The interplay between RNA binding proteins and cis
-acting regulatory elements within specific mRNA transcripts can regulate gene expression by stabilizing or destabilizing a particular transcript. Pub1 has been implicated in the stabilization of a group of mRNAs through binding to specific sequences in either the 5′- or 3′-UTR and thereby inhibiting different decay pathways (48
). In this report, we demonstrate that the poly(A) RNA binding protein Nab2 can bind directly to Pub1 and can also modulate the stability of a subset of Pub1 target mRNAs.
Since Pub1 is an mRNA binding protein and since we found that it can bind to Nab2, another RNA binding protein, we needed to determine if this interaction could be mediated by RNA. This point is particularly important because the essential CCCH domain on Nab2 that mediates binding to Pub1 has also been implicated in RNA binding (3
). We found that TAP-tagged Nab2 copurifies with Pub1 from yeast extract even when the lysate is pretreated with RNase A, suggesting that this interaction is not mediated by RNA. An in vitro binding assay confirmed that Pub1 can bind directly to Nab2 and showed that the zinc finger domain of Nab2 is both necessary and sufficient to directly interact with Pub1.
Classical zinc finger proteins often employ multiple zinc fingers to bind to nucleic acids (38
). Typically, aromatic residues within the zinc fingers interact with the nucleic acid to impart sequence specificity (38
). However, in addition to binding to nucleic acids, zinc fingers can also bind to proteins (35
). Furthermore, there are a number of proteins that contain multiple zinc fingers that mediate distinct binding interactions. For example, the transcription factor TFIIIA, a dual RNA/DNA binding protein, contains nine zinc fingers, and individual zinc fingers are used to recognize both RNA and DNA targets (6
). In addition, the transcription factor GATA-1 contains two zinc finger repeats, which not only act as the primary determinant of binding specificity for the DNA sequence GATA but also can mediate protein-protein interactions (38
). Similarly, our results suggest that the CCCH domain of Nab2, which contains seven zinc fingers, is a multitasking zinc finger domain required for both RNA-protein and protein-protein interactions.
Previously characterized Pub1 target mRNAs contain two distinct cis
-acting elements, the 5′-UTR STE or the 3′-UTR ARE (48
). A recent genome-wide survey identified a number of novel ARE-like and A-rich Pub1 binding motifs (16
). We found that nab2-1
cells show altered stability of the ARE-like Pub1 target transcripts but no effect on STE-containing Pub1 targets. It is not yet clear whether the effect on the stability of ARE transcripts is a consequence of the nuclear role of Nab2 or whether it reflects a distinct function for Nab2 in the cytoplasm. Given that the poly(A) RNA transcripts in nab2-1
mutant cells accumulate longer poly(A) tails than in wild-type cells (26
), it might be logical to assume that the mRNA half-life would be increased in nab2-1
cells. Instead, we found that target RNAs containing an ARE-like element have decreased stability in nab2-1
cells. Although this mutant does interact with Pub1, we suggest that the deletion of the Nab2 N terminus could impact Nab2 function in mRNA stabilization by affecting its interaction with another component of a multifunctional protein complex involved in mRNA export and/or mRNA decay. Consistent with this idea, the same defect in mRNA stability displayed by nab2-1
mutant cells was observed in the novel NAB2
, which encodes a Nab2 variant with a substantially decreased interaction with Pub1 (Fig. ).
Based on our results with the nab2-67
mutant, we suggest that the Nab2 function in stabilizing ARE-like sequence-containing transcripts is related to its interaction to Pub1, which acts by modulating the stability of this class of transcripts (16
). Consistent with an independent role for Nab2 in modulating mRNA stability through interactions with Pub1, we find that cells expressing the Nab2 mutant nab2-67
, which shows a decreased interaction with Pub1, do not display severe growth defects. However, these cells do show an altered stability of a subset of Pub1 target transcripts. These phenotypes for the nab2-67
allele are identical to the phenotypes, no growth defect and altered transcript stability, observed for cells lacking Pub1, strengthening the argument for a functional interaction between Nab2 and Pub1.
We propose that the specific effect of Nab2 on the ARE-like sequence-containing transcripts but not on the STE-containing transcripts could be due to the proximity of the Pub1 binding site, the ARE-like sequence within the 3′-UTR, to the poly(A) tail, which is where Nab2 associates with the transcript (25
; S. M. Kelly et al., unpublished data). Consistent with this idea, an interaction between the human poly(A) binding protein PABP and HuR, the apparent human Pub1 orthologue (45
), was recently described (42
). This interaction increased the stability of the ARE-containing β-casein mRNA transcript through the interaction of the poly(A) tail and the ARE element (42
), supporting the idea that these cis
-acting elements could work together. In contrast, the STE is located in the 5′-UTR of the GCN4
transcript, a site that should be physically distant from the poly(A) tail and thus not in close physical proximity to Nab2.
Although Nab2 is localized to the nucleus at steady state (3
), it shuttles between the nucleus and the cytoplasm (23
). Likewise, Pub1, which is primarily localized to the cytoplasm, can be detected in the nucleus (2
). Recently, evidence has emerged that a number of RNA binding proteins play distinct roles in the nucleus and the cytoplasm (18
). For example, Hrp1 is an hnRNP which localizes to the nucleus at steady state (27
), where it is required for proper mRNA 3′-end formation (33
). However, it can also shuttle to the cytoplasm, where it modulates the NMD pathway (21
). Other examples include Gbp2 and Hrb1, which bind to mRNA in the nucleus but are also associated with polysomes in the cytoplasm (62
). Additionally, eukaryotic translation initiation factor 4AIII, a nuclear protein that is loaded onto the mRNA during splicing in the nucleus, also functions in NMD and has properties related to the eukaryotic translation initiation factor 4A translation initiation factor (8
It is still not clear how Pub1 stabilizes transcripts containing 5′-UTR STE or 3′-UTR ARE elements. The NMD pathway is required for the degradation of upstream ORF-containing transcripts that contain the cis
-acting STE element (47
). In yeast, the major decay pathway for NMD involves 5′ decapping followed by 5′-3′ degradation bypassing deadenylation (12
). Previous work showed that the endogenous uORF-containing GCN4
transcript is protected from NMD-mediated decay by the binding of Pub1 to the STE sequence located in the 5′-UTR (48
). In the case of ARE-containing transcripts, Pub1 protects a hybrid transcript harboring the tumor necrosis factor alpha ARE from decay via the deadenylation-dependent pathway (56
). However, it is not clear whether endogenous ARE-containing transcripts are degraded by the same mechanism. Our results indicate that the stability of the RPS16B
transcript is not recovered in Δpub1
, or nab2-67
cells where the NMD pathway is impaired by the deletion of UPF1
. This result confirms that both Pub1 and Nab2 stabilize the ARE-like sequence-containing transcripts by modulating a pathway that is distinct from NMD.
Pub1 modulates RPS16B
mRNA decay through a direct interaction with an ARE-like sequence at the transcript 3′-UTR (16
). Our results indicate that the ARE-like sequence in RPS16B
mRNA is also necessary for the stabilization mediated by Nab2. The functional association of Nab2 with this specific cis
-acting mRNA element could help to coordinate different events involved in the control of gene expression, coupling the maturation and nuclear export steps of mRNA biogenesis to the mRNA decay machinery. Taken together, our study suggests that Nab2, a protein with defined nuclear functions, together with Pub1, plays a role in determining the cytoplasmic fate of some mRNA transcripts, adding Nab2 to a growing list of nuclear RNA binding proteins that modulate the destiny of mRNA transcripts in the cytoplasm.