Shuo Gu

Biological basis for restriction of microRNA targets to the 3¢ untranslated region in mammalian mRNAs

MicroRNAs (miRNAs) interact with target sites located in the 3′ untranslated regions (3′ UTRs) of mRNAs to downregulate their expression when the appropriate miRNA is bound to target mRNA. To establish the functional importance of target-site localization in the 3′ UTR, we modified the stop codon to extend the coding region of the transgene reporter through the miRNA target sequence. As a result, the miRNAs lost their ability to inhibit translation but retained their ability to function as small interfering RNAs in mammalian cells in culture and in vivo. The addition of rare but not optimal codons upstream of the extended opening reading frame (ORF) made the miRNA target site more accessible and restored miRNA-induced translational knockdown. Taken together, these results suggest that active translation impedes miRNA-programmed RISC association with target mRNAs and support a mechanistic explanation for the localization of most miRNA target sites in noncoding regions of mRNAs in mammals.

How do miRNAs mediate translational repression

Micro(mi)RNAs regulate gene expression by what are believed to be related but separate mechanistic processes. The relative contribution that each process plays, their mechanistic overlap, and the degree by which they regulate complex genetic networks is still being unraveled. One process by which miRNAs inhibit gene expression occurs through translational repression. In recent years, there has been a plethora of studies published, which have resulted in various molecular models of how miRNAs impair translation. At first evaluation, it appears that these models are quite different and incompatible with one another. In this paper, we focus on possible explanations for the various interpretations of these data sets, and provide a model that we believe is consistent with many of the observations published to date.

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

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.

Expression determinants of mammalian argonaute proteins in mediating gene silencing

RNA interference occurs by two main processes: mRNA site-specific cleavage and non-cleavage-based mRNA degradation or translational repression. Site-specific cleavage is carried out by argonaute-2 (Ago2), while all four mammalian argonaute proteins (Ago1–Ago4) can carry out non-cleavage-mediated inhibition, suggesting that Ago1, Ago3 and Ago4 may have similar but potentially redundant functions. It has been observed that in mammalian tissues, expression of Ago3 and Ago4 is dramatically lower compared with Ago1; however, an optimization of the Ago3 and Ago4 coding sequences to include only the most common codon at each amino acid position was able to augment the expression of Ago3 and Ago4 to levels comparable to that of Ago1 and Ago2. Thus, we examined whether particular sequence features exist in the coding region of Ago3 and Ago4 that may prevent a high level of expression. Swapping specific sub-regions of wild-type and optimized Ago sequence identified the portion of the coding region (nucleotides 1–1163 for Ago-3 and 1–1494 for Ago-4) that is most influential for expression. This finding has implications for the evolutionary conservation of Ago proteins in the mammalian lineage and the biological role that potentially redundant Ago proteins may have.

Weak base pairing in both seed and 3′ regions reduces RNAi off-targets and enhances si/shRNA designs

The use of RNA interference is becoming routine in scientific discovery and treatment of human disease. However, its applications are hampered by unwanted effects, particularly off-targeting through miRNA-like pathways. Recent studies suggest that the efficacy of such off-targeting might be dependent on binding stability. Here, by testing shRNAs and siRNAs of various GC content in different guide strand segments with reporter assays, we establish that weak base pairing in both seed and 3′ regions is required to achieve minimal off-targeting while maintaining the intended on-target activity. The reduced off-targeting was confirmed by RNA-Seq analyses from mouse liver RNAs expressing various anti-HCV shRNAs. Finally, our protocol was validated on a large scale by analyzing results of a genome-wide shRNA screen. Compared with previously established work, the new algorithm was more effective in reducing off-targeting without jeopardizing on-target potency. These studies provide new rules that should significantly improve on siRNA/shRNA design.

RNA interference-induced hepatotoxicity results from loss of the first synthesized isoform of microRNA-122 in mice

Small RNAs can be engineered to target and eliminate expression of disease-causing genes or infectious viruses, resulting in the preclinical and clinical development of RNA interference (RNAi) therapeutics using these small RNAs. To ensure the success of RNAi therapeutics, small hairpin RNAs (shRNAs) must co-opt sufficient quantities of the endogenous microRNA machinery to elicit efficient gene knockdown without impeding normal cellular function. We previously observed liver toxicity—including hepatocyte turnover, loss of gene repression and lethality—in mice receiving high doses of a recombinant adeno-associated virus (rAAV) vector expressing shRNAs (rAAV-shRNAs); however the mechanism by which toxicity ensues has not been elucidated. Using rAAV-shRNAs we have now determined that hepatotoxicity arises when exogenous shRNAs exceed 12% of the total amount of liver microRNAs. After this threshold was surpassed, shRNAs specifically reduced the initially synthesized 22-nucleotide isoform of microRNA (miR)-122-5p without substantially affecting other microRNAs, resulting in functional de-repression of miR-122 target mRNAs. Delivery of a rAAV-shRNA vector expressing mature miR-122-5p could circumvent toxicity, despite the exogenous shRNA accounting for 70% of microRNAs. Toxicity was also not observed in Mir122–knockout mice regardless of the level or sequence of the shRNA. Our study establishes limits to the microRNA machinery that is available for therapeutic siRNAs and suggests new paradigms for the role of miR-122 in liver homeostasis in mice.

737. RNAi Induced Hepatotoxicity Results from a Functional Depletion of the First Synthesized Isoform of miR-122

To ensure success of RNA interference (RNAi) therapeutics, small hairpin RNAs (shRNAs) must co-opt sufficient quantities of the endogenous microRNA machinery to elicit efficient gene knockdown without impeding normal cellular function or causing liver toxicity. Using several recombinant adeno-associated viral (rAAV) vectors expressing shRNAs followed by small RNA sequencing, we determined that hepatic toxicity arises when exogenous shRNA levels exceed 12% of liver microRNAs. High shRNA expression specifically reduced miR-122-5p without affecting any other microRNAs ultimately resulting in functional de-repression of miR-122 target mRNAs. Furthermore, we found that only one isoform of miR-122-5p, 22 nucleotides in length, is displaced in toxic liver samples and that this isoform is the first to be synthesized from miR-122. A causative link between miR-122 reduction and toxicity was established when delivery of an AAV-shRNA expressing miR-122-5p could circumvent toxicity despite reaching 70% of microRNA reads. Consistent with these results, toxicity was not observed in miR-122 knockout mice -which in part adapt to an absence of miR-122 reduction - regardless of the level or sequence of shRNA. Together these results establish the limit to expendable miRNA/RNAi machinery and providing new paradigms for the role of miR-122 in liver homeostasis.