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Tools & Techniques: Temporal Analysis of miRNA Expression; Enhancing RNAi via Ago2 Overexpression; and More


Aiming to address a key limitation with existing tools for studying microRNA expression, a team from the French National Center for Scientific Research has developed a new method for the temporal analysis of the small, non-coding RNAs in vivo.

"Current methods used to determine the expression of miRNAs have strongly impacted our knowledge of the biological roles that miRNAs play under physiological and pathophysiological conditions," according to the scientists.

However, these lack spatial and temporal resolution, and require complex tissue sampling and processing — all of which makes them "unsuitable for monitoring miRNA regulation during longitudinal studies," they noted. "This is particularly problematic as miRNAs are spatiotemporally regulated and subject to considerable interindividual variation."

In response to this hurdle, the investigators developed RILES — short for RNAi-Inducible luciferase expression system — with the goal of generating positive bioluminescence signals in mice that permit qualitative and quantitative measurements of endogenously expressed miRNAs "with sufficient sensitivity to monitor the dynamic regulation of miRNA expression during the development of a chronic disease," according to a paper appearing in Nucleic Acids Research.

RILES is based on an inducible expression system, also developed at CNRC, that uses an inducer capable of changing its conformation when bound to a repressor protein, which impedes its binding to an operator sequence located downstream from the translation start codon within a constitutive promoter. As a result, the expression of a transgene is turned on by the presence of the exogenous inducer.

"We reasoned that placing expression of the repressor molecule directly under the control of the endogenous RNAi machinery, rather than an exogenous molecule, would be an alternative way to switch on expression of the transgene," the researchers wrote. "Consequently, if the luciferase reporter gene is used as a transgene, the system will generate bioluminescence signals that will qualitatively and quantitatively reflect the expression pattern of miRNAs."

As detailed in Nucleic Acids Research, the researchers validated RILES in vitro using a panel of cell lines and in vivo in normal rodent organs including skeletal muscles and liver.

Lastly, the system was applied to a mouse model of muscular atrophy, in which the researchers were able to determine the kinetics of miR-206 expression during the muscle regeneration phase of the condition.

The bioluminescence data from this experiment indicated that the expression of miR-206 is "individual-dependent, finely regulated in a time-dependent manner, and characterized by individual heterogeneity during development of the pathology," the paper states.

Consistent with data generated from conventional qRT-PCR, the miRNA was found to be overexpressed. However, with RILES, its expression was found to be constant for 7 days before returning to the basal level — a discrepancy the scientists chalked up to qRT-PCR's need to complex tissue sampling and processing, which results in a set of information from a heterogeneous population collected at different time points.

Overall, RILES represents a new method to study the dynamic regulation of miRNAs in physiological and pathophysiological contexts, the study's authors concluded. Further, it represents a novel method to "program the expression of therapeutic transgenes … in specific target cells, with possible applications in the field of gene and cell therapy."

Driven by an interest in studying microRNAs in Drosophila, a group of researchers from the French National Center for Scientific Research have created a biosensor for both miRNA biogenesis and Argonaute 2-mediated silencing in vivo.

According to the scientists, the system, called AutomiG, may also be adapted for use in human cells to study the factors involved in miRNA biogenesis and activity, and potentially to identify therapeutics in miRNA-associated diseases.

"Although the majority of Drosophila miRNAs are preferentially loaded into Ago1, a subset of miRNA preferentially associates with Ago2," the investigators wrote in PLoS One. "In addition, miRNAs* strands, thus far considered as by-products of miRNA biogenesis, tend to accumulate in association with Ago2."

To date, several systems have been developed to screen in vivo for genes involved in miRNA silencing in Drosophila, but these rely on a single vector expressing an miRNA along with one vector expressing a reporter gene engineered to carry the corresponding miRNA target in its 3' UTR, according to the paper.

Such two-component systems, however, tend to generate both false positives and false negatives. "For instance, down regulation of the miR expression vector may be associated [with] false positives whereas hits associated to low reporter signal may be discarded during signal background filtering," the researchers wrote.

Speculating that a single-component system with a high dynamic range of response could sidestep these issues, the CNRC group generated a single-gene construct that simultaneously expresses green fluorescent protein, along with two artificial miRNAs perfectly matched to two distinct sites in the GFP coding sequence in order to maximize GFP silencing.

To test the AutomiG sensor's robustness, the investigators used it in a high-throughput chemical library screening experiment and were able to identify 29 compounds that strongly inhibited Ago2-mediated miRNA silencing. Five of the compounds also inhibited long dsRNA-mediated RNAi, which suggests that the compounds target components common to both the RNAi and miRNA mechanisms.

The system was also adapted for mammalian cells, with the scientists generating a cytomegalovirus-automiG variant construct under the control of the CMV promoter that drives transcription in mammalian cell.

When transfected alone or with a scramble siRNA control in HeLa cells, the CMV-automiG construct expressed "barely detectable levels of GFP protein," according to the PLoS One report. "In striking contrast, cotransfections with siRNAs targeting the human key components of miRNA biogenesis — Drosha and its cofactor DGCR8 — were associated with significant GFP induction, strongly suggesting that the CMV-automiG construct is indeed a biosensor for miRNA biogenesis in mammals."

In the end, the data show AutomiG to be a highly sensitive sensor system adaptable to high-throughput screening approaches, and potentially useful in mammalian cells, the paper concludes.

In a bid to enhance the gene-silencing effect of RNAi molecules, a group of researchers from Heidelberg University Hospital has developed a set of tools for enhancing RNAi via Argonaute 2 overexpression.

Given Ago2's key role in siRNA- and shRNA-mediated gene silencing, the scientists noted that many groups have explored its co-delivery with RNAi agents to enhance gene silencing. However, the need for a separate Ago2-expression construct has limited the strategies applicability, particularly in vivo.

To overcome this issue, the scientists tried three strategies for boosting siRNA and shRNA potency: one based on Ago2 overexpression from plasmids, another using viral vectors that co-encode an shRNA, and a third using human cell lines that can be transfected with any RNAi molecule, according to a paper appearing in Nucleic Acids Research.

All three avenues were able to increase target mRNA knockdowns by up to 10-fold using different classes of preexisting RNAi triggers. Specifically, the researchers tested a variety of existing or newly designed siRNAs and shRNAs and "consistently observed 2- to 10-fold Ago2-dependent enhancements of their activities with numerous targets and cell lines, and regardless of whether Ago2 was co-expressed from a plasmid, stably integrated into cells, or co-delivered in vitro or in vivo by a viral vector," they wrote.

Importantly, the team demonstrated the safety of the approaches, addressing both concerns over cellular perturbations arising from Ago2 overexpression, as well as Ago2-induced alleviation of toxic saturation effects caused by ectopic RNAi triggers. Notably, Ago2/shRNA co-expression from bi-cistronic viral vectors alleviated in vivo toxicities and provided more sustained inhibition of a hepatic transgene in adult mice.

All told, the three approaches should "broadly improve future in vitro and in vivo RNAi experiments in mammalian systems," the scientists wrote in Nucleic Acids Research.