Alternative cleavage and polyadenylation (APA) leads to mRNA isoforms with different coding sequences (CDS) and/or 3′ untranslated regions (3′UTRs). Using 3′ Region Extraction And Deep Sequencing (3′READS), a method which addresses the internal priming and oligo(A) tail issues that commonly plague polyA site (pA) identification, we comprehensively mapped pAs in the mouse genome, thoroughly annotating 3′ ends of genes and revealing over five thousand pAs (~8% of total) flanked by A-rich sequences, which have hitherto been overlooked. About 79% of mRNA genes and 66% of long non-coding RNA (lncRNA) genes have APA; but these two gene types have distinct usage patterns for pAs in introns and upstream exons. Promoter-distal pAs become relatively more abundant during embryonic development and cell differentiation, a trend affecting pAs in both 3′-most exons and upstream regions. Upregulated isoforms generally have stronger pAs, suggesting global modulation of the 3′ end processing activity in development and differentiation.

Alternative cleavage and polyadenylation (APA) results in mRNA isoforms containing different 3’ untranslated regions (3’UTRs) and/or coding sequences. How core cleavage/polyadenylation (C/P) factors regulate APA is not well understood. Using siRNA knockdown coupled with deep sequencing, we found that several C/P factors can play significant roles in 3’UTR-APA. Whereas Pcf11 and Fip1 enhance usage of proximal poly(A) sites (pAs), CFI-25/68, PABPN1 and PABPC1 promote usage of distal pAs. Strong cis element biases were found for pAs regulated by CFI-25/68 or Fip1, and the distance between pAs plays an important role in APA regulation. In addition, intronic pAs are substantially regulated by splicing factors, with U1 mostly inhibiting C/P events in introns near the 5’ end of gene and U2 suppressing those in introns with features for efficient splicing. Furthermore, PABPN1 inhibits expression of transcripts with pAs near the transcription start site (TSS), a property possibly related to its role in RNA degradation. Finally, we found that groups of APA events regulated by C/P factors are also modulated in cell differentiation and development with distinct trends. Together, our results support an APA code where an APA event in a given cellular context is regulated by a number of parameters, including relative location to the TSS, splicing context, distance between competing pAs, surrounding cis elements and concentrations of core C/P factors.

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...

MicroRNAs (miRNAs) silence the expression of their mRNA targets mainly through promoting mRNA decay. The mechanism, kinetics and participating enzymes for miRNA-mediated decay in mammalian cells remain largely unclear. Combining the approaches of transcriptional pulsing, RNA-tethering, over-expression of dominant-negative mutants, and siRNA-mediated gene knockdown, we show that let-7 miRNA-induced silencing complexes (miRISCs), which contain Argonaute (Ago) and TNRC6 (also known as GW182) proteins, trigger highly rapid mRNA decay by inducing accelerated biphasic deadenylation mediated via Pan2-Pan3 and Ccr4-Caf1 deadenylase complexes followed by Dcp1-Dcp2 complex-directed decapping in mammalian cells. When tethered to mRNAs, all four human Ago proteins and TNRC6C are each able to recapitulate the two deadenylation steps. Two conserved human Ago2 phenylalanines (F470 and F505) are critical for recruiting TNRC6 to promote deadenylation. These findings indicate that promoting biphasic deadenylation to trigger mRNA decay is an intrinsic property of miRISCs.

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...

PolyA_DB is a database cataloging cleavage and polyadenylation sites (PASs) in several genomes. Previous versions were based mainly on expressed sequence tags (ESTs), which had a limited amount and could lead to inaccurate PAS identification due to the presence of internal A-rich sequences in transcripts. Here, we present an updated version of the database based solely on deep sequencing data. First, PASs are mapped by the 3′ region extraction and deep sequencing (3′READS) method, ensuring unequivocal PAS identification. Second, a large volume of data based on diverse biological samples increases PAS coverage by 3.5-fold over the EST-based version and provides PAS usage information. Third, strand-specific RNA-seq data are used to extend annotated 3′ ends of genes to obtain more thorough annotations of alternative polyadenylation (APA) sites. Fourth, conservation information of PAS across mammals sheds light on significance of APA sites. The database (URL: http://www.polya-db.org/v3) currently holds PASs in human, mouse, rat and chicken, and has links to the UCSC genome browser for further visualization and for integration with other genomic data.

Most eukaryotic genes express alternative polyadenylation (APA) isoforms with different 3′UTR lengths, production of which is influenced by cellular conditions. Here, we show that arsenic stress elicits global shortening of 3′UTRs through preferential usage of proximal polyadenylation sites during stress and enhanced degradation of long 3′UTR isoforms during recovery. We demonstrate that RNA-binding protein TIA1 preferentially interacts with alternative 3′UTR sequences through U-rich motifs, correlating with stress granule association and mRNA decay of long 3′UTR isoforms. By contrast, genes with shortened 3′UTRs due to stress-induced APA can evade mRNA clearance and maintain transcript abundance post stress. Furthermore, we show that stress causes distinct 3′UTR size changes in proliferating and differentiated cells, highlighting its context-specific impacts on the 3′UTR landscape. Together, our data reveal a global, 3′UTR-based mRNA stability control in stressed cells and indicate that APA can function as an adaptive mechanism to preserve mRNAs in response to stress.

SUMMARYRegulation of cleavage and polyadenylation (CPA) affects gene expression and polyadenylation site (PAS) choice. Here, we report that the CPA and termination factor PCF11 modulates gene expression on the basis of gene size. Although downregulation of PCF11 leads to inhibition of short gene expression, long genes are upregulated because of suppressed intronic polyadenylation (IPA) enriched in large introns. We show that this regulatory scheme, named PCF11-mediated expression regulation through IPA (PEIPA), takes place in cell differentiation, during which downregulation of PCF11 is coupled with upregulation of long genes with functions in cell morphology, adhesion, and migration. PEIPA targets distinct gene sets in different cell contexts with similar rules. Furthermore, PCF11 is autoregulated through a conserved IPA site, the removal of which leads to global activation of PASs close to gene promotors. Therefore, PCF11 uses distinct mechanisms to regulate genes of different sizes, and its autoregulation maintains homeostasis of PAS usage in the cell.