Tob2 phosphorylation regulates global mRNA turnover to reshape transcriptome and impact cell proliferation.

Tob2, an anti-proliferative protein, promotes deadenylation through recruiting Caf1 deadenylase to the mRNA poly(A) tail by simultaneously interacting with both Caf1 and poly(A)-binding protein (PABP). Previously, we found that changes in Tob2 phosphorylation can alter its PABP-binding ability and deadenylation-promoting function. However, it remained unknown regarding the relevant kinase(s). Moreover, it was unclear whether Tob2 phosphorylation modulates the transcriptome and whether the phosphorylation is linked to Tob2's anti-proliferative function. In this study, we found that c-Jun amino-terminal kinase (JNK) increases phosphorylation of Tob2 at many Ser/Thr sites in the intrinsically disordered region (IDR) that contains two separate PABP-interacting PAM2 motifs. JNK-induced phosphorylation or phosphomimetic mutations at these sites weaken the Tob2-PABP interaction. In contrast, JNK-independent phosphorylation of Tob2 at serine 254 (S254) greatly enhances Tob2 interaction with PABP and its ability to promote deadenylation. We discovered that both PAM2 motifs are required for Tob2 to display these features. Combining mass spectrometry analysis, poly(A) size-distribution profiling, transcriptome-wide mRNA turnover analyses, and cell proliferation assays, we found that the phosphomimetic mutation at S254 (S254D) enhances Tob2's association with PABP, leading to accelerated deadenylation and decay of mRNAs globally. Moreover, the Tob2-S254D mutant accelerates the decay of many transcripts coding for cell cycle related proteins and enhances anti-proliferation function. Our findings reveal a novel mechanism by which Ccr4-Not complex is recruited by Tob2 to the mRNA 3' poly(A)-PABP complex in a phosphorylation dependent manner to promote rapid deadenylation and decay across the transcriptome, eliciting transcriptome reprogramming and suppressed cell proliferation.

Emerging Themes in Regulation of Global mRNA Turnover in cis.

mRNA is the molecule that conveys genetic information from DNA to the translation apparatus. mRNAs in all organisms display a wide range of stability, and mechanisms have evolved to selectively and differentially regulate individual mRNA stability in response to intracellular and extracellular cues. In recent years, three seemingly distinct aspects of RNA biology-mRNA N6-methyladenosine (m6A) modification, alternative 3' end processing and polyadenylation (APA), and mRNA codon usage-have been linked to mRNA turnover, and all three aspects function to regulate global mRNA stability in cis. Here, we discuss the discovery and molecular dissection of these mechanisms in relation to how they impact the intrinsic decay rate of mRNA in eukaryotes, leading to transcriptome reprogramming.

Protein segregase meddles in remodeling of mRNA-protein complexes.

Remodeling of RNA-protein complexes (mRNPs) plays a critical role in mRNA biogenesis and metabolism. However, relatively little is known about the underlying mechanism and regulation of the mRNP remodeling. In this issue of Genes & Development, Zhou and colleagues (pp. 1046-1058) report that a protein remodeling machine, the p97-UBXD8 complex, disassembles mRNPs containing the AU-rich elements (AREs) bound by HuR proteins in a nondegradative, ubiquitin signaling-dependent manner, revealing a novel mechanism to regulate mRNA turnover.

Antagonistic actions of two human Pan3 isoforms on global mRNA turnover.

Deadenylation is a fundamental process that regulates eukaryotic gene expression. Mammalian deadenylation exhibits biphasic kinetics, with the Pan2-Pan3 and Ccr4-Caf1 deadenylase complexes mediating the first and second phase, respectively; however, the significance of the biphasic nature of deadenylation in mRNA turnover remains unclear. In this study, we discovered that two distinct isoforms of human Pan3 display opposing properties necessary for coordinating the two phases of deadenylation. The shorter isoform (Pan3S) interacts more strongly with PABP than the longer isoform (Pan3L) does. Pan2 deadenylase activity is enhanced by Pan3S but suppressed by Pan3L. Knocking down individual Pan3 isoforms has opposing effects on the global poly(A) tail length profile, P-body formation, and different mRNA decay pathways. Transcriptome-wide analysis of Pan3 knockdown effects on mRNA turnover shows that depleting either Pan3 isoform causes profound and extensive changes in mRNA stability globally. These results reveal a new fundamental step governing mammalian mRNA metabolism. We propose that the first phase of deadenylation, coordinated through the interplay among the two Pan3 isoforms, Pan2, and PABP, represents a cytoplasmic mRNA maturation step important for proper mRNA turnover.

