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Pumilio turns on microRna functionrobinson triboulet and richard i. gregory1

Pumilio proteins PUM1 and PUM2 are shown to regulate microRna-dependent gene silencing by induction of a conformational switch in the 3 untranslated region of p27 mRna. This conformational change is required for efficient microRna-mediated repression of this cell-cycle regulator in rapidly proliferating cells.

MicroRNAs (miRNAs) are small (approxi-mately 22 nucleotides) noncoding RNAs in plants and animals that direct post-transcrip-tional gene silencing by base pairing with complementary sites preferentially found in the 3 untranslated regions (3 UTR) of target mRNA. Pairing is mediated through a critical sequence called the seed region encompass-ing nucleotides 27 of every miRNA1. This post-transcriptional gene repression involves the recruitment of the RNA-induced silencing complex (RISC), a core component of which is an Argonaute protein that binds directly to the miRNA2. Although this biological process has been extensively studied in the last decade, how post-transcriptional gene silencing is regulated remains incompletely understood. On page 1014 of this issue, Kedde et al. elucidate a new mechanism for regulating miRNA activity3. They find that Pumilio RNA-binding proteins are required for miR-221/miR-222-mediated repression of the p27 tumour suppressor. The binding of PUM1 induces a local conforma-tional change in the p27 transcript that exposes a miR-221/miR-222-binding site (Fig. 1).

p27, a cyclin-dependent kinase (CDK) inhibitor, interferes with cell-cycle progres-sion by blocking CDK2 activity. p27 is a well-characterized tumour suppressor gene that is downregulated in many human cancers4. In 2007, several groups identified p27 mRNA as a target for two vertebrate miRNAs, miR-221 and miR-222 (refs 57). In the p27 3 UTR there are two conserved sites for miR-221/miR-222 binding that mediate downregulation of p27 expression. This repression is essential for cell proliferation and may also have a role in can-cer, as miR-221/miR-222 are highly expressed in many different cancers, and high levels of miR-221/miR-222 correlate inversely with low levels of p27 in samples from glioblastoma

patients5. Conversely, high levels of p27 are detected in quiescent cells. But Kedde et al. report that miR-221/miR-222 levels are surpris-ingly unchanged in quiescent fibroblasts, com-pared with cycling fibroblasts3. Furthermore, although p27 mRNA is expressed at the same level in both dividing and nondividing cells, it is more actively translated in quiescent cells, suggesting that p27 transcripts somehow escape miR-221/miR-222-mediated silencing during quiescence.

The authors investigated the role of Pumilio proteins to understand why p27 transcripts escape miRNA-dependent silencing in qui-escent cells. Pumilio PUM1 and PUM2 pro-teins are well-characterized repressors of mRNA translation that recognize and bind to a specific sequence localized in the 3 UTR of certain mRNAs8. Recent screens for targets

of mammalian PUM1 and PUM2 identified two Pumilio-recognition elements (PRE) in the p27 3 UTR9,10. Indeed, Kedde et al. found that PUM1 and PUM2 depletion leads to an increase in p27, suggesting that these pro-teins act redundantly to inhibit p27 expres-sion. Furthermore, depletion of both proteins in quiescent cells delayed entry into S phase. Mutation of the PREs in the p27 UTR demon-strated that binding of PUM1 protein to the 3 UTR is required to silence p27. These results suggest that control of cell cycle re-entry by PUM 1 and PUM2 is, at least in part, mediated by controlling p27 expression.

As PUM1 is present in proliferating and quiescent cells, the authors addressed whether PUM1 RNA-binding activity might be differen-tially regulated in quiescent versus cycling cells. Using a fluorescence-based assay to monitor

Robinson Triboulet and Richard I. Gregory are in the Stem Cell Program, Childrens Hospital Boston, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Harvard Stem Cell Institute, Boston, MA 02115, USA.e-mail: rgregory@enders.tch.harvard.edu

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Figure 1 Pumilio-mediated regulation of p27 silencing by miR-221/miR-222. (a) In quiescent fibroblasts, p27 mRNA is actively translated to yield high levels of p27 protein. One of the two target sites for miR-221/miR-222 in the p27 3 UTR is embedded in a stable stem-loop structure together with one of the two conserved Pumilio recognition elements (PREs), thus preventing p27 silencing by miR-221/miR-222. (b) When cells re-enter the cell cycle on growth-factor stimulation, levels of Pumilio protein PUM1 increase and phosphorylation of the Ser 714 enhances its RNA-binding activity. PUM1 binds to the proximal PRE to induce a local change in the RNA that enables miR-221/miR-222 binding to its target site and repression of p27 translation. Ago; Argonaute.

928 nature cell biology VOLUME 12 | NUMBER 10 | OCTOBER 2010

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in vivo interactions, they show that PUM1 and its target RNA co-localize in cycling cells but not in quiescent cells. Co-immunoprecipitation experiments confirmed these observations and suggested that PUM1 RNA-binding activity may be differentially regulated. They uncov-ered two mechanisms that could account for this differential activity. First, PUM1 stability is increased in cycling cells and second, phos-phorylation of Ser 714 in PUM1 appears to enhance its RNA-binding activity. However, phosphorylation of PUM1 is probably not responsible for its stabilization as no differ-ences in expression were observed between a Ser 714 phospho-mutant and wild-type PUM1. Further work is required to understand how PUM1 phosphorylation is regulated, the iden-tity of the cell signalling pathways and kinase(s) involved, and how exactly phosphorylation modulates PUM1 activity.

