A key step in RNA interference (RNAi) is assembly of the RISC, the protein-siRNA complex that mediates target RNA cleavage. Here, we show that the two strands of an siRNA duplex are not equally eligible for assembly into RISC. Rather, both the absolute and relative stabilities of the base pairs at the 5' ends of the two siRNA strands determine the degree to which each strand participates in the RNAi pathway. siRNA duplexes can be functionally asymmetric, with only one of the two strands able to trigger RNAi. Asymmetry is the hallmark of a related class of small, single-stranded, noncoding RNAs, microRNAs (miRNAs). We suggest that single-stranded miRNAs are initially generated as siRNA-like duplexes whose structures predestine one strand to enter the RISC and the other strand to be destroyed. Thus, the common step of RISC assembly is an unexpected source of asymmetry for both siRNA function and miRNA biogenesis.

Small interfering RNAs (siRNAs) direct RNA interference (RNAi) in eukaryotes. In flies, somatic cells produce siRNAs from exogenous double-stranded RNA (dsRNA) as a defense against viral infection. We identified endogenous siRNAs (endo-siRNAs), 21 nucleotides in length, that correspond to transposons and heterochromatic sequences in the somatic cells of Drosophila melanogaster. We also detected endo-siRNAs complementary to messenger RNAs (mRNAs); these siRNAs disproportionately mapped to the complementary regions of overlapping mRNAs predicted to form double-stranded RNA in vivo. Normal accumulation of somatic endo-siRNAs requires the siRNA-generating ribonuclease Dicer-2 and the RNAi effector protein Argonaute2 (Ago2). We propose that endo-siRNAs generated by the fly RNAi pathway silence selfish genetic elements in the soma, much as Piwi-interacting RNAs do in the germ line.

Discovered in nematodes in 1993, microRNAs (miRNAs) are non-coding RNAs that are related to small interfering RNAs (siRNAs), the small RNAs that guide RNA interference (RNAi). miRNAs sculpt gene expression profiles during plant and animal development. In fact, miRNAs may regulate as many as one-third of human genes. miRNAs are found only in plants and animals, and in the viruses that infect them. miRNAs function very much like siRNAs, but these two types of small RNAs can be distinguished by their distinct pathways for maturation and by the logic by which they regulate gene expression. are characteristics of RNase III cleavage of dsRNA. Thus, Drosha cleavage defines either the 5Ј or the 3Ј end of the mature miRNA. (The mature miRNA resides in the 5Ј arm of some pre-miRNA and in the 3Ј arm in others.) The pre-miRNA is then exported from nucleus to cytoplasm by Exportin 5/RanGTP, which specifically recognizes the characteristic end structure of pre-miRNAs (Yi et al., 2003; Bohnsack et al., 2004;Lund et al., 2004;Zeng and Cullen, 2004).In the cytoplasm, a second RNase III, Dicer, together with its dsRBD protein partner, Loquacious (Loqs) in Drosophila or the trans-activator RNA (tar)-binding protein (TRBP) in humans, makes a pair of cuts that defines the other end of the mature miRNA, liberating an ~21-nucleotide RNA duplex (Bernstein et al., 2001;Grishok et al., 2001;Hutvagner et al., 2001;Ketting et al., 2001; Chendrimada et al., 2005;Forstemann et al., 2005;Jiang et al., 2005;Saito et al., 2005). This RNA duplex has essentially the same structure as a double-stranded siRNA, except that the mature miRNA is only partially paired to the miRNA* -the small RNA that resides on the side of the pre-miRNA stem opposite the miRNA -because the stems of pre-miRNAs are imperfectly double stranded. From the miRNA/miRNA* duplex, one strand, the miRNA, preferentially enters the protein complex that represses target gene expression, the RNA-induced silencing complex (RISC), whereas the other strand, the miRNA* strand, is degraded. The choice of strand relies on the local thermodynamic stability of the miRNA/miRNA* duplex -the strand whose 5Ј end is less stably paired is loaded into the RISC (Khvorova et al., 2003;Schwarz et al., 2003). This thermodynamic difference arises, in part, because miRNAs tend to begin with uracil, and, in part, because miRNA/miRNA* duplexes contain mismatches and bulges that favor the miRNA strand being loaded into the RISC.Plant microMaturation miRNA maturation in plants differs from the pathway in animals because plants lack a Drosha homolog (Fig. 2B). Instead, the RNase III enzyme DICER-LIKE 1 (DCL1), which is homologous to animal Dicer, is required for miRNA maturation (Park et al., 2002;Reinhart et al., 2002;Papp et al., 2003;Xie et al., 2004). In plants, DCL1 is localized in the nucleus and can make both the first pair of cuts made by Drosha and the second pair of cuts made by animal Dicer. As for animal Dicer, a dsRNA-binding domain protein partner, HYL1, has been implicated in DCL1 function in ...

We present primary results from the Sequencing Quality Control (SEQC) project, coordinated by the United States Food and Drug Administration. Examining Illumina HiSeq, Life Technologies SOLiD and Roche 454 platforms at multiple laboratory sites using reference RNA samples with built-in controls, we assess RNA sequencing (RNA-seq) performance for junction discovery and differential expression profiling and compare it to microarray and quantitative PCR (qPCR) data using complementary metrics. At all sequencing depths, we discover unannotated exon-exon junctions, with >80% validated by qPCR. We find that measurements of relative expression are accurate and reproducible across sites and platforms if specific filters are used. In contrast, RNA-seq and microarrays do not provide accurate absolute measurements, and gene-specific biases are observed, for these and qPCR. Measurement performance depends on the platform and data analysis pipeline, and variation is large for transcript-level profiling. The complete SEQC data sets, comprising >100 billion reads (10Tb), provide unique resources for evaluating RNA-seq analyses for clinical and regulatory settings.