MiR-26 down-regulates TNF-α/NF-κB signalling and IL-6 expression by silencing HMGA1 and MALT1.

MiR-26 has emerged as a key tumour suppressor in various cancers. Accumulating evidence supports that miR-26 regulates inflammation and tumourigenicity largely through down-regulating IL-6 production, but the underlying mechanism remains obscure. Here, combining a transcriptome-wide approach with manipulation of cellular miR-26 levels, we showed that instead of directly targeting IL-6 mRNA for gene silencing, miR-26 diminishes IL-6 transcription activated by TNF-α through silencing NF-κB signalling related factors HMGA1 and MALT1. We demonstrated that miR-26 extensively dampens the induction of many inflammation-related cytokine, chemokine and tissue-remodelling genes that are activated via NF-κB signalling pathway. Knocking down both HMGA1 and MALT1 by RNAi had a silencing effect on NF-κB-responsive genes similar to that caused by miR-26. Moreover, we discovered that poor patient prognosis in human lung adenocarcinoma is associated with low miR-26 and high HMGA1 or MALT1 levels and not with levels of any of them individually. These new findings not only unravel a novel mechanism by which miR-26 dampens IL-6 production transcriptionally but also demonstrate a direct role of miR-26 in down-regulating NF-κB signalling pathway, thereby revealing a more critical and broader role of miR-26 in inflammation and cancer than previously realized.

HuD stimulates translation via eIF4A.

In this issue of Molecular Cell, Fukao et al. (2009) report that HuD upregulates mRNA translation through direct interaction with eIF4A in the 5' cap-binding complex, revealing a posttranscriptional role for HuD in neuronal development and plasticity.

Mammalian miRNA RISC recruits CAF1 and PABP to affect PABP-dependent deadenylation.

MicroRNAs (miRNAs) inhibit mRNA expression in general by base pairing to the 3'UTR of target mRNAs and consequently inhibiting translation and/or initiating poly(A) tail deadenylation and mRNA destabilization. Here we examine the mechanism and kinetics of miRNA-mediated deadenylation in mouse Krebs-2 ascites extract. We demonstrate that miRNA-mediated mRNA deadenylation occurs subsequent to initial translational inhibition, indicating a two-step mechanism of miRNA action, which serves to consolidate repression. We show that a let-7 miRNA-loaded RNA-induced silencing complex (miRISC) interacts with the poly(A)-binding protein (PABP) and the CAF1 and CCR4 deadenylases. In addition, we demonstrate that miRNA-mediated deadenylation is dependent upon CAF1 activity and PABP, which serves as a bona fide miRNA coactivator. Importantly, we present evidence that GW182, a core component of the miRISC, directly interacts with PABP via its C-terminal region and that this interaction is required for miRNA-mediated deadenylation.

ROCK inhibition enhances microRNA function by promoting deadenylation of targeted mRNAs via increasing PAIP2 expression.

The reduced expression levels and functional impairment of global miRNAs are related to various human diseases, including cancers. However, relatively little is known about how global miRNA function may be upregulated. Here, we report that global miRNA function can be enhanced by Rho-associated, coiled-coil-containing protein kinase (ROCK) inhibitors. The regulation of miRNA function by ROCK inhibitors is mediated, at least in part, by poly(A)-binding protein-interacting protein 2 (PAIP2), which enhances poly(A)-shortening of miRNA-targeted mRNAs and leads to global upregulation of miRNA function. In the presence of a ROCK inhibitor, PAIP2 expression is enhanced by the transcription factor hepatocyte nuclear factor 4 alpha (HNF4A) through increased ROCK1 nuclear localization and enhanced ROCK1 association with HNF4A. Our data reveal an unexpected role of ROCK1 as a cofactor of HNF4A in enhancing PAIP2 transcription. ROCK inhibitors may be useful for the various pathologies associated with the impairment of global miRNA function.

