Pumilio and Nanos control important functions, including development (30
), stem cell proliferation (2
), and learning (12
). Previous analyses of Pumilio and Nanos function were restricted to mutant or transgenic Drosophila
. The experiments presented in this work build upon these elegant studies to elucidate the mechanism of regulation. We developed a reporter assay that recapitulates Nanos-dependent repression. Nanos is not detectable in D.mel-2 cells (B) (7
), but expression of Nanos confers potent repression of an mRNA bearing Hunchback NREs (C and D). Pumilio is essential for Nanos repression (E) (3
). Therefore, Nanos activates Pumilio. Acting together, Nanos and Pumilio inhibit protein expression and cause a corresponding decrease in mRNA level (D). This data is consistent with Nanos and Pumilio collaborating to repress Hunchback mRNA in the Drosophila
Pumilio also represses independently of Nanos (). Without Nanos, endogenous Pumilio in D.mel-2 cells minimally represses the NRE-bearing reporter (see Fig. S1B in the supplemental material); however, efficient repression was elicited by increasing the concentration of Pumilio (A; see Fig. S3 in the supplemental material). A likely explanation is that the amount of endogenous Pumilio is insufficient to efficiently repress. Like Nanos-dependent repression, Pumilio potently decreased reporter protein and mRNA levels (B). Several facts support the conclusion that Nanos was not necessary for repression by Pumilio. First, Nanos is not detectable in D.mel-2 cells. Also, RNAi of Nanos did not affect Pumilio repression (D). Furthermore, a mutation that blocks Nanos binding to Pumilio (F1367S) did not alleviate repression (C). Nanos-inde-pendent Pumilio repression is supported by observations that Pumilio regulates Bicoid and cyclin B mRNAs in the anterior of Drosophila
embryos, where Nanos is below the limit of detection (16
The finding that Pumilio represses independently of Nanos raises the question: what is the function of Nanos? One logical answer is that Nanos strengthens Pumilio repression. The observation that Nanos activates endogenous Pumilio supports a model wherein Nanos enhances the RNA-binding activity of Pumilio. Previous work strengthens this hypothesis: Nanos and Pumilio interact with each other and both contact the NRE RNA through a network of protein-protein and protein-RNA interactions that may cooperate to enhance binding (47
). The necessity of Nanos could be obviated by increasing the level of Pumilio (), likely resulting from increased occupancy of the NRE reporter. Another hypothesis is that binding of Nanos to Pumilio might displace a negative regulatory factor, resulting in activation of endogenous Pumilio. Nanos may also collaborate with Pumilio to recruit corepressors. The Nanos-Pumilio-NRE complex is thought to recruit Brain Tumor and 4EHP to refine regulation of the Hunchback gradient (8
). However, RNAi depletion of Brain Tumor and 4EHP did not abrogate Nanos-dependent repression (). We interpret this as evidence that Nanos and Pumilio repress mRNAs through additional mechanisms (see below), with the caveat that residual Brain Tumor and 4EHP might be sufficient to support Nanos-dependent repression. As an alternative model, Nanos and Pumilio may collaborate to recruit the Ccr4-Not deadenylase complex through interactions with Not4 and Pop2 subunits, respectively (22
). Future research is necessary to address these models.
Potent Pumilio repression in the absence of Nanos indicates that Pumilio independently inhibits protein expression and enhances mRNA decay. Involvement of known corepressors Brain Tumor and 4EHP is improbable because recruitment of these proteins depends on Nanos (8
). Furthermore, a mutant Pumilio (G1330D) that cannot bind Brain Tumor is fully active for Nanos-independent repression (C). In addition, depletion of Brain Tumor by RNAi did not affect Pumilio repression (D). These findings reveal that mechanisms other than Brain Tumor-4EHP-mediated inhibition of 5′-cap-dependent translation are utilized by Pumilio. Previous studies concluded that Brain Tumor, and therefore 4EHP, are dispensable for Pumilio repression in certain contexts. For instance, Pumilio repression of cyclin B in embryonic pole cells does not require Brain Tumor (26
). Furthermore, while Brain Tumor is necessary for Pumilio repression in motor neurons, it is not essential in other neurons (37
). Finally, the G1330D mutant Pumilio, which is deficient for recruitment of Brain Tumor, is functional for regulation of dendritic morphology (62
). We do not dismiss the importance of Brain Tumor and 4EHP in embryonic development. Instead, these findings illustrate that while Brain Tumor and 4EHP facilitate repression of Hunchback in the embryo, in other contexts, Pumilio represses by other means.
