MiRAR—miRNA Activity Reporter for Living Cells

microRNA (miRNA) activity and regulation are of increasing interest as new therapeutic targets. Traditional approaches to assess miRNA levels in cells rely on RNA sequencing or quantitative PCR. While useful, these approaches are based on RNA extraction and cannot be applied in real-time to observe miRNA activity with single-cell resolution. We developed a green fluorescence protein (GFP)-based reporter system that allows for a direct, real-time readout of changes in miRNA activity in live cells. The miRNA activity reporter (MiRAR) consists of GFP fused to a 3′ untranslated region containing specific miRNA binding sites, resulting in miRNA activity-dependent GFP expression. Using qPCR, we verified the inverse relationship of GFP fluorescence and miRNA levels. We demonstrated that this novel optogenetic reporter system quantifies cellular levels of the tumor suppressor miRNA let-7 in real-time in single Human embryonic kidney 293 (HEK 293) cells. Our data shows that the MiRAR can be applied to detect changes in miRNA levels upon disruption of miRNA degradation pathways. We further show that the reporter could be adapted to monitor another disease-relevant miRNA, miR-122. With trivial modifications, this approach could be applied across the miRNome for quantification of many specific miRNA in cell cultures, tissues, or transgenic animal models.


Introduction
Human gene expression and RNA transcript stability can be regulated before, during, and after transcription. MicroRNAs (miRNAs) regulate transcript stability by binding to messenger RNAs (mRNAs) in complementary regions, inducing endonuclease-mediated cleavage or inhibiting protein synthesis [1]. In mammals, miRNAs usually contain sequence homology to their target transcript in their 5 and 3 untranslated regions (UTR) [2,3]. Genes encoding miRNAs are transcribed as long precursor transcripts and processed to yield short 18-24-nucleotide-long miRNAs. These miRNAs are subsequently integrated into protein complexes to induce mRNA silencing. While translational aspects of miRNA research are primarily focused on cancer, deregulation of miRNA stability and activity has relevance to many diseases. Dysfunctional miRNA expression, processing, and degradation have been found in diseases including breast cancer [4], acute myeloid leukemia [5], ovarian cancer [6], and hepatocellular carcinoma [7], but links between miRNAs and Alzheimer's disease [8], diabetes [9], and schizophrenia [10] are also emerging.
The miRNA let-7 is often implicated in disease and the let-7 miRNA sequence and timing of expression during development are highly conserved amongst vertebrates [11]. In normal cells and USA) and 10% fetal bovine serum (FBS). Cells from half of a 150 mm plate (~10 7 cells) were resuspended in 2.4 mL lysis buffer (0.6% sodium dodecyl sulfate (SDS), 10 mM ethylenediaminetetraacetic acid (EDTA), 10 mM Tris-HCl pH 8.0) and 25 units RNase I (NEB M0243S) and incubated at 37 • C for 1 h. This was followed by the addition of 240 µL 5 M NaCl (6 mmol) and 1 h of incubation on ice. The solution was centrifuged at 10,000× g for 30 min at 4 • C and the DNA was extracted from the supernatant via phenol chloroform extraction. The extracted DNA was stored at −20 • C until further use.