Phosphorylation at intrinsically disordered regions of PAM2 motif-containing proteins modulates their interactions with PABPC1 and influences mRNA fate.

Cytoplasmic poly(A)-binding protein (PABP) C1 recruits different interacting partners to regulate mRNA fate. The majority of PABP-interacting proteins contain a PAM2 motif to mediate their interactions with PABPC1. However, little is known about the regulation of these interactions or the corresponding functional consequences. Through in silico analysis, we found that PAM2 motifs are generally embedded within an extended intrinsic disorder region (IDR) and are located next to cluster(s) of potential serine (Ser) or threonine (Thr) phosphorylation sites within the IDR. We hypothesized that phosphorylation at these Ser/Thr sites regulates the interactions between PAM2-containing proteins and PABPC1. In the present study, we have tested this hypothesis using complementary approaches to increase or decrease phosphorylation. The results indicate that changing the extent of phosphorylation of three PAM2-containing proteins (Tob2, Pan3, and Tnrc6c) alters their ability to interact with PABPC1. Results from experiments using phospho-blocking or phosphomimetic mutants in PAM2-containing proteins further support our hypothesis. Moreover, the phosphomimetic mutations appreciably affected the functions of these proteins in mRNA turnover and gene silencing. Taken together, these results provide a new framework for understanding the roles of intrinsically disordered proteins in the dynamic and signal-dependent control of cytoplasmic mRNA functions.

BTG/TOB factors impact deadenylases.

BTG/TOB factors are a family of antiproliferative proteins whose expression is altered in numerous cancers. They have been implicated in cell differentiation, development and apoptosis. Although proposed to affect transcriptional regulation, these factors interact with CAF1, a subunit of the main eukaryotic deadenylase, and with poly(A)-binding-proteins, strongly suggesting a role in post-transcriptional regulation of gene expression. The recent determination of the structures of BTG2, TOB1 N-terminal domain (TOB1N138) and TOB1N138-CAF1 complexes support a role for BTG/TOB proteins in mRNA deadenylation, a function corroborated by recently published functional characterizations. We highlight molecular mechanisms by which BTG/TOB proteins influence deadenylation and discuss the need for a better understanding of BTG/TOB physiological functions.

Unraveling regulation and new components of human P-bodies through a protein interaction framework and experimental validation.

The cellular factors involved in mRNA degradation and translation repression can aggregate into cytoplasmic domains known as GW bodies or mRNA processing bodies (P-bodies). However, current understanding of P-bodies, especially the regulatory aspect, remains relatively fragmentary. To provide a framework for studying the mechanisms and regulation of P-body formation, maintenance, and disassembly, we compiled a list of P-body proteins found in various species and further grouped both reported and predicted human P-body proteins according to their functions. By analyzing protein-protein interactions of human P-body components, we found that many P-body proteins form complex interaction networks with each other and with other cellular proteins that are not recognized as P-body components. The observation suggests that these other cellular proteins may play important roles in regulating P-body dynamics and functions. We further used siRNA-mediated gene knockdown and immunofluorescence microscopy to demonstrate the validity of our in silico analyses. Our combined approach identifies new P-body components and suggests that protein ubiquitination and protein phosphorylation involving 14-3-3 proteins may play critical roles for post-translational modifications of P-body components in regulating P-body dynamics. Our analyses provide not only a global view of human P-body components and their physical interactions but also a wealth of hypotheses to help guide future research on the regulation and function of human P-bodies.

Deadenylation and P-bodies.

