Genome-wide analysis of poly(A) site selection in Schizosaccharomyces pombe

Polyadenylation of pre-mRNAs, a critical step in eukaryotic gene expression, is mediated by cis elements collectively called the polyadenylation signal. Genome-wide analysis of such polyadenylation signals was missing in fission yeast, even though it is an important model organism. We demonstrate that the canonical AATAAA motif is the most frequent and functional polyadenylation signal in Schizosaccharomyces pombe. Using analysis of RNA-Seq data sets from cells grown under various physiological conditions, we identify 3' UTRs for nearly 90% of the yeast genes. Heterogeneity of cleavage sites is common, as is alternative polyadenylation within and between conditions. We validated the computationally identified sequence elements likely to promote polyadenylation by functional assays, including qRT-PCR and 3'RACE analysis. The biological importance of the AATAAA motif is underlined by functional analysis of the genes containing it. Furthermore, it has been shown that convergent genes require trans elements, like cohesin for efficient transcription termination. Here we show that convergent genes lacking cohesin (on chromosome 2) are generally associated with longer overlapping mRNA transcripts. Our bioinformatic and experimental genome-wide results are summarized and can be accessed and customized in a user-friendly database Pomb(A).

[1]  Hans D. Ochs,et al.  A rare polyadenylation signal mutation of the FOXP3 gene (AAUAAA→AAUGAA) leads to the IPEX syndrome , 2001, Immunogenetics.

[2]  N. Proudfoot,et al.  A sequence downstream of AAUAAA is required for rabbit β-globin mRNA 3′-end formation , 1984, Nature.

[3]  M. Edmonds,et al.  A poly(A) polymerase from calf thymus. Characterization of the reaction product and the primer requirement. , 1973, The Journal of biological chemistry.

[4]  Chun Liang,et al.  Unique Features of Nuclear mRNA Poly(A) Signals and Alternative Polyadenylation in Chlamydomonas reinhardtii , 2008, Genetics.

[5]  J. McLauchlan,et al.  The consensus sequence YGTGTTYY located downstream from the AATAAA signal is required for efficient formation of mRNA 3' termini. , 1985, Nucleic acids research.

[6]  Guoli Ji,et al.  Genome level analysis of rice mRNA 3′-end processing signals and alternative polyadenylation , 2008, Nucleic acids research.

[7]  Marc Dreyfus,et al.  The Poly(A) Tail of mRNAs Bodyguard in Eukaryotes, Scavenger in Bacteria , 2002, Cell.

[8]  C. Norbury,et al.  Decapping is preceded by 3' uridylation in a novel pathway of bulk mRNA turnover. , 2009, Nature structural & molecular biology.

[9]  Bin Tian,et al.  A large-scale analysis of mRNA polyadenylation of human and mouse genes , 2005, Nucleic acids research.

[10]  M. Edmonds,et al.  A poly(A) polymerase from calf thymus. Purification and properities of the enzyme. , 1973, The Journal of biological chemistry.

[11]  R. Aebersold,et al.  Quantitative Analysis of Fission Yeast Transcriptomes and Proteomes in Proliferating and Quiescent Cells , 2012, Cell.

[12]  Larry N. Singh,et al.  U1 snRNP Determines mRNA Length and Regulates Isoform Expression , 2012, Cell.

[13]  Qingshun Quinn Li,et al.  Compilation of mRNA Polyadenylation Signals in Arabidopsis Revealed a New Signal Element and Potential Secondary Structures1[w] , 2005, Plant Physiology.

[14]  Letian Kuai,et al.  Polyadenylation of rRNA in Saccharomyces cerevisiae. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[15]  Job Harms,et al.  THE LANDSCAPE OF , 2010 .

[16]  H. Lange,et al.  Polyadenylation-assisted RNA degradation processes in plants. , 2009, Trends in plant science.

[17]  A. Krämer,et al.  Generation of histone mRNA 3′ ends by endonucleolytic cleavage of the pre‐mRNA in a snRNP‐dependent in vitro reaction. , 1986, The EMBO journal.

[18]  Temple F. Smith,et al.  Probabilistic prediction of Saccharomyces cerevisiae mRNA 3'-processing sites. , 2002, Nucleic acids research.

[19]  C R Cantor,et al.  Genomic detection of new yeast pre-mRNA 3'-end-processing signals. , 1999, Nucleic acids research.

[20]  T. Marr,et al.  alternative polyadenylation in human , mouse , and rat Computational analysis of 3 '-ends of ESTs shows four classes of data , 2005 .

[21]  Jing Zhao,et al.  Formation of mRNA 3′ Ends in Eukaryotes: Mechanism, Regulation, and Interrelationships with Other Steps in mRNA Synthesis , 1999, Microbiology and Molecular Biology Reviews.

[22]  N. Proudfoot.  How RNA polymerase II terminates transcription in higher eukaryotes. , 1989, Trends in biochemical sciences.

