Thermodynamic stability of small hairpin RNAs highly influences the loading process of different mammalian Argonautes

Abstract

MicroRNAs and siRNAs interact with target sequences in mRNAs, inducing cleavage-and non-cleavage-based gene repression through the RNA-induced silencing complex (RISC) that consists of one of four mammalian Argonaute proteins, Ago1-Ago4. The process of how Dicer substrate small hairpin RNAs (shRNAs) are loaded into different mammalian Agos in vivo is not well established. Here we report that shRNAs are loaded into mammalian Agos in two stepwise processes, physical association and activation, with the latter being the rate-limiting step with noncleaving RISC. We establish that, although RNA duplexes processed from shRNAs bind to Agos in cells with similar affinity, the degree by which the complexes are activated (coupled with the removal of the passenger strand) correlates with the thermodynamic instability of RNA duplexes being loaded rather than the structure of the RNA, as was previously demonstrated in Drosophila. Interestingly, Ago loading of siRNAs is less sensitive to thermostability than that of their shRNA equivalents. These results may have important implications for the future design of RNAi-based therapeutics.RNA interference | gene silencing M icroRNAs (miRNAs) and 21-to 23-nt siRNAs regulate gene expression in almost all eukaryotic organisms. Small RNA-mediated gene regulation is sequence-specific and believed to regulate at least one-half of all protein-encoding mRNAs (reviewed in refs. 1-4).Argonaute proteins (Agos), which directly associate with small RNAs, are the core of all known RNA-induced silencing complexes (RISCs). These proteins are functionally specialized into distinct RNA silencing pathways. In Drosophila, Ago2-RISC is involved in target mRNA cleavage, and Ago1-RISC mediates translational repression (5, 6). The human genome encodes four Ago-like subfamily proteins (Ago1-Ago4) that have redundant functions as negative regulators of gene expression, but only Ago2 has cleavage activity (7-9).In Drosophila, siRNAs and miRNAs are actively sorted into functionally distinct Ago-RISCs based on differences in structure rather than on their biogenesis (10, 11). Perfectly matched duplexes (siRNA-like) are preferentially incorporated into Ago2, and the loading is achieved by cleaving the passenger strand, whereas duplexes with central mismatched bulges (miRNA-like) are sorted to Ago1, and the unselected strand (asterisk strand) is eliminated through a "bypass" pathway, which is sensitive to the structure of the small RNA duplex and independent of the weak cleavage activity of Ago1 (12). A similar sorting mechanism exists in Caenorhabditis elegans, whereby small RNA duplexes with perfectly matched or bulged stems are channeled into RDE-1 and ALG-1, respectively (13). In plants, a different sorting mechanism has been reported. The composition and phosphorylation status of the 5′ nucleotide of small RNA duplexes influences their binding by the different Agos (14,15). In mammalian cells, several studies have shown that miRNAs are similarly associated with four human Agos (7,8,16), suggesting the abse...

Studies of Efficacy and Liver Toxicity Related to Adeno-Associated Virus–Mediated RNA Interference

“…2), a result consistent with that in our previous report (Chen et al, 2009). The mechanisms that make HBV-S1 such a potent shRNA are not entirely clear but may be because of its high affinity to RNA-induced silencing complex determined by the thermodynamic stability, which is thought to influence the loading process of the guide strand for incorporation into RNA-induced silencing complex (Schwarz et al, 2003;Gu et al, 2011), or cis-elements in small RNA sequences and RNA-binding proteins impacting small RNA stability ( Ji and Chen, 2012). Importantly, AAV-H1/HBV-S1 does not induce detectable liver injury, even over a 1-year observation period (Chen et al, 2009.…”

The Multiple Functions of TRBP, at the Hub of Cell Responses to Viruses, Stress, and Cancer

“…Whereas two studies suggested that this complex remains intact with multiple rounds of cleavage (60,90), others indicated that Ago2 is associated more with mature RNA sequences and tends to dissociate after miRNA loading, suggesting that TRBP and Dicer are essential for processing shRNAs/miRNAs but do not participate in target cleavage (63,97,99). While few in vivo studies have been carried out, results indicate that the presence of TRBP is crucial for RNAi activity.…”

Alleviation of off-target effects from vector-encoded shRNAs via codelivered RNA decoys

“…strand can inhibit reporter constructs with artificial complementary binding sites (11)(12)(13). These observations are accompanied by accumulating data indicating that naturally occurring miRNAs also use both strands and that the miRNA sense (also called the "star") strand can play essential roles in cellular physiology and pathology (14)(15)(16).…”

“…Complementing early notions that strands with a lower 5′ thermodynamic stability are preferentially incorporated into RISC (17)(18)(19), recent data now suggest that many other parameters, such as the position of the loop, the sequence and structure of the stem, the relative abundance of different Argonaute proteins, or competitive Argonaute-2 binding of the 5′ nucleotide of either strand, influence strand selectivity (12,14,15,20). Moreover, there is ongoing controversy over whether Dicer and associated proteins are required or dispensable for asymmetric RISC loading (21)(22)(23).…”