Deadenylation is the major step in triggering mRNA decay and results in mRNA translation inhibition in eukaryotic cells. Therefore, it is plausible that deadenylation also induces the mRNP remodeling required for formation of GW bodies or RNA processing bodies (P-bodies), which harbor translationally silenced mRNPs. In this chapter, we discuss several examples to illustrate the roles of deadenylation in regulating gene expression. We highlight several lines of evidence indicating that even though non-translatable mRNPs may be prepared and/or assembled into P-bodies in different ways, deadenylation is always a necessary, and perhaps the earliest, step in mRNA decay pathways that enable mRNP remodeling required for P-body formation. Thus, deadenylation and the participating deadenylases are not simply required for preparing mRNA substrates; they play an indispensable role both structurally and functionally in P-body formation and regulation.

Concerted action of poly(A) nucleases and decapping enzyme in mammalian mRNA turnover.

In mammalian cells, the enzymatic pathways involved in cytoplasmic mRNA decay are incompletely defined. In this study, we have used two approaches to disrupt activities of deadenylating and/or decapping enzymes to monitor effects on mRNA decay kinetics and trap decay intermediates. Our results show that deadenylation is the key first step that triggers decay of both wild-type stable and nonsense codon-containing unstable beta-globin mRNAs in mouse NIH3T3 fibroblasts. PAN2 and CCR4 are the major poly(A) nucleases active in cytoplasmic deadenylation that have biphasic kinetics, with PAN2 initiating deadenylation followed by CCR4-mediated poly(A) shortening. DCP2-mediated decapping takes place after deadenylation and may serve as a backup mechanism for triggering mRNA decay when initial deadenylation by PAN2 is compromised. Our findings reveal a functional link between deadenylation and decapping and help to define in vivo pathways for mammalian cytoplasmic mRNA decay.

Rapid deadenylation triggered by a nonsense codon precedes decay of the RNA body in a mammalian cytoplasmic nonsense-mediated decay pathway.

Nonsense-mediated mRNA decay (NMD) is an RNA surveillance pathway that detects and destroys aberrant mRNAs containing nonsense or premature termination codons (PTCs) in a translation-dependent manner in eukaryotes. In yeast, the NMD pathway bypasses the deadenylation step and directly targets PTC-containing messages for decapping, followed by 5'-to-3' exonuclease digestion of the RNA body. In mammals, most PTC-containing mRNAs are subject to active nucleus-associated NMD. Here, using two distinct transcription-pulsing approaches to monitor mRNA deadenylation and decay kinetics, we demonstrate the existence of an active cytoplasmic NMD pathway in mammalian cells. In this pathway, a nonsense codon triggers accelerated deadenylation that precedes decay of the PTC-containing mRNA body. Transcript is stabilized when accelerated deadenylation is impeded by blocking translation initiation; by ectopically expressing two RNA-binding proteins, UNR and NSAP1; or by ectopically expressing a UPF1 dominant-negative mutant. These results are consistent with the notion that the nonsense codon can function in the cytoplasm by promoting rapid removal of the poly(A) tail as a necessary first step in the decay process.

Highly selective actions of HuR in antagonizing AU-rich element-mediated mRNA destabilization.

Human RNA-binding protein HuR, a nucleocytoplasmic shuttling protein, is a ubiquitously expressed member of the family of Hu proteins, which consist of two N-terminal RNA recognition motifs (RRM1 and RRM2), a hinge region, and a C-terminal RRM (RRM3). Although in vitro experiments showed indiscriminate binding of Hu proteins synthesized in bacterial systems to many different AU-rich elements (AREs), in vivo studies have pointed to a cytoplasmic role for HuR protein in antagonizing the rapid decay of some specific ARE-containing mRNAs, depending on physiological situations. By ectopically overexpressing HuR and its mutant derivatives in NIH 3T3 cells to mimic HuR upregulation of specific ARE-containing mRNAs in other systems, we have examined the in vivo ARE-binding specificity of HuR and dissected its functionally critical domains. We show that in NIH 3T3 cells, HuR stabilizes reporter messages containing only the c-fos ARE and not other AREs. Two distinct binding sites were identified within the c-fos ARE, the 5' AUUUA-containing domain and the 3' U-stretch-containing domain. These actions of HuR are markedly different from those of another ARE-binding protein, hnRNP D (also termed AUF1), which in vivo recognizes AUUUA repeats found in cytokine AREs and can exert both stabilizing and destabilizing effects. Further experiments showed that any combination of two of the three RRM domains of HuR is sufficient for strong binding to the c-fos ARE in vitro and to exert an RNA stabilization effect in vivo comparable to that of intact HuR and that the hinge region containing nucleocytoplasmic shuttling signals is dispensable for the stabilization effect of HuR. Our data suggest that the ARE-binding specificity of HuR in vivo is modulated to interact only with and thus regulate specific AREs in a cell type- and physiological state-dependent manner.

