Ghildiyal, M., Xu, J., Seitz, H., Weng, Z. & Zamore, P. D. Sorting of Drosophila small silencing RNAs partitions microRNA* strands into the RNA interference pathway. RNA 16, 43-56

Howard Hughes Medical Institute, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School,Worcester, Massachusetts 01605, USA.
RNA (Impact Factor: 4.94). 11/2009; 16(1):43-56. DOI: 10.1261/rna.1972910
Source: PubMed


In flies, small silencing RNAs are sorted between Argonaute1 (Ago1), the central protein component of the microRNA (miRNA) pathway, and Argonaute2 (Ago2), which mediates RNA interference. Extensive double-stranded character-as is found in small interfering RNAs (siRNAs)-directs duplexes into Ago2, whereas central mismatches, like those found in miRNA/miRNA* duplexes, direct duplexes into Ago1. Central to this sorting decision is the affinity of the small RNA duplex for the Dcr-2/R2D2 heterodimer, which loads small RNAs into Ago2. Here, we show that while most Drosophila miRNAs are bound to Ago1, miRNA* strands accumulate bound to Ago2. Like siRNA loading, efficient loading of miRNA* strands in Ago2 favors duplexes with a paired central region and requires both Dcr-2 and R2D2. Those miRNA and miRNA* sequences bound to Ago2, like siRNAs diced in vivo from long double-stranded RNA, typically begin with cytidine, whereas Ago1-bound miRNA and miRNA* disproportionately begin with uridine. Consequently, some pre-miRNA generate two or more isoforms from the same side of the stem that differentially partition between Ago1 and Ago2. Our findings provide the first genome-wide test for the idea that Drosophila small RNAs are sorted between Ago1 and Ago2 according to their duplex structure and the identity of their first nucleotide.

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Available from: Megha Ghildiyal, May 16, 2014
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    • "Repression correlates similarly with abundance in the Ago-1 dataset and microRNA expression level, and so the rate of Ago-1 association is unable to explain variations in microRNA repression. Argonaute association has been reported to be dependent upon a number of factors including microRNA nucleotide composition, and thermodynamic stability of microRNA duplexes and microRNA:target complexes [10]–[14], [38]–[40]. Though these factors are undoubtedly relevant, our data do not support a significant correlation of any of these factors with microRNA repressive ability. We do, however, see a weak negative correlation between microRNA/target duplex stability and repressive ability. "
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    ABSTRACT: MicroRNAs are small RNAs that regulate protein levels. It is commonly assumed that the expression level of a microRNA is directly correlated with its repressive activity - that is, highly expressed microRNAs will repress their target mRNAs more. Here we investigate the quantitative relationship between endogenous microRNA expression and repression for 32 mature microRNAs in Drosophila melanogaster S2 cells. In general, we find that more abundant microRNAs repress their targets to a greater degree. However, the relationship between expression and repression is nonlinear, such that a 10-fold greater microRNA concentration produces only a 10% increase in target repression. The expression/repression relationship is the same for both dominant guide microRNAs and minor mature products (so-called passenger strands/microRNA* sequences). However, we find examples of microRNAs whose cellular concentrations differ by several orders of magnitude, yet induce similar repression of target mRNAs. Likewise, microRNAs with similar expression can have very different repressive abilities. We show that the association of microRNAs with Argonaute proteins does not explain this variation in repression. The observed relationship is consistent with the limiting step in target repression being the association of the microRNA/RISC complex with the target site. These findings argue that modest changes in cellular microRNA concentration will have minor effects on repression of targets.
    PLoS ONE 08/2014; 9(8):e104286. DOI:10.1371/journal.pone.0104286 · 3.23 Impact Factor
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    • "In addition to Clr4, the CLRC complex consists of the cullin scaffold protein Cul4, the β-propeller protein Rik1, the RING box protein Rbx1, the WD-40 protein Raf1/Dos1 and Raf2/Dos2 [19], [20], [21], [22]. We have previously shown that members of the CLRC complex (Cul4, Rik1, and Raf1) are predicted to adopt a structure similar to the conserved Cul4-DDB1-DDB2 E3 ubiquitin ligase and recent structural analysis of Raf1 has confirmed this prediction [23], [24]. "
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    PLoS ONE 08/2014; 9(8):e104161. DOI:10.1371/journal.pone.0104161 · 3.23 Impact Factor
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    • "Curiously, while there are also some reads mapped uniquely to the 5p arm, the 21-to 22-nt 5p species have 14 and 17 genomic matches, respectively, indicating a relationship to a repetitive element despite clear evidence for small RNA production from this particular locus. We note that its duplex region is nearly completely paired, including within the central duplex region that is predicted to promote loading to AGO2 (Czech et al. 2009; Okamura et al. 2009; Ghildiyal et al. 2010). Indeed, a high fraction of reads from both arms derived specifically from an AGO2-IP library from S2-R+ cells, with very few reads in the corresponding AGO1-IP library (Fig. 3C). "
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