MicroRNA Quantification by Real-Time Quantitative Polymerase Chain Reaction
RT-qPCR was performed as described previously [35]. Briefly, a primer with an internal stem loop structure was designed to target mature let-7 miRNA (5 -GTTGGCTCTGGTGCAGG GTCCGAGGTATTCGCACCAGAGCCAACAACTAT-3 ) or miR-122 (5 -GTCGTATGCAGAGC AGGGTCCGAGGTATTCGCACTGCATACGACCAAACA-3 ). This primer was unfolded for 5 min at 65 • C and then refolded for 2 min on ice to form a stem loop structure. The primer was then incubated with total purified cellular RNA. Then, 0.125 pmol RNA was synthesized into complementary DNA (cDNA) using SuperScript III RT (200 units/µL) and stem loop primers. cDNA synthesis was carried out for both RNA extracted from wildtype and Tut4-knockdown cell lines. The reaction was incubated in a thermocycler for 30 min at 16 • C, followed by pulsed RT of 60 cycles at 30 • C for 30 s, 42 • C for 30 s, and 50 • C for 1 min. The cDNA generated was later diluted 10-fold, and quantitative PCR was conducted using SYBR Green qPCR MasterMix (Thermo Fisher Scientific) and qPCR primers (300 nM). Forward primers were designed for miR-122 (5 -AGGCTGGAGTGTGACAATG-3 ), let-7 (5 -TGAGGTAGTAGGTTGTATAGTTGTTGG-3 ) and universal miR reverse (5-GAGCAGGGTCCGAGGT-3 ). Samples were amplified for 35 cycles with Eppendorf Realplex (Eppendorf, Hamburg, Germany), and miRNA levels were extrapolated using a comparative C T (Cycle Threshold)method described previously [35].

Let-7 micro RNA Reduces Green Fluorescent Proteins Fluorescence in Live Cells
To assess miRNA levels in live cells, we generated a GFP-based reporter system. The 3 -UTR of KRas was cloned downstream of a gfp gene into pcDNA3.1 to generate a reporter system where GFP fluorescence is responsive to changes in let-7 concentration in the cell (Figure 1a). Cells transfected with the reporter construct pMiRAR-let-7 expressed GFP (Figure 2a,c), indicating that endogenous let-7 levels do not entirely silence gfp expression. Background fluorescence of cells without miRAR (1858 ± 44 RFU (relative fluorescence units)) was subtracted from the fluorescence intensities. To evaluate the responsiveness of GFP production, we co-transfected the reporter pMiRAR-let-7 with 80 pMlet-7 miRNA or separately with anti-miR RNA complementary to let-7 (anti-let-7). Supplementing cells with exogeneous let-7 effectively inhibited GFP translation, reducing fluorescence by more than 3-fold (Figure 2a,c). In contrast, supplementing cells with anti-let-7, which binds to and de-activates cellular let-7, led to a marked decrease in active let-7 molecules in the cell as reported by a 1.3-fold increase in GFP production and fluorescence (Figure 2a,c).

Visualizing Let-7 Accumulation due to Inhibition of Let-7 Degradative Enzymes
We further tested the reporter system by assessing miRNA levels in cells depleted for the let-7 degradative enzyme Tut4, which has been shown previously to affect miRNA degradation [1,29,36,37]. Tut4 polyuridylates let-7 miRNAs, marking them for degradation by the exonuclease Dis3L2 ( Figure 1b) [1]. Tut4 was knocked down using siRNA, and partial knockdown of~50% of Tut4 was confirmed by western blotting (Figure 2b). As expected, the depletion of Tut4 resulted in a decrease of GFP fluorescence by 2.4-fold, confirming an increase in cellular let-7 levels (Figure 2a,c). Thus, elevation of miRNA concentrations caused by inhibition of the uridylyltransferase Tut4, and the subsequent lack of U-dependent let-7 degradation can be measured using our GFP reporter system. let-7 levels do not entirely silence gfp expression. Background fluorescence of cells without miRAR (1858 ± 44 RFU (relative fluorescence units)) was subtracted from the fluorescence intensities. To evaluate the responsiveness of GFP production, we co-transfected the reporter pMiRAR-let-7 with 80 pMlet-7 miRNA or separately with anti-miR RNA complementary to let-7 (anti-let-7). Supplementing cells with exogeneous let-7 effectively inhibited GFP translation, reducing fluorescence by more than 3-fold (Figure 2a,c). In contrast, supplementing cells with anti-let-7, which binds to and de-activates cellular let-7, led to a marked decrease in active let-7 molecules in the cell as reported by a 1.3-fold increase in GFP production and fluorescence (Figure 2a

