A protein sensor for siRNA asymmetry
ABSTRACT To act as guides in the RNA interference (RNAi) pathway, small interfering RNAs (siRNAs) must be unwound into their component strands, then assembled with proteins to form the RNA-induced silencing complex (RISC), which catalyzes target messenger RNA cleavage. Thermodynamic differences in the base-pairing stabilities of the 5' ends of the two approximately 21-nucleotide siRNA strands determine which siRNA strand is assembled into the RISC. We show that in Drosophila, the orientation of the Dicer-2/R2D2 protein heterodimer on the siRNA duplex determines which siRNA strand associates with the core RISC protein Argonaute 2. R2D2 binds the siRNA end with the greatest double-stranded character, thereby orienting the heterodimer on the siRNA duplex. Strong R2D2 binding requires a 5'-phosphate on the siRNA strand that is excluded from the RISC. Thus, R2D2 is both a protein sensor for siRNA thermodynamic asymmetry and a licensing factor for entry of authentic siRNAs into the RNAi pathway.
SourceAvailable from: Mehmet Volkan Cakir[Show abstract] [Hide abstract]
ABSTRACT: RNA-mediated silencing was first described in plants and became famous by studies in Caenorhabditis elegans. RNA interference (RNAi) is the mechanism through which an RNA interferes with the production of other RNAs in a sequence specific manner. MiRNAs are a type of RNA which originate from the genome with their active form being ss-RNAs of 21-23 nucleotides in length. They are being transcribed as pri-miRNAs then processed in the nucleus by Drosha to pre-miRNAs with a stem-loop structure and ~70 nucleotides in length. This stem-loop containing pre-miRNAs is then processed in the cytoplasm to ds-RNA one strand of which will serve as interfering RNA. Toxoplasma gondii is a species of parasitic protozoa which causes several diseases. T.gondii emerges as a good candidate for computational efforts with its small genome size, publicly available genome files and extensive information about its gene structure, either based on experimental data or the prediction with several gene finders in parallel. Therefore, it seems important to establish the regulatory network composed of RNAi which may be beneficial for the Toxoplasma community. Within this context the pool of possible stem-loop constitutive transcripts are produced, further analysis of this pool for desired 2D structure is integrated and mapping of possible RNAi regulation to T.gondii’s genome is established. In connection with computational assessment and mapping, the derived information is provided as a database for quick lookup using a convenient web interface for experimental studies of RNAi regulation in Toxoplasma, thus reduce time and money costs in such studies.12/2009, Degree: M.Sc., Supervisor: Jens Allmer
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ABSTRACT: Steady state cellular microRNA (miRNA) levels represent the balance between miRNA biogenesis and turnover. The kinetics and sequence determinants of mammalian miRNA turnover during and after miRNA maturation are not fully understood. Through a large-scale study on mammalian miRNA turnover, we report the co-existence of multiple cellular miRNA pools with distinct turnover kinetics and biogenesis properties and reveal previously unrecognized sequence features for fast turnover miRNAs. We measured miRNA turnover rates in eight mammalian cell types with a combination of expression profiling and deep sequencing. While most miRNAs are stable, a subset of miRNAs, mostly miRNA*s, turnovers quickly, many of which display a two-step turnover kinetics. Moreover, different sequence isoforms of the same miRNA can possess vastly different turnover rates. Fast turnover miRNA isoforms are enriched for 5' nucleotide bias against Argonaute-(AGO)-loading, but also additional 3' and central sequence features. Modeling based on two fast turnover miRNA*s miR-222-5p and miR-125b-1-3p, we unexpectedly found that while both miRNA*s are associated with AGO, they strongly differ in HSP90 association and sensitivity to HSP90 inhibition. Our data characterize the landscape of genome-wide miRNA turnover in cultured mammalian cells and reveal differential HSP90 requirements for different miRNA*s. Our findings also implicate rules for designing stable small RNAs, such as siRNAs. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.Nucleic Acids Research 02/2015; 43(4). DOI:10.1093/nar/gkv057 · 8.81 Impact Factor
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ABSTRACT: In plants and animals, a large number of double-stranded RNA binding proteins (DRBs) have been shown to act as non-catalytic cofactors of DICERs and to participate in the biogenesis of small RNAs involved in RNA silencing. We have previously shown that the loss of Arabidopsis thaliana's DRB2 protein results in a significant increase in the population of RNA polymerase IV (p4) dependent siRNAs, which are involved in the RNA-directed DNA methylation (RdDM) process. Surprisingly, despite this observation, we show in this work that DRB2 is part of a high molecular weight complex that does not involve RdDM actors but several chromatin regulator proteins, such as MSI4, PRMT4B and HDA19. We show that DRB2 can bind transposable element (TE) transcripts in vivo but that drb2 mutants do not have a significant variation in TE DNA methylation. We propose that DRB2 is part of a repressive epigenetic regulator complex involved in a negative feedback loop, adjusting epigenetic state to transcription level at TE loci, in parallel of the RdDM pathway. Loss of DRB2 would mainly result in an increased production of TE transcripts, readily converted in p4-siRNAs by the RdDM machinery.BMC Plant Biology 03/2015; 15(1). DOI:10.1186/s12870-015-0455-z · 3.94 Impact Factor