Analysis of the accessibility of CLIP bound sites reveals that nucleation of the miRNA:mRNA pairing occurs preferentially at the 3'-end of the seed match.

To find out whether the AGO-miRNA complex is more sensitive to the accessibility of a particular region inside the seed match, we analyze in detail the accessibility of a wide set of miRNA binding sites validated by PAR-CLIP and HITS-CLIP experiments. Our analysis reveals that nucleotides at the 3'-end of bound seed matches are significantly more accessible than nucleotides at the 5'-end as well as nucleotides at any positions in the unbound seed matches. We show that the accessibility of a single nucleotide at the 3'-end is more effective than the accessibility of several nucleotides at the 5'-end in discriminating between functional and nonfunctional binding sites. Analysis of mRNA and protein fold changes induced by miRNA overexpression demonstrates that genes with accessible nucleation regions at the 3'-end are down-regulated more strongly than genes whose accessible nucleation regions are located elsewhere within the seed match. We also observed an increase in the precision of the miRNA target prediction algorithm PACMIT when accessibility toward the 3'-end of the seed match was required. The pronounced sensitivity of the AGO-miRNA complex to the accessibility of the 3'-end of the seed match suggests that, in most cases, nucleation occurs in this region. We show that this conclusion is consistent with previous experimental studies.

[1]  Y. Li,et al.  Incorporating structure to predict microRNA targets. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[2]  T. Tuschl,et al.  Structure of the guide-strand-containing argonaute silencing complex , 2008, Nature.

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

[4]  Peter F. Stadler,et al.  Local RNA base pairing probabilities in large sequences , 2006, Bioinform..

[5]  R. Giegerich,et al.  Fast and effective prediction of microRNA/target duplexes. , 2004, RNA.

[6]  Scott B. Dewell,et al.  Transcriptome-wide Identification of RNA-Binding Protein and MicroRNA Target Sites by PAR-CLIP , 2010, Cell.

[7]  G. Nuovo,et al.  Experimental validation of miRNA targets. , 2008, Methods.

[8]  Dong Yue,et al.  Improving performance of mammalian microRNA target prediction , 2010, BMC Bioinformatics.

[9]  Oliver Hobert,et al.  Perfect seed pairing is not a generally reliable predictor for miRNA-target interactions , 2006, Nature Structural &Molecular Biology.

[10]  William H Press,et al.  Human microRNAs target a functionally distinct population of genes with AT-rich 3′ UTRs , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[11]  Stefan L Ameres,et al.  Molecular Basis for Target RNA Recognition and Cleavage by Human RISC , 2007, Cell.

[12]  John G Doench,et al.  Specificity of microRNA target selection in translational repression. , 2004, Genes & development.

[13]  G. Obernosterer,et al.  Target site effects in the RNA interference and microRNA pathways. , 2008, Biochemical Society transactions.

[14]  Jirí Vanícek,et al.  Efficient use of accessibility in microRNA target prediction , 2010, Nucleic Acids Res..

[15]  Jirí Vanícek,et al.  Suppression of immediate-early viral gene expression by herpesvirus-coded microRNAs: Implications for latency , 2008, Proceedings of the National Academy of Sciences.

[16]  Stefan L Ameres,et al.  The impact of target site accessibility on the design of effective siRNAs , 2008, Nature Biotechnology.

[17]  Mihaela Zavolan,et al.  Relative contribution of sequence and structure features to the mRNA binding of Argonaute/EIF2C-miRNA complexes and the degradation of miRNA targets. , 2009, Genome research.

[18]  N. Rajewsky,et al.  Widespread changes in protein synthesis induced by microRNAs , 2008, Nature.

[19]  S. P. Walton,et al.  Impact of target mRNA structure on siRNA silencing efficiency: A large‐scale study , 2008, Biotechnology and bioengineering.

[20]  Anders Krogh,et al.  MicroRNA transfection and AGO-bound CLIP-seq data sets reveal distinct determinants of miRNA action. , 2011, RNA.

[21]  Martin Reczko,et al.  The database of experimentally supported targets: a functional update of TarBase , 2008, Nucleic Acids Res..

[22]  M. Kiebler,et al.  Faculty Opinions recommendation of Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps. , 2009 .

[23]  Karin Musier-Forsyth,et al.  Mechanistic insights into the kinetics of HIV-1 nucleocapsid protein-facilitated tRNA annealing to the primer binding site. , 2004, Journal of molecular biology.

[24]  Ray M. Marín,et al.  Optimal Use of Conservation and Accessibility Filters in MicroRNA Target Prediction , 2012, PloS one.

[25]  W. Filipowicz,et al.  Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? , 2008, Nature Reviews Genetics.

[26]  Tongbin Li,et al.  miRecords: an integrated resource for microRNA–target interactions , 2008, Nucleic Acids Res..

[27]  Louis M. Smith.  An Alternative Model , 1983 .

[28]  Anjali J. Koppal,et al.  Supplementary data: Comprehensive modeling of microRNA targets predicts functional non-conserved and non-canonical sites , 2010 .

[29]  Michael Kertesz,et al.  The role of site accessibility in microRNA target recognition , 2007, Nature Genetics.

[30]  I. MacRae,et al.  The Crystal Structure of Human Argonaute2 , 2012, Science.

[31]  R. Russell,et al.  Principles of MicroRNA–Target Recognition , 2005, PLoS biology.

[32]  Oliver Hobert,et al.  Molecular architecture of a miRNA-regulated 3' UTR. , 2008, RNA.

[33]  Dang D. Long,et al.  Potent effect of target structure on microRNA function , 2007, Nature Structural &Molecular Biology.

[34]  C. Bracken,et al.  Experimental strategies for microRNA target identification , 2011, Nucleic acids research.

[35]  Molly Hammell,et al.  Computational methods to identify miRNA targets. , 2010, Seminars in cell & developmental biology.

[36]  U. A. Ørom,et al.  Experimental identification of microRNA targets. , 2010, Gene.

[37]  D. Bartel,et al.  The impact of microRNAs on protein output , 2008, Nature.

[38]  S. Chi,et al.  An alternative mode of microRNA target recognition , 2012, Nature Structural &Molecular Biology.

[39]  Nikolaus Rajewsky,et al.  The Impact of miRNA Target Sites in Coding Sequences and in 3′UTRs , 2011, PloS one.

[40]  Rolf Backofen,et al.  IntaRNA: efficient prediction of bacterial sRNA targets incorporating target site accessibility and seed regions , 2008, Bioinform..

[41]  D. Bartel,et al.  Structure of yeast Argonaute with guide RNA , 2012, Nature.

[42]  Quaid Morris,et al.  Predicting in vivo binding sites of RNA-binding proteins using mRNA secondary structure. , 2010, RNA.

[43]  Peter F. Stadler,et al.  Thermodynamics of RNA-RNA Binding , 2006, German Conference on Bioinformatics.