Visualizing Let-7 Accumulation due to Inhibition of Let-7 Degradative Enzymes
We further tested the reporter system by assessing miRNA levels in cells depleted for the let-7 degradative enzyme Tut4, which has been shown previously to affect miRNA degradation [1,29,36,37]. Tut4 polyuridylates let-7 miRNAs, marking them for degradation by the exonuclease Dis3L2 (Figure 1b) [1]. Tut4 was knocked down using siRNA, and partial knockdown of ~50% of Tut4 was confirmed by western blotting (Figure 2b). As expected, the depletion of Tut4 resulted in a decrease of GFP fluorescence by 2.4-fold, confirming an increase in cellular let-7 levels (Figure 2a,c). Thus, elevation of miRNA concentrations caused by inhibition of the uridylyltransferase Tut4, and the subsequent lack of U-dependent let-7 degradation can be measured using our GFP reporter system.

Mutation of Let-7 Binding Sites Abolishes the Sensitivity of the pMiRAR to Changes in Let-7 Levels
To confirm that the changes in fluorescence were indeed exclusively due to let-7 binding to the KRas-UTR in our reporter, we generated a variant of the reporter gene construct with mutated let-7 binding sites. Mutations in the let-7 binding sites abolish the regulatory effect of let-7 on gene expression. The let-7 un-responsive KRas-3 UTR mutant was described previously [34], and cloned downstream of the gfp coding sequence. As before, we observed a significant decrease in florescence in cells co-transfected with the wildtype pMiRAR-let-7 construct with Tut4 siRNA compared to untreated cells (Figure 3a,b). In contrast, GFP fluorescence in cells carrying a plasmid with a mutated KRas-UTR fused to GFP (pMiRAR-let-7-mutant) did not respond to a Tut4 knockdown (Figure 3a,b). These data confirm that the change in fluorescence is indeed due to binding of let-7 to the KRas-3 -UTR. As a control experiment, a second plasmid coding for tdTomato was co-transfected to probe for differences in transfection efficiency. No significant changes in transfection efficiency of tdTomato were observed (Figure 3a), indicating that variation in transfection efficiency does not account for the decrease or increase of GFP fluorescence. To further confirm that the Tut4 knockdown decreases let-7 levels in the cell, we quantified let-7 miRNA levels by qPCR ( Figure 4). Indeed, in the Tut4 knockdown, a 2.7-fold increase in let-7 miRNA levels was observed, correlating with the 2.4-fold decrease in GFP fluorescence in the pMiRAR-let-7 reporter. These data further confirm that depletion of Tut4 leads to an increase of let-7 levels in the cells, as described previously [29]. We here show that a 2.4-fold decrease in GFP fluorescence corresponds to a 2.7-fold increase in cellular let-7 levels, showing direct inverse reporting of let-7 levels by pMiRAR-let-7.

Mutation of Let-7 Binding Sites Abolishes the Sensitivity of the pMiRAR to Changes in Let-7 Levels
To confirm that the changes in fluorescence were indeed exclusively due to let-7 binding to the KRas-UTR in our reporter, we generated a variant of the reporter gene construct with mutated let-7 binding sites. Mutations in the let-7 binding sites abolish the regulatory effect of let-7 on gene expression. The let-7 un-responsive KRas-3′UTR mutant was described previously [34], and cloned downstream of the gfp coding sequence. As before, we observed a significant decrease in florescence in cells co-transfected with the wildtype pMiRAR-let-7 construct with Tut4 siRNA compared to untreated cells (Figure 3a,b). In contrast, GFP fluorescence in cells carrying a plasmid with a mutated KRas-UTR fused to GFP (pMiRAR-let-7-mutant) did not respond to a Tut4 knockdown (Figure 3a,b). These data confirm that the change in fluorescence is indeed due to binding of let-7 to the KRas-3′-UTR. As a control experiment, a second plasmid coding for tdTomato was co-transfected to probe for differences in transfection efficiency. No significant changes in transfection efficiency of tdTomato were observed (Figure 3a), indicating that variation in transfection efficiency does not account for the decrease or increase of GFP fluorescence. To further confirm that the Tut4 knockdown decreases let-7 levels in the cell, we quantified let-7 miRNA levels by qPCR ( Figure 4). Indeed, in the Tut4 knockdown, a 2.7-fold increase in let-7 miRNA levels was observed, correlating with the 2.4-fold decrease in GFP fluorescence in the pMiRAR-let-7 reporter. These data further confirm that depletion of Tut4 leads to an increase of let-7 levels in the cells, as described previously [29]. We here show that a 2.4-fold decrease in GFP fluorescence corresponds to a 2.7-fold increase in cellular let-7 levels, showing direct inverse reporting of let-7 levels by pMiRAR-let-7.

