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Evidence that poly(A) binding protein C1 binds nuclear pre-mRNA poly(A) tails

Department of Biochemistry and Biophysics , University of Rochester, Rochester, New York, United States
Molecular and Cellular Biology (Impact Factor: 5.04). 05/2006; 26(8):3085-97. DOI: 10.1128/MCB.26.8.3085-3097.2006
Source: PubMed

ABSTRACT In mammalian cells, poly(A) binding protein C1 (PABP C1) has well-known roles in mRNA translation and decay in the cytoplasm. However, PABPC1 also shuttles in and out of the nucleus, and its nuclear function is unknown. Here, we show that PABPC1, like the major nuclear poly(A) binding protein PABPN1, associates with nuclear pre-mRNAs that are polyadenylated and intron containing. PABPC1 does not bind nonpolyadenylated histone mRNA, indicating that the interaction of PABPC1 with pre-mRNA requires a poly(A) tail. Consistent with this conclusion, UV cross-linking results obtained using intact cells reveal that PABPC1 binds directly to pre-mRNA poly(A) tails in vivo. We also show that PABPC1 immunopurifies with poly(A) polymerase, suggesting that PABPC1 is acquired by polyadenylated transcripts during poly(A) tail synthesis. Our findings demonstrate that PABPC1 associates with polyadenylated transcripts earlier in mammalian mRNA biogenesis than previously thought and offer insights into the mechanism by which PABPC1 is recruited to newly synthesized poly(A). Our results are discussed in the context of pre-mRNA processing and stability and mRNA trafficking and the pioneer round of translation.

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    • "This suggests that PAF1c plays a role in mRNA nuclear export in addition to its role in 3′ end processing. In this regard, immunopurified PAF1c associates with the cytoplasmic poly(A) binding protein PABPC1 (our unpublished data), which is thought to function in mRNA nuclear export in yeast and mammals (Brune et al. 2005; Hosoda et al. 2006). PAF1c depletion might cause inefficient PABPC1 recruitment to poly(A) tails and defective nuclear export (Fig. 7). "
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    ABSTRACT: Polyadenylation of mRNA precursors is frequently coupled to transcription by RNA polymerase II. Although this coupling is known to involve interactions with the C-terminal domain of the RNA polymerase II largest subunit, the possible role of other factors is not known. Here we show that a prototypical transcriptional activator, GAL4-VP16, stimulates transcription-coupled polyadenylation in vitro. In the absence of GAL4-VP16, specifically initiated transcripts accumulated but little polyadenylation was observed, while in its presence polyadenylation was strongly enhanced. We further show that this stimulation requires the transcription elongation-associated PAF complex (PAF1c), as PAF1c depletion blocked GAL4-VP16-stimulated polyadenylation. Furthermore, knockdown of PAF subunits by siRNA resulted in decreased 3' cleavage, and nuclear export, of mRNA in vivo. Finally, we show that GAL4-VP16 interacts directly with PAF1c and recruits it to DNA templates. Our results indicate that a transcription activator can stimulate transcription-coupled 3' processing and does so via interaction with PAF1c.
    Molecular cell 02/2011; 41(4):409-18. DOI:10.1016/j.molcel.2011.01.022 · 14.46 Impact Factor
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    • "PABPC1 is mostly cytoplasmic but also nuclear (Hosoda et al. 2006), and its cellular distribution was not altered by hemin or Df treatment (Fig. 2). Like PABPN1, PABPC1 coimmunoprecipitates with CBP80 (Chiu et al. 2004) and begins associating with newly synthesized transcripts prior to splicing (Hosoda et al. 2006). Furthermore, PABPC1 appears to activate pioneer rounds of translation similarly to how it activates steady-state rounds of translation, since Paip2, which inhibits the interaction of PABPC1 with poly(A), inhibits NMD (Chiu et al. 2004). "
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    ABSTRACT: Mammalian mRNAs lose and acquire proteins throughout their life span while undergoing processing, transport, translation, and decay. How translation affects messenger RNA (mRNA)-protein interactions is largely unknown. The pioneer round of translation uses newly synthesized mRNA that is bound by cap-binding protein 80 (CBP80)-CBP20 (also known as the cap-binding complex [CBC]) at the cap, poly(A)-binding protein N1 (PABPN1) and PABPC1 at the poly(A) tail, and, provided biogenesis involves pre-mRNA splicing, exon junction complexes (EJCs) at exon-exon junctions. Subsequent rounds of translation engage mRNA that is bound by eukaryotic translation initiation factor 4E (eIF4E) at the cap and PABPC1 at the poly(A) tail, but that lacks detectable EJCs and PABPN1. Using the level of intracellular iron to regulate the translation of specific mRNAs, we show that translation promotes not only removal of EJC constituents, including the eIF4AIII anchor, but also replacement of PABPN1 by PABPC1. Remarkably, translation does not affect replacement of CBC by eIF4E. Instead, replacement of CBC by eIF4E is promoted by importin beta (IMPbeta): Inhibiting the binding of IMPbeta to the complex of CBC-IMPalpha at an mRNA cap using the IMPalpha IBB (IMPbeta-binding) domain or a RAN variant increases the amount of CBC-bound mRNA and decreases the amount of eIF4E-bound mRNA. Our studies uncover a previously unappreciated role for IMPbeta and a novel paradigm for how newly synthesized messenger ribonucleoproteins (mRNPs) are matured.
    Genes & development 11/2009; 23(21):2537-50. DOI:10.1101/gad.1817109 · 12.64 Impact Factor
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    • "At present, analyzing NMD provides the only functional assay for this round of translation, which constitutes only a very small fraction of cellular translation (Ishigaki et al., 2001; Lejeune et al., 2002). However, the pioneer translation initiation complex shares many constituents with the translation initiation complex that supports the bulk of cellular protein synthesis, including eIF2, eIF3, eIF4G, PABPC1, and ribosomes (Chiu et al., 2004; Hosoda et al., 2006; Ishigaki et al., 2001; Lejeune et al., 2002, 2004). Moreover, NMD, like the bulk of cellular translation, is inhibited by suppressor tRNA, anisomysin, cycloheximide, emetine, pactamycin, and puromycin (Isken and Maquat, 2007). "
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    ABSTRACT: In mammalian cells, nonsense-mediated mRNA decay (NMD) generally requires that translation terminates sufficiently upstream of a post-splicing exon junction complex (EJC) during a pioneer round of translation. The subsequent binding of Upf1 to the EJC triggers Upf1 phosphorylation. We provide evidence that phospho-Upf1 functions after nonsense codon recognition during steps that involve the translation initiation factor eIF3 and mRNA decay factors. Phospho-Upf1 interacts directly with eIF3 and inhibits the eIF3-dependent conversion of 40S/Met-tRNA(i)(Met)/mRNA to translationally competent 80S/Met-tRNA(i)(Met)/mRNA initiation complexes to repress continued translation initiation. Consistent with phospho-Upf1 impairing eIF3 function, NMD fails to detectably target nonsense-containing transcripts that initiate translation independently of eIF3 from the CrPV IRES. There is growing evidence that translational repression is a key transition that precedes mRNA delivery to the degradation machinery. Our results uncover a critical step during NMD that converts a pioneer translation initiation complex to a translationally compromised mRNP.
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