889048

Wahle, E. and Rüegsegger, U. (1999). 3'-end processing of pre-mRNA in eukaryotes, FEMS Microbiol. Rev. 23: 277-295. Medline abstract: 10371034

date revised: 15 February 2008

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