Evidence that Poly(A) Binding Protein C1 Binds Nuclear Pre-mRNA Poly(A) Tails

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.The 3Ј ends of almost all eukaryotic mRNAs and their precursors consist of homopolymeric tails of adenosine, or poly(A), that are added by poly(A) polymerase (PAP) during the process of 3Ј end formation. There are two classes of poly(A) binding proteins (PABPs) in mammalian cells (32,40). One class is exemplified by PABPN1, formerly called PABP2. PABPN1 is primarily nuclear and plays a role in the synthesis of poly(A) tails, but it also shuttles between the nucleus and cytoplasm (5,8,9,31,53). The other class consists of the primarily cytoplasmic PABPs, of which PABPC1, formerly called PABI or PABP1, is the major form in mammalian somatic cells (40). In humans, at least four separate PABPC genes and four pseudogenes have been identified (40). PABPC1 influences mRNA translation and decay (18,20,23,27,29,30,50,(54)(55)(56), and it shuttles between the nucleus and cytoplasm of at least some mammalian cells (1,57,58). Consistent with the preferential compartmentalization of PABPN1 to the nucleus and PABPC1 to the cytoplasm, a physical interaction has been detected between PAP and PABPN1 but not PABPC1 (28). Furthermore, PABPN1 associates with RNA polymerase II during transcription and accompanies the released transcript to the nuclear pore (2). Given that PABPC1 can exist within nuclei, a key issue is whether PABPC1 binds to transcripts inside the nucleus, and if it does, at which step in mRNA maturation.The 5Ј ends of eukaryotic mRNAs and their precursors are capped, and the cap is initially bound by the mostly nuclear cap binding protein (CBP) heterodimer of CBP80 and CBP20 (25, 35, 38), which will be called CBP80/CBP20. CBP80/CBP20 is detectably replaced by the mostly cytoplasmic eukaryotic translation initiation factor 4E (eIF4E) only after introns have been removed by splicing (35). Evidence indicates that PABPC1 is a component...

Regulation of the Nuclear Poly(A)-binding Protein by Arginine Methylation in Fission Yeast

“…It is therefore possible that in pab2-null fission yeast, Pab1 complements some of the essential nuclear functions normally performed by Pab2. Such a model is consistent with: (i) pab1 being a high-copy suppressor of nab2 mutant alleles in budding yeast (11) and (ii) recent data indicating that the mammalian Pab1 homolog copurifies with poly(A) polymerase and associates with intron-containing polyadenylated transcripts (71). PABP2 is required for cell viability and embryonic development in Drosophila (18).…”

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).…”

Crossing the borders: Poly(A)-binding proteins working on both sides of the fence

“…26 Furthermore, the canonical poly(A)-polymerase copurifies with PABPC1, 26 suggesting the presence of PABPC1 in the vicinity of the 3' end processing complex. Consistently, a proteomic study that characterized the protein content of the mRNA 3' processing complex identified several peptides corresponding to PABPC1.…”

“…First, fractionation experiments using different human cell types indicate the presence of PABPC1 in the nucleus. 26 Furthermore, cell biological experiments demonstrate that PABPC1 actively shuttles between the cytoplasm and nucleus. 27 What is the functional significance for the presence of the cytosolic PABP in the nucleus?…”