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.

Abbreviations used in this paper: ARE, AU-rich element; miRNA, microRNA; NMD, nonsense-mediated decay; PTC, premature translation-termination codon.The online version of this paper contains supplemental material. IntroductionRegulation of mRNA turnover plays an essential role in modulating gene expression Parker and Song, 2004 ;Garneau et al., 2007 ). For all major paths of mRNA decay yet recognized in mammalian cells, including mRNA decay directed by AU-rich elements (AREs) in the 3 Ј untranslated region ( Chen and Shyu, 1995 ), decay mediated by destabilizing elements in protein-coding regions ( Grosset et al., 2000 ; Chang et al., 2004 ), nonsense-mediated decay (NMD;Chen and Shyu, 2003 ), decay directed by microRNAs (miRNAs; Wu et al., 2006 ), and decay of stable mRNAs such as ␤ -globin mRNA ( Yamashita et al., 2005 ), the fi rst major step is deadenylation.Mammalian deadenylation is mediated by the concerted action of two different poly(A) nuclease complexes ( Yamashita et al., 2005 ). Poly(A) tails are fi rst shortened to ‫ف‬ 110 nt by Pan2 in association with Pan3. In the second phase of deadenylation, a complex composed of Ccr4 and Caf1 catalyze further shortening of the poly(A) tail to oligo(A). Decapping by the Dcp1 -Dcp2 complex ( Lykke-Andersen, 2002 ;van Dijk et al., 2002 ;Wang et al., 2002 ;Piccirillo et al., 2003 ) may occur during and/or after the second phase of deadenylation ( Yamashita et al., 2005 ). Although Pan3 and Caf1 associate with Pan2 and Ccr4 poly(A) nucleases, respectively ( Brown et al., 1996 ;Albert et al., 2000 ;Tucker et al., 2001 ;Temme et al., 2004 ;Uchida et al., 2004 ), their in vivo roles in mammalian mRNA turnover remain unclear. In yeast, Pan3 does not exhibit poly(A) nuclease activity but its association with Pan2 is required for proper function of Pan2 ( Brown et al., 1996 ;Mangus et al., 2004 ). In vitro experiments using recombinant human Pan2 and Pan3 proteins ( Uchida et al., 2004 ) suggest that Pan3 plays a role in enhancing the poly(A) nuclease activity of Pan2 in mammalian cells. However, ectopic overexpression of Pan2 alone in mouse NIH3T3 cells results in highly rapid and processive deadenylation of an otherwise stable reporter mRNA or a premature translation-termination codon (PTC) -containing mRNA ( Yamashita et al., 2005 ), indicating that Pan3 is not required for the nuclease activity of Pan2 for mammalian mRNA turnover. Instead, Pan3 may modulate the activity of Pan2 poly(A) nuclease or link deadenylation to subsequent decay of the mRNA body. Unlike Pan3, Caf1 exhibits poly(A) nuclease activity ( Daugeron et al., 2001 ;Dupressoir et al., 2001 ;Temme et al., 2004 ;Bianchin et al., 2005 ; Molin and Puisieux, 2005 ). However, studies in yeast show that Caf1 poly(A) nuclease activity per se is not required for general deadenylation in vivo, although the presence of Caf1 is necessary for proper deadenylation by D eadenylation is the major step triggering mammalian mRNA decay. One consequence of deadenylation is the formation of nontranslatable messenger RNA (m...

The underlying genetic etiology of rhabdomyolysis remains elusive in a significant fraction of individuals presenting with recurrent metabolic crises and muscle weakness. Using exome sequencing, we identified bi-allelic mutations in TANGO2 encoding transport and Golgi organization 2 homolog (Drosophila) in 12 subjects with episodic rhabdomyolysis, hypoglycemia, hyperammonemia, and susceptibility to life-threatening cardiac tachyarrhythmias. A recurrent homozygous c.460G>A (p.Gly154Arg) mutation was found in four unrelated individuals of Hispanic/Latino origin, and a homozygous ∼34 kb deletion affecting exons 3-9 was observed in two families of European ancestry. One individual of mixed Hispanic/European descent was found to be compound heterozygous for c.460G>A (p.Gly154Arg) and the deletion of exons 3-9. Additionally, a homozygous exons 4-6 deletion was identified in a consanguineous Middle Eastern Arab family. No homozygotes have been reported for these changes in control databases. Fibroblasts derived from a subject with the recurrent c.460G>A (p.Gly154Arg) mutation showed evidence of increased endoplasmic reticulum stress and a reduction in Golgi volume density in comparison to control. Our results show that the c.460G>A (p.Gly154Arg) mutation and the exons 3-9 heterozygous deletion in TANGO2 are recurrent pathogenic alleles present in the Latino/Hispanic and European populations, respectively, causing considerable morbidity in the homozygotes in these populations.