Adapting the Optogenetic Reporter for Quantifying miR-122
To investigate whether the reporter system is applicable and useful for the reporting of other miRNAs, we generated a similar reporter system for miR-122. The CPEB 3′-UTR encodes 2 miR-122 binding sites and CPEB translation is regulated by cellular miR-122 levels [38]. We fused the CPEB 3′-UTR to gfp to generate a reporter system for miR-122 (pMiRAR-miR-122). Low concentrations of co-transfected miR-122 and anti-miR-122 showed no significant change in fluorescence and transfected RNA concentrations were increased to 100 nM. This led to significant changes in GFP fluorescence (Figure 5a). Transfection with miR-122 at 100 nM led to a 1.2-fold decrease in fluorescence, while the anti-miR-122 led to a 1.3-fold increase in fluorescence (Figure 5a,b). Thus, a reporter with two miRNA binding sites is applicable to more pronounced changes in miRNA activity, while a reporter with five miRNA binding sites may be useful for subtle changes. This demonstrates that with minor modification the MiRAR can be adapted to likely any desired target miRNA or range of sensitivity.

Adapting the Optogenetic Reporter for Quantifying miR-122
To investigate whether the reporter system is applicable and useful for the reporting of other miRNAs, we generated a similar reporter system for miR-122. The CPEB 3 -UTR encodes 2 miR-122 binding sites and CPEB translation is regulated by cellular miR-122 levels [38]. We fused the CPEB 3 -UTR to gfp to generate a reporter system for miR-122 (pMiRAR-miR-122). Low concentrations of co-transfected miR-122 and anti-miR-122 showed no significant change in fluorescence and transfected RNA concentrations were increased to 100 nM. This led to significant changes in GFP fluorescence (Figure 5a). Transfection with miR-122 at 100 nM led to a 1.2-fold decrease in fluorescence, while the anti-miR-122 led to a 1.3-fold increase in fluorescence (Figure 5a,b). Thus, a reporter with two miRNA binding sites is applicable to more pronounced changes in miRNA activity, while a reporter with five miRNA binding sites may be useful for subtle changes. This demonstrates that with minor modification the MiRAR can be adapted to likely any desired target miRNA or range of sensitivity.

Discussion
The level of miRNA in cells is usually determined by next-generation sequencing or qPCR, which are methods that require cell lysis and subsequent RNA extraction. To observe and quantify miRNA activity in live cells and to capture real-time responses to environmental or chemical changes, a genetically encoded reporter system that allows for a time-resolved quantification of miRNA activity is required. Previously, several luciferase-based reporter systems were generated [34,[39][40][41]. These reporters allowed for the quantification of miRNA levels without cell lysis but required the addition of luciferin or a luciferin synthesis plasmid. Luciferase-based reporters provide a snapshot of cellular miRNA levels, rather than the ability to continuously report miRNA activity over an uninterrupted time course.
To generate a reporter system that is independent of external supplementation with chemicals and allows for a time-resolved quantification of miRNA activity in the cell, we developed a GFPbased miRNA activity reporter. The 3′-UTR of KRas contains several let-7 miRNA binding sites (Figure 1a), and KRas mRNA stability is well known to be regulated by let-7 miRNA [16]. We showed that our reporter accurately reports cellular miRNA concentrations via an inverse response from MiRAR fluorescence. Co-transfected miRNA let-7 or anti-let-7 efficiently reduced or elevated GFP fluorescence in the anticipated inverse relationship. The let-7 reporter displayed high sensitivity, with

