The poly(A) nucleases involved in deadenylation of mRNA in various organisms have started to be identified and characterized in the last decade. The CCR4-CAF1 complex accounts for the major deadenylation activity in Saccharomyces cerevisiae
and Drosophila melanogaster
). In mammals, a second poly(A) nuclease complex, PAN2-PAN3, has recently been shown to initiate deadenylation prior to CCR4-CAF1 action (61
). Given that deadenylation of mRNA has the important consequence of shutting off translation of the transcript, a void in our understanding of how deadenylation is controlled, particularly in mammalian somatic cells during mRNA turnover, has prompted us to examine the underlying mechanisms.
Previous studies (29
) suggested that communication between poly(A) nuclease complexes PAN2-PAN3 and CCR4-CAF1 and the 3′ PABP-poly(A) tail complex of the mRNA in mammalian cells can greatly influence the poly(A) nuclease activity and, thus, the rate and extent of deadenylation. In this study, we set out to identify protein factors that mediate or modulate the interaction between the CCR4-CAF1 complex and the PABP-poly(A) complex. Our results led to three main findings. First, TOB, previously considered a transcription factor, can function as a positive, PABP-dependent regulatory factor in the deadenylation of both wild-type stable and nonsense-containing unstable β-globin mRNAs. Second, TOB can associate simultaneously with cytoplasmic PABP via TOB's C-terminal PAM2 motifs and with CAF1 through TOB's N-terminal domain. Third, TOB colocalizes with P-bodies in the G1
phase but not in the G2
or M phase.
Although it was previously reported that TOB can separately interact with CAF1 (25
) and bind PABP (25
), those studies did not determine whether TOB can simultaneously interact with both PABP and CAF1. Moreover, the functional significance of these interactions for regulating deadenylation remains unclear. The present results (Fig. and ) not only demonstrate a direct interaction between TOB and PABPC1 but also reveal that the PAM2 motifs in TOB and the PABC motif in PABPC1 are necessary for the interaction, thus establishing TOB as a genuine PABP-interacting protein.
Our observation of an enhancing effect of TOB on deadenylation raises an important question as to whether TOB directly destabilizes the poly(A)-PABP complex. In a gel mobility shift assay, we showed that while TOB does supershift the PABP-poly(A) complex, it has little effect on the complex formed between poly(A) and a truncated PABP that contains only the first RNA-binding domain (Fig. ). These results suggest that instead of destabilizing the poly(A)-PABP complex, TOB promotes deadenylation through its interaction with the poly(A)-PABP complex, possibly by recruiting poly(A) nucleases, such as CCR4-CAF1, to the 3′ poly(A) tail and/or by modulating their poly(A) nuclease activity. It is of particular interest that TOB's deadenylation-enhancing effect is dependent on its interaction with PABP (Fig. ; see also Fig. S1 in the supplemental material). When a double point mutation was introduced into the two PAM2 motifs in TOB proteins, their ability to interact with PABPC1 was greatly diminished. This finding is consistent with the above notions.
The ability of TOB to modulate deadenylation via the CCR4-CAF1 complex is reminiscent of the actions of yeast PUF proteins, a family of RNA-binding proteins that interact with the 3′ untranslated regions of specific mRNAs and stimulate their deadenylation (57
). One of the yeast PUF proteins, Mpt5p, has been shown to physically bind Pop2p, a homolog of mammalian CAF1 (21
). By binding Pop2p, Mpt5p recruits the cytoplasmic Ccr4p-Pop2p deadenylase complex to the target transcript, thus promoting removal of its poly(A) tails and consequent repression of its translation. Thus, it appears that both mammalian TOB and yeast Mpt5p proteins regulate mRNA decay through recruiting the CCR4-CAF1 complex to stimulate deadenylation. Yet, there are distinct differences between the yeast and mammalian systems. Whereas the PUF proteins act in an RNA sequence-specific manner, TOB proteins interact with the PABPC1 protein of the 3′ poly(A)-PABP complex and thus are likely to function in an RNA sequence-independent way. Another difference is that there is no known TOB ortholog in yeast, but the interaction between PUF and POP2 is conserved in yeast, Drosophila
, and humans (24
). Moreover, TOB belongs to a family of antiproliferative proteins whose levels of expression change during the cell cycle, while expression of PUF proteins is not known to be related to the cell cycle. Thus, two distinct families of unrelated proteins, TOB and PUF, that share no homology and play different roles in embryogenesis, cell growth, and cell differentiation appear to have evolved independently to accomplish their functions by converging on deadenylation.
What may be the biological significance or consequence of enhanced deadenylation by TOB? Our experiments show that TOB enhances deadenylation without significantly changing the overall decay rate of the deadenylated RNA body. Several observations in this and other studies suggest a role for the TOB proteins in facilitating the exit of mRNPs from the translation pool to the nontranslated pool residing in P-bodies. First, the enhancement of deadenylation observed with TOB could help to stop translation initiation by abolishing the interaction between the 5′ cap and the 3′ poly(A) tail. Second, TOB interacts with the C-terminal PABC domain of PABP, a region that is also used during translation for interactions with Paip1, Paip2, eukaryotic initiation factor 4B, eRF3, and the 60S ribosomal subunit (2
). One might imagine that TOB could compete with these factors and thus decrease translation efficiency. The above notions are consistent with the role for TOB in translation repression implicated in a recent report that overexpression of TOB in an NIH 3T3 cell line, stably expressing exogenous inducible PABP, results in reduction of interleukin-2 protein expression from a simultaneously transfected interleukin-2 plasmid (43
). Third, TOB colocalizes in the G1
phase with P-bodies, structures linked to silencing translation and/or for storing translationally repressed mRNPs (for a recent review, see reference 17
). One possibility is that after promoting deadenylation, TOB may facilitate remodeling of mRNPs and their movement into P-bodies, thus repressing translation.
In summary, our data suggest that TOB provides functional links among deadenylation, P-bodies, and cell growth arrest. Consistent with this notion is a study that showed that overexpression of CAF1 leads to cell growth arrest (7
). It will be interesting to learn how knockdown of TOB expression may change the mRNA expression profile in cells arrested in the G0
phase. The information gathered has the potential to provide important insight into TOB's function as an antiproliferative factor. Given that phosphorylation of TOB at specific serines inactivates its antiproliferative function when cells exit the G0
), it will also be important to address whether and how those posttranslational modifications may regulate TOB's function in modulating deadenylation.