In mammalian cells, mRNA decay begins with deadenylation, which involves two consecutive phases mediated by the PAN2-PAN3 and the CCR4-CAF1 complexes, respectively. The regulation of the critical deadenylation step and its relationship with RNA-processing bodies (P-bodies), which are thought to be a site where poly(A)-shortened mRNAs get degraded, are poorly understood. Using the Tet-Off transcriptional pulsing approach to investigate mRNA decay in mouse NIH 3T3 fibroblasts, we found that TOB, an antiproliferative transcription factor, enhances mRNA deadenylation in vivo. Results from glutathione S-transferase pull-down and coimmunoprecipitation experiments indicate that TOB can simultaneously interact with the poly(A) nuclease complex CCR4-CAF1 and the cytoplasmic poly(A)-binding protein, PABPC1. Combining these findings with those from mutagenesis studies, we further identified the protein motifs on TOB and PABPC1 that are necessary for their interaction and found that interaction with PABPC1 is necessary for TOB's deadenylation-enhancing effect. Moreover, our immunofluorescence microscopy results revealed that TOB colocalizes with P-bodies, suggesting a role of TOB in linking deadenylation to the P-bodies. Our findings reveal a new mechanism by which the fate of mammalian mRNA is modulated at the deadenylation step by a protein that recruits poly(A) nuclease(s) to the 3 poly(A) tail-PABP complex.Deadenylation is the first major step that triggers mRNA decay in eukaryotic cells (reviewed in references 19, 41, and 44). Computational modeling of eukaryotic mRNA turnover indicates that changes in levels of mRNA are highly leveraged to the rate of deadenylation (8). The importance of deadenylation in regulating mammalian mRNA turnover can be observed in several modes of mRNA decay, including decay directed by AU-rich elements in the 3Ј untranslated region (4, 10), the rapid decay mediated by destabilizing elements in protein-coding regions (9, 23), the surveillance mechanism that detects and degrades nonsense-containing mRNA (11), and the decay directed by microRNA (59). Shortening of the 3Ј poly(A) tail also plays a critical role in rendering mRNAs nontranslatable (26,46,58), thus inactivating gene expression. In spite of the importance of deadenylation, relatively little is known about the mechanisms that control it.Recent progress in identifying key mammalian poly(A) nucleases involved in deadenylation (1,6,13,16,20,38,53,55) has offered the opportunity to examine the regulation of deadenylation and to characterize the participating regulatory proteins. In mammalian cells, shortening of the poly(A) tail is mediated by the consecutive activities of two different poly(A) nuclease complexes (61). During the first phase, PAN2, presumably complexed with PAN3 (53, 61), shortens the poly(A) tails to ϳ110 A nucleotides. In the second phase, CCR4, presumably complexed with CAF1 (6, 55, 61), further shortens the poly(A) tail to oligo(A). Decapping mediated by the DCP1-DCP2 complex (36, 54, 56) was found to occur after ei...

Messenger RNA decay mediated by the c-fos major protein coding-region determinant of instability (mCRD) is a useful system for studying translationally coupled mRNA turnover. Among the five mCRD-associated proteins identified previously, UNR was found to be an mCRD-binding protein and also a PABP-interacting protein. Interaction between UNR and PABP is necessary for the full destabilization function of the mCRD. By testing different classes of mammalian poly(A) nucleases, we identified CCR4 as a poly(A) nuclease involved in the mCRD-mediated rapid deadenylation in vivo and also associated with UNR. Blocking either translation initiation or elongation greatly impeded poly(A) shortening and mRNA decay mediated by the mCRD, demonstrating that the deadenylation step is coupled to ongoing translation of the message. These findings suggest a model in which the mCRD/UNR complex serves as a "landing/assembly" platform for formation of a deadenylation/decay mRNA-protein complex on an mCRD-containing transcript. The complex is dormant prior to translation. Accelerated deadenylation and decay of the transcript follows ribosome transit through the mCRD. This study provides new insights into a mechanism by which interplay between mRNA turnover and translation determines the lifespan of an mCRD-containing mRNA in the cytoplasm.[Keywords: CCR4; UNR; PABP; translational control; mRNA turnover; poly(A) nuclease] Supplemental material is available at http://www.genesdev.org.