The authors also investigated how Pumilio proteins influence miR-221/miR-222 activity towards p27 mRNA. No interactions between PUM1 and Argonaute were detected, arguing against a role for PUM1 recruiting RISC to the 3 UTR of p27 mRNA. Rather, the authors evidence suggests that Pumilio proteins induce a switch in RNA conformation leading to an increase in the accessibility of miR-221/miR-222-associated RISC to its target site. Indeed,

their data suggest that the proximal PRE and distal miR-221/miR-222 target site adopt a sta-ble hairpin conformation in quiescent cells and this conformation is weakened in cycling cells. Finally, knockdown of Pumilio demonstrated that Pumilio proteins control this conforma-tional switch.

In summary, Kedde et al. reveal how an RNA-binding protein can promote miRNA activity by inducing a conformation switch in the RNA (Fig. 1). Their data support a model whereby p27 expression is high in quiescent cells because the interaction of miR-221/miR-222 with the target site of p27 mRNA is hindered. When cells re-enter the cell cycle, PUM1 is both upregulated and phosphorylated leading to increased RNA-binding activity. Activated PUM1 binds to the proximal PRE of p27 mRNA to allow RISC recognition of the miR-221/miR-222-target site that leads to miRNA-mediated repression of p27 expres-sion. Questions remain about the role of the second miR-221/miR-222 target site present in the p27 3 UTR. Although there is no evi-dence that accessibility of this site is regulated by Pumilio protein, as multiple miRNA target sites have been suggested to cooperate to medi-ate efficient target repression, the proximal target site for miR-221/miR-222 may require Pumilio-mediated activation of the distal site

to achieve maximal repression of p27 mRNA. Further work will be needed to elucidate the role of the distal PRE in controlling p27 expres-sion and miRNA function.

A functional link between a Caenorhabditis elegans Pumilio homologue puf9, and the miRNA let7, in repressing hbl1 expression has been proposed and genome-wide analysis of human PRE motifs has revealed an enrich-ment around predicted miRNA binding sites, suggesting evolutionary conserved interactions between Pumilio proteins and the miRNA reg-ulatory system10,11. Deciphering to what extent this interaction contributes to the control of gene expression is an important next step.

CoMpeting FinAnCiAl interestsThe authors declare no competing financial interests.

1. Bartel, D. P. Cell 136, 215233 (2009).2. Fabian, M. R., Sonenberg, N. & Filipowicz, W. Annu.

Rev. Biochem. 79, 351379 (2010).3. Kedde, M. et al. Nat. Cell Biol. 12, 10141020

(2010).4. le Sage, C., Nagel, R. & Agami, R. Cell Cycle 6, 2742

2749 (2007).5. le Sage, C. et al. EMBO J. 26, 36993708 (2007).6. Galardi, S. et al. J. Biol. Chem. 32, 2371623724

(2007).7. Gillies, J. K. & Lorimer, I. A. Cell Cycle 16, 20052009

(2007).8. Spassov, D. S. & Jurecic, R. IUBMB Life 55, 359,366

(2003).9. Morris, A. R., Mukherjee, N. & Keene, J. D. Mol. Cell.

Biol. 28, 40934103 (2008).10. Galgano, A. et al. PLoS One 3, e3164 (2008).11. Nolde, M. J. et al. Dev. Biol. 305, 551563 (2007).

Turning down the volume on transcriptional noiseDaniel neems and steven t. Kosak

Transcriptional noise has an important role in generating diversity in cellular populations that are seemingly identical. as this noise stems from the inherent stochasticity of gene expression, it has been unclear whether it is directly controlled. dig1, a regulator of the budding yeast mating pathway, is now shown to prevent transcriptional noise by regulating the spatial organization of downstream gene targets.

Transcription is controlled by activators, silenc-ers and basal transcription factors binding to promoters and enhancers. It is now also known that gene expression can be further modulated by chromatin modification, RNA interference and even spatial positioning of genetic loci within the nucleus. In all of these forms of regu-lation, there is an underlying stochastic behav-

iour inherent in complex systems. The outcome of these probabilistic events is defined as tran-scriptional noise, which has been shown to be important in establishing patterns of expres-sion that can yield varied cellular outcomes. An early study illustrated this phenomenon by showing that in an isogenic population of bacteria under uniform conditions individual cells still exhibited unique phenotypes1.

An elegant system, in which two green fluorescent protein (GFP) variants under the control of identical inducible promoters are introduced into defined genomic positions,

was used for the delineation of transcrip-tional noise into two components2,3. The first type of noise occurs at the level of the pro-moter. The events leading up to transcription initiation promoter binding and activa-tion occur through stochastic interactions of the regulatory and mechanistic components that, even in a completely synchronous popu-lation, will not happen exactly the same, even at identical promoters. Appropriately called intrinsic, this type of noise reveals itself with different levels of the two GFP variants in the same cell. Intrinsic noise is believed to have

Daniel Neems and Steven T. Kosak are in the

Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, 303 East Chicago Ave., Chicago, IL 60611, USA.e-mail: s-kosak@northwestern.edu

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