[23]  K. Nishida,et al.  Mechanisms and consequences of alternative polyadenylation. , 2011, Molecules and Cells.

[24]  D. Gautheret,et al.  Patterns of variant polyadenylation signal usage in human genes. , 2000, Genome research.

[25]  T. Jensen,et al.  The exosome: a multipurpose RNA-decay machine. , 2008, Trends in biochemical sciences.

[26]  N. Proudfoot,et al.  Cohesin Complex Promotes Transcriptional Termination between Convergent Genes in S. pombe , 2008, Cell.

[27]  D. Spector,et al.  An unexpected ending: noncanonical 3' end processing mechanisms. , 2010, RNA.

[28]  Larry N. Singh,et al.  U1 snRNP protects pre-mRNAs from premature cleavage and polyadenylation , 2010, Nature.

[29]  B. Futcher,et al.  The Fission Yeast RNA Binding Protein Mmi1 Regulates Meiotic Genes by Controlling Intron Specific Splicing and Polyadenylation Coupled RNA Turnover , 2011, PloS one.

[30]  C. K. Schmidt,et al.  Conserved features of cohesin binding along fission yeast chromosomes , 2009, Genome Biology.

[31]  G. Barton,et al.  Direct Sequencing of Arabidopsis thaliana RNA Reveals Patterns of Cleavage and Polyadenylation , 2012, Nature Structural &Molecular Biology.

[32]  K. Venkataraman,et al.  Analysis of a noncanonical poly(A) site reveals a tripartite mechanism for vertebrate poly(A) site recognition. , 2005, Genes & development.

[33]  N. Proudfoot.  Ending the message: poly(A) signals then and now. , 2011, Genes & development.

[34]  C. Cole,et al.  Patterns of polyadenylation site selection in gene constructs containing multiple polyadenylation signals , 1988, Molecular and cellular biology.

[35]  Nicole Cremona,et al.  A meiotic gene regulatory cascade driven by alternative fates for newly synthesized transcripts , 2011, Molecular biology of the cell.

[36]  D. Bartel.  MicroRNAs: Target Recognition and Regulatory Functions , 2009, Cell.

[37]  C. Mayr,et al.  Widespread Shortening of 3′UTRs by Alternative Cleavage and Polyadenylation Activates Oncogenes in Cancer Cells , 2009, Cell.

[38]  C. Burge,et al.  Prediction of Mammalian MicroRNA Targets , 2003, Cell.

[39]  C. V. Jongeneel,et al.  Similarities and differences of polyadenylation signals in human and fly , 2006, BMC Genomics.

[40]  P. Kapranov,et al.  Comprehensive Polyadenylation Site Maps in Yeast and Human Reveal Pervasive Alternative Polyadenylation , 2010, Cell.

[41]  Matthias W. Hentze,et al.  Increased efficiency of mRNA 3′ end formation: a new genetic mechanism contributing to hereditary thrombophilia , 2001, Nature Genetics.

[42]  P. Philippsen,et al.  Heterologous modules for efficient and versatile PCR‐based gene targeting in Schizosaccharomyces pombe , 1998, Yeast.

[43]  T. Hohn,et al.  The contribution of AAUAAA and the upstream element UUUGUA to the efficiency of mRNA 3′‐end formation in plants. , 1994, The EMBO journal.

[44]  D. Moazed,et al.  Autoregulation of convergent RNAi genes in fission yeast. , 2011, Genes & development.

[45]  Juan Mata,et al.  Genome-wide mapping of polyadenylation sites in fission yeast reveals widespread alternative polyadenylation , 2013, RNA biology.

[46]  D. Steege.  Emerging features of mRNA decay in bacteria. , 2000, RNA.

[47]  Sebastian D. Mackowiak,et al.  The Landscape of C. elegans 3′UTRs , 2010, Science.

[48]  I. Goodhead,et al.  Dynamic repertoire of a eukaryotic transcriptome surveyed at single-nucleotide resolution , 2008, Nature.

[49]  D. Gautheret,et al.  Sequence determinants in human polyadenylation site selection , 2003, BMC Genomics.

[50]  H. Nojima,et al.  Comprehensive isolation of meiosis-specific genes identifies novel proteins and unusual non-coding transcripts in Schizosaccharomyces pombe. , 2001, Nucleic acids research.

[51]  V. Wood,et al.  The Genome and Beyond , 2004 .

[52]  Y. Sakai,et al.  In silico Analysis of 3′-End-Processing Signals in Aspergillus oryzae Using Expressed Sequence Tags and Genomic Sequencing Data , 2011, DNA research : an international journal for rapid publication of reports on genes and genomes.

[53]  C. Birse,et al.  RNA 3′ end signals of the S.pombe ura4 gene comprise a site determining and efficiency element. , 1994, The EMBO journal.