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.

Download full-text


Available from: Megha Ghildiyal, May 16, 2014
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    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.
    Full-text · Article · Aug 2014 · PLoS ONE
  • Source
    • "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]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Centromeric heterochromatin assembly in fission yeast is critical for faithful chromosome segregation at mitosis. Its assembly requires a concerted pathway of events whereby the RNA interference (RNAi) pathway guides H3K9 methylation to target sequences. H3K9 methylation, a hallmark of heterochromatin structure, is mediated by the single histone methyltransferase Clr4 (equivalent to metazoan Suv3-9), a component of the CLRC complex. Loss of or defects in CLRC components disrupts heterochromatin formation due to loss of H3K9 methylation, thus an intact, fully functional CLRC complex is required for heterochromatin integrity. Despite its importance, little is known about the contribution of the CLRC component Raf2 to H3K9 methylation and heterochromatin assembly. We demonstrate that Raf2 is concentrated at centromeres and contrary to other analyses, we find that loss of Raf2 does not affect CENP-ACnp1 localisation or recruitment to centromeres. Our sequence alignments show that Raf2 contains a Replication Foci Targeting Sequence (RFTS) domain homologous to the RFTS domain of the human DNA methyltransferase DNMT1. We show that the Raf2 RFTS domain is required for centromeric heterochromatin formation as its mutation disrupts H3K9 methylation but not the processing of centromeric transcripts into small interfering RNAs (siRNAs) by the RNAi pathway. Analysis of biochemical interactions demonstrates that the RFTS domain mediates an interaction between Raf2 and the CLRC component Cul4. We conclude that the RFTS domain of Raf2 is a protein interaction module that plays an important role in heterochromatin formation at centromeres.
    Full-text · Article · Aug 2014 · PLoS ONE
  • Source
    • "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). "
    [Show abstract] [Hide abstract]
    ABSTRACT: We expanded the knowledge base for Drosophila cell line transcriptomes by deeply sequencing their small RNAs. In total, we analyzed more than 1 billion raw reads from 53 libraries across 25 cell lines. We verify reproducibility of biological replicate data sets, determine common and distinct aspects of miRNA expression across cell lines, and infer the global impact of miRNAs on cell line transcriptomes. We next characterize their commonalities and differences in endo-siRNA populations. Interestingly, most cell lines exhibit enhanced TE-siRNA production relative to tissues, suggesting this as a common aspect of cell immortalization. We also broadly extend annotations of cis-NAT-siRNA loci, identifying ones with common expression across diverse cells and tissues, as well as cell-restricted loci. Finally, we characterize small RNAs in a set of ovary-derived cell lines, including somatic cells (OSS and OSC) and a mixed germline/somatic cell population (fGS/OSS) that exhibits ping-pong piRNA signatures. Collectively, the ovary data reveal new genic piRNA loci, including unusual configurations of piRNA-generating regions. Together with the companion analysis of mRNAs described in a previous study, these small RNA data provide comprehensive information on the transcriptional landscape of diverse Drosophila cell lines. These data should encourage broader usage of fly cell lines, beyond the few that are presently in common usage.
    Full-text · Article · Jul 2014 · Genome Research
Show more

Similar Publications