Emerging mechanisms of mRNP remodeling regulation.

The assembly and remodeling of the components of messenger ribonucleoprotein particles (mRNPs) are important in determining the fate of a messenger RNA (mRNA). A combination of biochemical and cell biology research, recently complemented by genome-wide high-throughput approaches, has led to significant progress on understanding the formation, dynamics, and function of mRNPs. These studies also advanced the challenging process of identifying the evolving constituents of individual mRNPs at various stages during an mRNA's lifetime. While research on mRNP remodeling in general has been gaining momentum, there has been relatively little attention paid to the regulatory aspect of mRNP remodeling. Here, we discuss the results of some new studies and potential mechanisms for regulation of mRNP remodeling.

Evidence providing new insights into TOB-promoted deadenylation and supporting a link between TOB's deadenylation-enhancing and antiproliferative activities.

The mammalian TOB1 and TOB2 proteins have emerged as key players in repressing cell proliferation. Accumulating evidence indicates that TOBs regulate mRNA deadenylation. A recruitment model was proposed in which TOBs promote deadenylation by recruiting CAF1-CCR4 deadenylase complex to the 3' end of mRNAs by simultaneously binding CAF1 and PABP. However, the exact molecular mechanism underlying TOB-promoted deadenylation remains unclear. It is also unclear whether TOBs' antiproliferative and deadenylation-promoting activities are connected. Here, we combine biochemical analyses with a functional assay directly monitoring deadenylation and mRNA decay to characterize the effects of tethering TOBs or their mutant derivatives to mRNAs. The results provide direct evidence supporting the recruitment model and reveal a link between TOBs' antiproliferative and deadenylation-promoting activities. We also find that TOBs' actions in deadenylation are independent of the phosphorylation state of three serines known to regulate antiproliferative actions, suggesting that TOBs arrest cell growth through at least two different mechanisms. TOB1 and TOB2 were interchangeable in the properties tested here, indicating considerable functional redundancy between the two proteins. We propose that their multiple modes of modulating mRNA turnover and arresting cell growth permit the TOB proteins to coordinate their diverse roles in controlling cell growth and differentiation.

Mechanisms of deadenylation-dependent decay.

Degradation of messenger RNAs (mRNAs) plays an essential role in modulation of gene expression and in quality control of mRNA biogenesis. Nearly all major mRNA decay pathways characterized thus far in eukaryotes are initiated by deadenylation, i.e., shortening of the mRNA 3(') poly(A) tail. Deadenylation is often a rate-limiting step for mRNA degradation and translational silencing, making it an important control point for both processes. In this review, we discuss the fundamental principles that govern mRNA deadenylation in eukaryotes. We use several major mRNA decay pathways in mammalian cells to illustrate mechanisms and regulation of deadenylation-dependent mRNA decay, including decay directed by adenine/uridine-rich elements (AREs) in the 3(') -untranslated region (UTR), the rapid decay mediated by destabilizing elements in protein-coding regions, the surveillance mechanism that detects and degrades nonsense-containing mRNA [i.e., nonsense-mediated decay (NMD)], the decay directed by miRNAs, and the default decay pathway for stable messages. Mammalian mRNA deadenylation involves two consecutive phases mediated by the PAN2-PAN3 and the CCR4-CAF1 complexes, respectively. Decapping takes place after deadenylation and may serve as a backup mechanism to trigger mRNA decay if initial deadenylation is compromised. In addition, we discuss how deadenylation impacts the dynamics of RNA processing bodies (P-bodies), where nontranslatable mRNAs can be degraded or stored. Possible models for mechanisms of various deadenylation-dependent mRNA decay pathways are also discussed.