Dinghai Zheng

Analysis of alternative cleavage and polyadenylation by 3′ region extraction and deep sequencing

Alternative cleavage and polyadenylation (APA) generates diverse mRNA isoforms. We developed 3′ region extraction and deep sequencing (3′READS) to address mispriming issues that commonly plague poly(A) site (pA) identification, and we used the method to comprehensively map pAs in the mouse genome. Thorough annotation of gene 3′ ends revealed over 5,000 previously overlooked pAs (∼8% of total) flanked by A-rich sequences, underscoring the necessity of using an accurate tool for pA mapping. About 79% of mRNA genes and 66% of long noncoding RNA genes undergo APA, but these two gene types have distinct usage patterns for pAs in introns and upstream exons. Quantitative analysis of APA isoforms by 3′READS indicated that promoter-distal pAs, regardless of intron or exon locations, become more abundant during embryonic development and cell differentiation and that upregulated isoforms have stronger pAs, suggesting global modulation of the 3′ end–processing activity in development and differentiation.

Deadenylation is prerequisite for P-body formation and mRNA decay in mammalian cells

Deadenylation is the major step triggering mammalian mRNA decay. One consequence of deadenylation is the formation of nontranslatable messenger RNA (mRNA) protein complexes (messenger ribonucleoproteins [mRNPs]). Nontranslatable mRNPs may accumulate in P-bodies, which contain factors involved in translation repression, decapping, and 5′-to-3′ degradation. We demonstrate that deadenylation is required for mammalian P-body formation and mRNA decay. We identify Pan2, Pan3, and Caf1 deadenylases as new P-body components and show that Pan3 helps recruit Pan2, Ccr4, and Caf1 to P-bodies. Pan3 knockdown causes a reduction of P-bodies and has differential effects on mRNA decay. Knocking down Caf1 or overexpressing a Caf1 catalytically inactive mutant impairs deadenylation and mRNA decay. P-bodies are not detected when deadenylation is blocked and are restored when the blockage is released. When deadenylation is impaired, P-body formation is not restorable, even when mRNAs exit the translating pool. These results support a dynamic interplay among deadenylation, mRNP remodeling, and P-body formation in selective decay of mammalian mRNA.

Ago–TNRC6 triggers microRNA-mediated decay by promoting two deadenylation steps

MicroRNAs (miRNAs) silence the expression of their mRNA targets mainly by 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, overexpression of dominant-negative mutants, and siRNA-mediated gene knockdown, we show that let-7 miRNA-induced silencing complexes (miRISCs), which contain the proteins Argonaute (Ago) and TNRC6 (also known as GW182), trigger very rapid mRNA decay by inducing accelerated biphasic deadenylation mediated by 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 (Phe470 and Phe505) are critical for recruiting TNRC6 to promote deadenylation. These findings indicate that promotion of biphasic deadenylation to trigger mRNA decay is an intrinsic property of miRISCs.MicroRNAs (miRNAs) silence the expression of their mRNA targets mainly by 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, overexpression of dominant-negative mutants, and siRNA-mediated gene knockdown, we show that let-7 miRNA-induced silencing complexes (miRISCs), which contain the proteins Argonaute (Ago) and TNRC6 (also known as GW182), trigger very rapid mRNA decay by inducing accelerated biphasic deadenylation mediated by 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 (Phe470 and Phe505) are critical for recruiting TNRC6 to promote deadenylation. These findings indicate that promotion of biphasic deadenylation to trigger mRNA decay is an intrinsic property of miRISCs.

Human TOB, an Antiproliferative Transcription Factor, Is a Poly(A)-Binding Protein-Dependent Positive Regulator of Cytoplasmic mRNA Deadenylation

ABSTRACT 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 TOBs 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.

Systematic Profiling of Poly(A)+ Transcripts Modulated by Core 3’ End Processing and Splicing Factors Reveals Regulatory Rules of Alternative Cleavage and Polyadenylation

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.

