The Human Poly(A)-binding Protein 1 Shuttles between the Nucleus and the Cytoplasm

Abstract

We have studied the intracellular localization of poly(A)-binding protein 1 (PABP1) by indirect immunofluorescence as well as by tagging with the green fluorescent protein (GFP) in living cells. We show that PABP1 is able to enter the nucleus. Accumulation of PABP1 in the nuclei was observed upon transcription inhibition, suggesting that active transcription is required for PABP1 export. The nuclear import of PABP1 is an energy-dependent process since PABP1 fails to enter the nucleus upon ATP depletion and at low temperature. Transfection of PABP1 or PABP1-GFP resulted in heterogeneity of intracellular distribution of the protein. In the low expressing cells, PABP1 was localized in the cytoplasm, whereas in the high expressors, we observed accumulation of the protein in the nucleus. Nuclear PABP1 observed either after overexpression or after transcription inhibition was found in speckles and colocalized with splicing factor SC35. The ability of PABP1 to shuttle between nucleus and cytoplasm was also shown by heterokaryon formation upon cell fusion. Deletion mutagenesis showed that the minimal part of PABP1 retaining the ability to shuttle consists of the first two RNA-binding domains. This mutant interacted with poly(A) RNA with high affinity and accumulated in the nucleus. Deletion mutants exhibiting reduced RNA binding affinity did not accumulate in the nucleus. PABP1 has been proposed to participate at various steps of mRNA utilization. Our results suggest involvement of PABP1 in nuclear events associated with the formation and transport of mRNP to the cytoplasm and identify a new trafficking pattern for RNA-binding proteins.Eukaryotic mRNAs are organized in ribonucleoprotein complexes (1, 2). One predominant protein of these complexes is the poly(A)-binding protein 1 (PABP1) 1 , which associates with the 3Ј poly(A) tail of mRNA (3-6). PABP1 is an essential protein in yeast (7) and is highly conserved among eukaryotic organisms (3, 4). PABP1 is clearly a multifunctional protein, proposed to participate in 3Ј end formation of mRNA, translation initiation, mRNA stabilization, protection of poly(A) from nuclease activity, mRNA deadenylation, inhibition of mRNA decapping, and mRNP maturation (8, 9). PABP1 may be a key factor in mediating regulation of mRNA turnover through the inhibition of mRNA decapping by the poly(A) tail or by influencing the rate of deadenylation (10 -13). Several lines of evidence argue that PABP1 plays a role in stimulating translation initiation (14 -16), suggesting that the interaction of this protein with the 3Ј poly(A) sequence can influence events at the 5Ј end of mRNA. PABP1 availability for interaction with processed mRNA is very important. Recent studies have shown that PABP1 is involved in the processing of 3Ј end of premessenger RNA (9). These results imply that interaction of PABP1 with poly(A) tail occurs in the nucleus. Mature mRNA is exported from the nucleus accompanied by several hnRNP proteins (17-19). Presence of PABP1 on the poly(A) tails of exported mRNA could be advant...

Structure and function of the C-terminal PABC domain of human poly(A)-binding protein

“…Early work suggested that the C terminus is important in structuring the mRNA tail and for the cellular localization of PABP. This work was confirmed by more recent studies that describe PABP dimerization through the C-terminal domain and a requirement of the C terminus for proper nuclear shuttling (11,12). The C-terminal half of PABP contains binding sites for eRF3 (5), Paip1 (13), Paip2 (4), Pbp1p (7), and a viral RNA polymerase (14).…”

Human cytomegalovirus UL69 protein facilitates translation by associating with the mRNA cap-binding complex and excluding 4EBP1

“…Alternatively, the presence of pUL69 in the complex could make 4EBP1 more susceptible to phosphorylation, which would then cause its exclusion. Although primarily cytoplasmic proteins, a portion of 4EBP1 (30), eIF4E (31), PABPC1 (32,33), and pUL69 (16) are present in the nucleus, so it is possible that pUL69 prevents association of 4EBP1 with cap-associated proteins in the nucleus, even before the mRNA is transported to the cytoplasm. An interaction of pUL69 with nuclear RNAs through PABPC1 could also contribute to its role in mRNA nucleocytoplasmic transport (12).…”

Deciphering the cellular pathway for transport of poly(A)-binding protein II

“…Comparison of mammalian and yeast pre-mRNA 39-end-processing components reveals that, although many homologies can be found, the proteins are not function-ally interchangeable (for a recent review, see )+ An apparent case of divergence during evolution occurs with poly(A)-binding proteins+ Yeast contains a single poly(A)-binding protein (termed Pab1p), which is an essential protein present in both the nucleus and the cytoplasm (Sachs et al+, 1986)+ In the nucleus it stimulates polyadenylation and controls poly(A) tail length (Amrani et al+, 1997;MinvielleSebastia et al+, 1997;Brown & Sachs, 1998), whereas in the cytoplasm it is involved in translation initiation (Sachs & Davis, 1989;Sachs & Deardorff, 1992;Tarun & Sachs, 1995, 1996Le et al+, 1997) and mRNA degradation (Caponigro & Parker, 1995;Boeck et al+, 1998;Coller et al+, 1998)+ In contrast, mammalian cells contain two distinct proteins called poly(A)-binding protein I (PABP1) and poly(A)-binding protein II (PAB II or PABP2)+ PABP1 is predominantly detected in the cytoplasm (Görlach et al+, 1994), whereas PABP2 is localized in the nucleus (Wahle, 1991;Krause et al+, 1994)+ PABP1, which is the mammalian counterpart of yeast Pab1p (51% identity), is apparently involved in both cytoplasmic mRNA stability (Bernstein et al+, 1989;Wormington et al+, 1996;Afonina et al+, 1997;Ford et al+, 1997;Körner & Wahle, 1997) and translation (Craig et al+, 1998), but to date there is no evidence that it participates in nuclear pre-mRNA 39-end processing (see )+ Consistent with its nuclear localization, PABP2 is the mammalian protein involved in polyadenylation+ PABP2 stimulates processive poly(A) addition and controls the size of the tail to be ;250 nt in length (Wahle, 1991(Wahle, , 1995Bienroth et al+, 1993)+ How PABP2 bound to poly(A) tails in the nucleus is replaced by PABP1 in the cytoplasm remains unknown, but recent evidence indicates that both PABP1 and PABP2 shuttle between nucleus and cytoplasm (Afonina et al+, 1998;Chen et al+, 1999)+ Because it is not known when PABP1 exchanges with PABP2, one possibility is that PABP2 crosses the nuclear pores in association with the mRNA and dissoci...…”