Our results provide the first evidence that PABPC1, like PABPN1, can associate in vivo with intron-containing, polyadenylated transcripts that go on to be productively spliced. Furthermore, PABPC1, like PABPN1, binds directly to the poly(A) tail of these transcripts. First, PABPC1 is present within the nuclei of both cell types that we examined (Fig. ; data not shown). Second, anti-PABPC1, like anti-PABPN1, immunopurifies every unspliced pre-mRNA, partially spliced pre-mRNA, and fully spliced mRNA that we tested (Fig. and ). Third, cellular PABPC1 binding to these transcripts is dependent on a poly(A) tail, as evidenced by the failure of anti-PABPC1 to immunopurify nonadenylated histone H4 mRNA (Fig. and ). Fourth, anti-PABPC1 immunopurifies pre-mRNA, largely as a consequence of binding in vivo and not because of an experimental artifact (Fig. ). Fifth, cellular PABPC1 remains UV cross-linked to pre-mRNA under conditions that dissociate noncovalent bonds, and cross-linking depends on the presence of a poly(A) tail (Fig. ). Sixth, cellular PAP-γ coimmunopurifies with cellular PABPC1, and Myc-PABPC1 coimmunopurifies with cellular PAP-γ (Fig. ). Our ability to detect an interaction between PAP-γ and PABPC1 contrasts with data demonstrating that purified bovine PAP-γ and Xenopus laevis
PABPC1 do not detectably interact (28
). However, Xenopus
PABPC1 may be unable to interact with human PAP because of incompatibility between species, because mammalian PABPC1 functions in ways that Xenopus
PABPC1 does not, or because in vitro binding conditions were insufficient to support the interaction of PABPC1 with PAP-γ.
We also find that E. coli
-produced and purified PABPC1 copurifies with E. coli
-produced and purified GST-PABPN1 but not GST alone (data not shown). However, considering there are several examples of proteins that interact in vitro but not apparently in cells (e.g., Staufen1 and Staufen2) (49
; H. A. Kuzmiak and L. E. Maquat, unpublished data), it would be premature to conclude that PABPN1 and PABPC1 interact directly in mammalian cells. We favor the interpretation that PABPC1 associates with unspliced pre-mRNA directly via the poly(A) tail because PABPC1 can be UV cross-linked to pre-mRNA in intact cells. Nevertheless, we cannot exclude the possibility that PABPC1 also associates via PABPN1.
In view of the unexpectedly early step at which PABPC1 is acquired during mRNA biogenesis, it became important to determine how long PABPN1 remains associated with mRNA, given that PABPN1 is ultimately replaced by PABPC1. We have found that PABPN1 remains associated with mRNA during the pioneer round of translation, for which CBP80/CBP20-bound mRNA serves as a template. This was evidenced by the ability of anti-PABPN1 to immunopurify nonsense-containing mRNA that had already been reduced in abundance by NMD (Fig. ). Even though anti-CBP80 immunopurifies PABPN1 and PABPC1 (11
) and anti-eIF4E immunopurifies only PABPC1 (11
), we cannot be certain that PABPN1 is no longer present after CBP80 and CBP20 are replaced by eIF4E, since the fraction of eIF4E-bound mRNA that is bound by PABPN1 may be too small to detect.
We conclude that both PABPN1 and PABPC1 bind to the poly(A) tail of newly synthesized transcripts in the nucleus, at least in some cases prior to splicing. Consistent with this, not all splicing occurs cotranscriptionally in mammalian cells and, consequently, unspliced or partially spliced polyadenylated transcripts do exist (6
; data not shown).
PABPN1 has been shown to function in nuclear polyadenylation by recruiting PAP to transcripts and, by so doing, increasing the processivity of PAP and controlling the length of the newly synthesized poly(A) tail (5
). PABPN1 has also been functionally implicated in mRNA transport, since it shuttles between the nucleus and the cytoplasm (9
), and immunoelectron microscopy demonstrates that it is present on nuclear messenger ribonucleoprotein particles (mRNPs) that transit the nuclear envelope (2
). A functional homolog to mammalian PABPN1 in Saccharomyces cerevisiae
has not been found. The S. cerevisiae
homolog to mammalian PABPC1, Pab1p, is known to play a role in both cytoplasmic mRNA metabolism, including mRNA translation and decay, as well as in nuclear RNA metabolism, including polyadenylation (32
) and mRNA export (7
). Recent studies have shown that PABPC1, like yeast Pab1p (7
), shuttles between the nucleus and the cytoplasm (1
Our data indicate PABPC1 associates with nuclear pre-mRNP prior to intranuclear transport by directly binding poly(A), most likely simultaneously with PABPN1. PABPC1 bound to newly synthesized poly(A) tails may function in pre-mRNA metabolism. For example, it could protect nuclear pre-mRNA from decapping, which is known to occur within nuclei, much as it protects cytoplasmic mRNA from decapping (30
). As another example, PABPC1 could promote nuclear poly(A) nuclease 2 (Pan2)-mediated pre-mRNA poly(A) trimming, as it does in the cytoplasm (51
), since both Pan2 and Pan3, the latter of which tethers Pan2 to PABPC1, are known to shuttle between the nucleus and the cytoplasm (59
). In fact, Pan2p and Pan3p, the S. cerevisiae
orthologs of mammalian Pan2 and Pan3, are known to regulate nuclear poly(A) tail length (39
PABPC1 may also influence the metabolism of nuclear mRNP. For example, as polyadenylated mRNP approaches the nuclear pore and is unfolded for transit through the nuclear pore complex (13
), poly(A)-bound PABPC1 may play a role during transit by further nucleating PABPC1 assembly and concomitantly removing PABPN1, a process that would be completed after the pioneer round of translation. Another possibility is that PABPC1 primarily assembles with RNP and functions in the nucleus at a step that is much earlier than transit across the pore. In either scenario, PABPC1 may also be involved in the localization of particular mRNAs to specific regions of the cytoplasm. For example, PABPC1 was recently shown to bind directly to paxillin, which is an abundant protein of focal complexes at the leading edges of migrating cells (57
). The PABPC1-paxillin complex localizes to the perinuclear endoplasmic reticulum and the leading edge of the migrating cell plasma membrane in mouse fibroblasts. Thus, nuclear PABPC1 could play a role in assembling proteins at the 3′ untranslated region of an mRNP that are subsequently required for site-specific cytoplasmic localization of that mRNP.
PABPC1 function within nuclei would occur prior to its earliest certified role, which is during the pioneer round of translation. This round of translation involves CBP80/CBP20-bound mRNA, supports the decay of nonsense-containing mRNAs, precedes the translation of eIF4E-bound mRNA, and most often occurs in association with nuclei, probably during mRNA export to the cytoplasm (11
). It is likely that PABPC1 augments the translation of CBP80/CBP20-bound mRNA in a manner similar to its augmentation of the translation of eIF4E-bound mRNA. PABPC1 binding to eIF4G increases the efficiency of translation initiation (27
), and eIF4G is a functional component of the pioneer translation initiation complex (37
). Furthermore, PABPC1 binding to eukaryotic translation release factor 3 (21
) may increase the efficiency of translation termination.
Future studies aim to determine at what step in RNA metabolism the bulk of PABPC1 binding to poly(A) tails occurs and specifically how PABPC1 functions prior to its role in the pioneer round of translation.