Comparative Analysis of mRNA Isoform Expression in Cardiac Hypertrophy and Development Reveals Multiple Post-Transcriptional Regulatory Modules

Cardiac hypertrophy is enlargement of the heart in response to physiological or pathological stimuli, chiefly involving growth of myocytes in size rather than in number. Previous studies have shown that the expression pattern of a group of genes in hypertrophied heart induced by pressure overload resembles that at the embryonic stage of heart development, a phenomenon known as activation of the “fetal gene program”. Here, using a genome-wide approach we systematically defined genes and pathways regulated in short- and long-term cardiac hypertrophy conditions using mice with transverse aortic constriction (TAC), and compared them with those regulated at different stages of embryonic and postnatal development. In addition, exon-level analysis revealed widespread mRNA isoform changes during cardiac hypertrophy resulting from alternative usage of terminal or internal exons, some of which are also developmentally regulated and may be attributable to decreased expression of Fox-1 protein in cardiac hypertrophy. Genes with functions in certain pathways, such as cell adhesion and cell morphology, are more likely to be regulated by alternative splicing. Moreover, we found 3′UTRs of mRNAs were generally shortened through alternative cleavage and polyadenylation in hypertrophy, and microRNA target genes were generally de-repressed, suggesting coordinated mechanisms to increase mRNA stability and protein production during hypertrophy. Taken together, our results comprehensively delineated gene and mRNA isoform regulation events in cardiac hypertrophy and revealed their relations to those in development, and suggested that modulation of mRNA isoform expression plays an importance role in heart remodeling under pressure overload.

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.

Alternative cleavage and polyadenylation in spermatogenesis connects chromatin regulation with post-transcriptional control

BackgroundMost mammalian genes display alternative cleavage and polyadenylation (APA). Previous studies have indicated preferential expression of APA isoforms with short 3’ untranslated regions (3’UTRs) in testes.ResultsBy deep sequencing of the 3’ end region of poly(A) + transcripts, we report widespread shortening of 3’UTR through APA during the first wave of spermatogenesis in mouse, with 3’UTR size being the shortest in spermatids. Using genes without APA as a control, we show that shortening of 3’UTR eliminates destabilizing elements, such as U-rich elements and transposable elements, which appear highly potent during spermatogenesis. We additionally found widespread regulation of APA events in introns and exons that can affect the coding sequence of transcripts and global activation of antisense transcripts upstream of the transcription start site, suggesting modulation of splicing and initiation of transcription during spermatogenesis. Importantly, genes that display significant 3’UTR shortening tend to have functions critical for further sperm maturation, and testis-specific genes display greater 3’UTR shortening than ubiquitously expressed ones, indicating functional relevance of APA to spermatogenesis. Interestingly, genes with shortened 3’UTRs tend to have higher RNA polymerase II and H3K4me3 levels in spermatids as compared to spermatocytes, features previously known to be associated with open chromatin state.ConclusionsOur data suggest that open chromatin may create a favorable cis environment for 3’ end processing, leading to global shortening of 3’UTR during spermatogenesis. mRNAs with shortened 3’UTRs are relatively stable thanks to evasion of powerful mRNA degradation mechanisms acting on 3’UTR elements. Stable mRNAs generated in spermatids may be important for protein production at later stages of sperm maturation, when transcription is globally halted.

PolyA_DB 3 catalogs cleavage and polyadenylation sites identified by deep sequencing in multiple genomes

Abstract 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.

Activity-Dependent Regulation of Alternative Cleavage and Polyadenylation During Hippocampal Long-Term Potentiation

Long-lasting forms of synaptic plasticity that underlie learning and memory require new transcription and translation for their persistence. The remarkable polarity and compartmentalization of neurons raises questions about the spatial and temporal regulation of gene expression within neurons. Alternative cleavage and polyadenylation (APA) generates mRNA isoforms with different 3′ untranslated regions (3′UTRs) and/or coding sequences. Changes in the 3′UTR composition of mRNAs can alter gene expression by regulating transcript localization, stability and/or translation, while changes in the coding sequences lead to mRNAs encoding distinct proteins. Using specialized 3′ end deep sequencing methods, we undertook a comprehensive analysis of APA following induction of long-term potentiation (LTP) of mouse hippocampal CA3-CA1 synapses. We identified extensive LTP-induced APA changes, including a general trend of 3′UTR shortening and activation of intronic APA isoforms. Comparison with transcriptome profiling indicated that most APA regulatory events were uncoupled from changes in transcript abundance. We further show that specific APA regulatory events can impact expression of two molecules with known functions during LTP, including 3′UTR APA of Notch1 and intronic APA of Creb1. Together, our results reveal that activity-dependent APA provides an important layer of gene regulation during learning and memory.