We identified Pumilio domains that mediate Nanos-inde-pendent repression. The Pumilio RBD has modest repressive activity compared to the full-length protein (C and D; see Fig. S3 in the supplemental material), indicating that regions outside the RBD must confer repressive activity. Previous analysis of the ability of Pumilio transgenes to rescue abdominal segmentation defects in a pumilio
mutant embryo support this conclusion. Whereas overexpression of the Pumilio RBD partially rescued segmentation defects, the full-length Pumilio fully restored proper embryonic development (3
). Indeed, we discovered three repression domains within the N-terminal two-thirds of Pumilio that provide the major repressive activity (). These unique repression domains (i.e., aa 1 to 378, 548 to 776, and 848 to 1090) do not share sequence homology. Each functions autonomously when tethered to mRNA (). Because all known Pumilio cofactors (i.e., Nanos, Brain Tumor, 4EHP, and Pop2) interact with the RBD (8
), the N-terminal repression domains likely function through novel mechanisms. While full-length Pumilio affects both mRNA and protein levels almost equally (D and B), the individual repression domains affect protein expression more than mRNA levels (A and B). This suggests translational inhibition may be their predominant function.
The repressive function of the Pumilio N terminus may be evolutionarily conserved. Sequence alignments indicated that the N terminus of vertebrate PUMs, including human PUM1 and PUM2, are related to the Pumilio N terminus (A). When tethered, the N-terminal portions of human PUM1 and PUM2 repressed, providing evidence that human PUFs are repressors (B). Two regions in human PUM1 are autonomous repression domains (C). These regions are small (region 2, 152 amino acids; region 3, 240 amino acids), share 19% and 15% identity with Pumilio, and do not contain previously identified motifs. We propose that they may contact novel corepressors, which remain to be identified.
We compared the Pumilio N terminus to other PUF proteins; no detectable relationship could be found with six Saccharomyces cerevisiae
PUFs or 12 C. elegans
PUFs. Instead, these PUFs have evolved unique sequences, appended to their RNA-binding domains, whose function remains unknown. We also searched the nonredundant protein sequence database (www.ncbi.nlm.nih.gov
) using the BLAST algorithm (1
) to identify protein sequences similar to the Pumilio N terminus: no proteins, other than PUF family members, share homology. The broad implication is that members of the PUF family have evolved unique domains, appended to the evolutionarily conserved PUF repeat RNA-binding module, which may confer unique regulatory activities to individual PUFs. Consistent with this idea, specific PUFs have been shown to affect translation, mRNA degradation, mRNA localization, and, for one PUF, activation of target mRNAs (10
We identified two sequence motifs in the N terminus of Pumilio, designated PCMa and PCMb, which are conserved between insects (e.g., Drosophila
) and vertebrates (e.g., humans) (A). PCMb encompasses a motif in Xenopus
PUM2 proposed to interfere with cap-dependent translation (4
). However, when tethered, PCMb does not repress (), nor does deletion of PCMb diminish Pumilio repression (see Fig. S4A in the supplemental material). In addition, mutation of a PCMb tryptophan residue (W783) proposed to contact the 5′ cap (4
) had no effect (data not shown). Therefore, we find no evidence that the putative cap binding motif of PCMb is important for Pumilio repression. Instead, PCMb negatively affects repression domains aa 548 to 776 (region 2) and aa 848 to 1090 (region 3) (E). While PCMa had weak repressive activity on its own, it could counteract the inhibitory effect of PCMb (D), and PCMa deletion of PCMa caused a minor but significant drop in repression (Fig. see S4A in the supplemental material). The precise roles of PCMa and PCMb remain to be determined; we speculate that they may have autoregulatory functions.
We successfully programmed Pumilio to repress a new target mRNA. By changing the RNA recognition amino acids in the sixth PUF repeat of Pumilio from NYQ to SYE, the specificity was altered from uridine to guanosine, thereby conferring repression to an mRNA with an altered binding site (Rn3×NRE UGG) (D). This experiment provides the proof of principle that PUF proteins with programmed RNA-binding specificity can be engineered to repress new mRNAs. While programmed Pumilio fully represses its new target, a similarly programmed RBD lacks substantial activity (), further emphasizing the importance of the N-terminal repression domains. This finding has important implications for future engineering of PUFs. The Pumilio RBD provides a protein module with low intrinsic regulatory activity that can be programmed to bind new RNA sequences. Functional domains—either repression or activation domains—can be attached to this module to create novel RNA regulators. Consistent with this idea, a recent study reported that addition of splicing effector domains and a nuclear localization signal transformed a PUF RBD into a splicing regulator (56
An important question for future research is how do the Pumilio repression domains function? A probable hypothesis is that the repression domains inhibit the translation machinery. Alternatively, the repression domains may activate enzymes that degrade mRNAs. In future experiments, we seek to identify corepressors that interact with these domains. Also worth consideration is why Pumilio possesses multiple repression domains. These domains may recruit the same corepressor, either acting redundantly or collaboratively. Alternatively, each repression domain could bind to a different corepressor, perhaps affecting different steps in the gene expression pathway (e.g., translation initiation or mRNA degradation). In this case, their individual repressive activities would collaborate to increase the efficiency of repression. Addressing these crucial questions will help reveal how Pumilio regulates mRNAs to control diverse biological functions.