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Nucleation, propagation and cleavage of target RNAs in Ago silencing complexes.

ABSTRACT: The slicer activity of the RNA-induced silencing complex resides within its Argonaute (Ago) component, in which the PIWI domain provides the catalytic residues governing guide-strand mediated site-specific cleavage of target RNA. Here we report on structures of ternary complexes of Thermus thermophilus Ago catalytic mutants with 5'-phosphorylated 21-nucleotide guide DNA and complementary target RNAs of 12, 15 and 19 nucleotides in length, which define the molecular basis for Mg(2+)-facilitated site-specific cleavage of the target. We observe pivot-like domain movements within the Ago scaffold on proceeding from nucleation to propagation steps of guide-target duplex formation, with duplex zippering beyond one turn of the helix requiring the release of the 3'-end of the guide from the PAZ pocket. Cleavage assays on targets of various lengths supported this model, and sugar-phosphate-backbone-modified target strands showed the importance of structural and catalytic divalent metal ions observed in the crystal structures.

SUBMITTER: Wang Y

PROVIDER: S-EPMC2880917 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:MicroRNAs (miRNAs) are a class of small RNAs which typically function by guiding cleavage of target messenger RNAs. They have been shown to play major roles in a variety of plant processes including development, and responses to pathogens and environmental stresses. To identify new miRNAs and regulation in Arabidopsis thaliana, 27 small RNA libraries were constructed and sequenced from various tissues, stresses and small RNA biogenesis mutants, resulting in 95 million genome-matched sequences. The use of rdr2 to enrich the miRNA population greatly enhanced this analysis and led to the discovery of 44 new miRNAs arising from both known and new precursors. Parallel Analysis of RNA Ends (PARE) data provide evidence that the majority guide target cleavage. The inclusion of novel stress/tissue conditions, such as submergence-stressed flowers, enabled identification of new stress regulation of both miRNAs and their targets, all of which were validated in wild type plants. By combining small RNA expression analysis with ARGONAUTE (AGO) immunoprecipitation data and global target cleavage data from PARE, a much more complete picture of Arabidopsis miRNAs was obtained. This combinatorial approach led to the discovery of AGO loading and target cleavage biases, which gave important insights into tissue-specific expression patterns, pathogen responses and the role of sequence variation among closely related miRNA family members. Examination of various tissues, stresses and small RNA biogenesis mutants in Arabidopsis by high-throughput sequencing for small RNA profiling. We have used AGO-IP and PARE data from the published data, which were downloaded from NCBI GEO with the following accession number. AGO-IP from GSM253622, GSM707682, GSM642335, GSM642336, GSM512703, GSM512702, GSM707683, GSM707684, GSM707685, GSM149080, GSM253623, GSM304283, GSM642337, GSM642338, GSM253624, GSM415788, GSM707686, GSM707687, GSM707688, GSM707689, GSM415787, GSM149081, GSM253625, GSM415789, GSM415790, GSM304285, GSM415791, GSM415792 PARE sequencing data from xrn4 flowers were obtained from Gene Expression Omnibus with accession number GSM280227.

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Nucleation, propagation and cleavage of target RNAs in Ago silencing complexes.

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