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 recognition of the importance of mRNA turnover in regulating eukaryotic gene expression has mandated the development of reliable, rigorous, and "user-friendly" methods to accurately measure changes in mRNA stability in mammalian cells. Frequently, mRNA stability is studied indirectly by analyzing the steady-state level of mRNA in the cytoplasm; in this case, changes in mRNA abundance are assumed to reflect only mRNA degradation, an assumption that is not always correct. Although direct measurements of mRNA decay rate can be performed with kinetic labeling techniques and transcriptional inhibitors, these techniques often introduce significant changes in cell physiology. Furthermore, many critical mechanistic issues as to deadenylation kinetics, decay intermediates, and precursor-product relationships cannot be readily addressed by these methods. In light of these concerns, we have previously reported transcriptional pulsing methods based on the c-fos serum-inducible promoter and the tetracycline-regulated (Tet-off) promoter systems to better explain mechanisms of mRNA turnover in mammalian cells. In this chapter, we describe and discuss in detail different protocols that use these two transcriptional pulsing methods. The information described here also provides guidelines to help develop optimal protocols for studying mammalian mRNA turnover in different cell types under a wide range of physiologic conditions.
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...
Proper gene expression relies on a class of ubiquitously expressed, uridine-rich small nuclear RNAs (snRNAs) transcribed by RNA polymerase II (RNAPII). Vertebrate snRNAs are transcribed from a unique promoter, which is required for proper 3-end formation, and cleavage of the nascent transcript involves the activity of a poorly understood set of proteins called the Integrator complex. To examine 3-end formation in Drosophila melanogaster, we developed a cell-based reporter that monitors aberrant 3-end formation of snRNA through the gain in expression of green fluorescent protein (GFP). We used this reporter in Drosophila S2 cells to determine requirements for U7 snRNA 3-end formation and found that processing was strongly dependent upon nucleotides located within the 3 stem-loop as well as sequences likely to comprise the Drosophila equivalent of the vertebrate 3 box. Substitution of the actin promoter for the snRNA promoter abolished proper 3-end formation, demonstrating the conserved requirement for an snRNA promoter in Drosophila. We tested the requirement for all Drosophila Integrator subunits and found that Integrators 1, 4, 9, and 11 were essential for 3-end formation and that Integrators 3 and 10 may be dispensable for processing. Depletion of cleavage and polyadenylation factors or of histone pre-mRNA processing factors did not affect U7 snRNA processing efficiency, demonstrating that the Integrator complex does not share components with the mRNA 3-end processing machinery. Finally, flies harboring mutations in either Integrator 4 or 7 fail to complete development and accumulate significant levels of misprocessed snRNA in the larval stages.In eukaryotes, the major transcripts produced by RNA polymerase II (RNAPII) include the polyadenylated [poly (A) ϩ ] mRNAs, the replication-dependent histone mRNAs, and the Sm class of small nuclear RNAs (snRNAs). The 3Ј ends of these three general classes of RNAs are all formed by cotranscriptional cleavage, but each one has a distinct mechanism for 3Ј-end formation (for reviews, see references 29 and 32). In poly(A) ϩ and histone pre-mRNAs there are conserved upstream and downstream sequences that flank the cleavage site; factors bind to these sites and then recruit additional factors that initiate cleavage (53). In the case of poly(A) ϩ pre-mRNA, the upstream element is the canonical AAUAAA polyadenylation signal (PAS) and the downstream sequence is the G/Urich downstream element (DSE). Recognition of the PAS is carried out by the cleavage and polyadenylation specificity complex (CPSF) component CPSF160 via its RNA recognition motifs (RRM) (36), whereas the DSE is bound by the RRM of the cleavage stimulation factor (CstF) component CstF64 (28). Subsequent to this recognition event is recruitment of additional factors that activate the endonucleolytic cleavage between the PAS and the DSE.Histone pre-mRNA contains a distinct set of flanking elements. Upstream of the cleavage site is a conserved stem-loop structure (SL) and downstream a purine-rich element called the ...
Formation of the 39 end of RNA polymerase II-transcribed snRNAs requires a poorly understood group of proteins called the Integrator complex. Here we used a fluorescence-based read-through reporter that expresses GFP in response to snRNA misprocessing and performed a genome-wide RNAi screen in Drosophila S2 cells to identify novel factors required for snRNA 39-end formation. In addition to the known Integrator complex members, we identified Asunder and CG4785 as additional Integrator subunits. Functional and biochemical experiments revealed that Asunder and CG4785 are additional core members of the Integrator complex. We also identified a conserved requirement in both fly and human snRNA 39-end processing for cyclin C and Cdk8 that is distinct from their function in the Mediator Cdk8 module. Moreover, we observed biochemical association between Integrator proteins and cyclin C/Cdk8, and that overexpression of a kinase-dead Cdk8 causes snRNA misprocessing. These data functionally define the Drosophila Integrator complex and demonstrate an additional function for cyclin C/Cdk8 unrelated to its function in Mediator.
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