Discussion
The level of miRNA in cells is usually determined by next-generation sequencing or qPCR, which are methods that require cell lysis and subsequent RNA extraction. To observe and quantify miRNA activity in live cells and to capture real-time responses to environmental or chemical changes, a genetically encoded reporter system that allows for a time-resolved quantification of miRNA activity is required. Previously, several luciferase-based reporter systems were generated [34,[39][40][41]. These reporters allowed for the quantification of miRNA levels without cell lysis but required the addition of luciferin or a luciferin synthesis plasmid. Luciferase-based reporters provide a snapshot of cellular miRNA levels, rather than the ability to continuously report miRNA activity over an uninterrupted time course.
To generate a reporter system that is independent of external supplementation with chemicals and allows for a time-resolved quantification of miRNA activity in the cell, we developed a GFP-based miRNA activity reporter. The 3 -UTR of KRas contains several let-7 miRNA binding sites (Figure 1a), and KRas mRNA stability is well known to be regulated by let-7 miRNA [16]. We showed that our reporter accurately reports cellular miRNA concentrations via an inverse response from MiRAR fluorescence. Co-transfected miRNA let-7 or anti-let-7 efficiently reduced or elevated GFP fluorescence in the anticipated inverse relationship. The let-7 reporter displayed high sensitivity, with changes in fluorescence corresponding to pM of miRNA transfected. Thus, we can utilize the reporter to observe small changes in miRNA activity resulting from increasing and decreasing let-7 levels.
Our MiRAR system can also monitor changes in cellular miRNA metabolism. A 2.4-fold decrease in fluorescence corresponds to a 2.7-fold increase in miRNA content upon depletion of the let-7 degradative enzyme Tut4. MiRAR may thus be a highly useful tool to screen small molecule inhibitor libraries for compounds active in altering miRNA metabolism. Previous studies relied on biochemical assays to identify inhibitors of let-7 degradative enzymes Lin28 [27,28] and Tut4 [29]. Utilizing a live-cell reporter to quantify the impact of small molecule inhibitors may lead to a more efficient screening in a physiologically relevant model system. Screening drugs with MiRAR can circumvent time and effort spent on in vitro hits that subsequently fail to permeate the cell or increase let-7 levels in the cell.
For the miRNA let-7, we observed a less pronounced change in GFP fluorescence when adding anti-miR RNA complementary to let-7. This decrease in sensitivity compared to supplementing additional let-7 is most likely due to reaching the level of endogenous let-7 levels in HEK 293 cells. Supplementation of the anti-miRNA will eventually bind and deactivate free cellular let-7, and if provided in excess will not further decrease GFP fluorescence. Thus, the reporter system can also be utilized as a tool to not only quantify changes in let-7 levels, but our data suggest that MiRAR may also be a valuable tool to quantify absolute let-7 levels by titrating increasing concentrations of anti-let-7 until no further increase in GFP fluorescence is observed.
To demonstrate the generality of our approach, we generated a MiRAR for another miRNA, miR-122. miR-122 is associated with both Hepatitis B and C infections [42][43][44] and hepatic cancer [45][46][47][48][49]. We used an miR-122-responsive CBEB 3 -UTR in generating this MiRAR contruct. The CBEB 3 -UTR contains only two miR-122 binding sites [38], compared to the five binding sites of let-7 in our KRas-derived MiRAR. Consistent with a reduced number of miRNA binding sites, we observed that a significant change in GFP fluorescence was only observed upon an increase to 100 nM of transfected miR-122 compared to 80 pM in the MiRAR-let-7. The MiRAR-miR-122 reporter is thus approximately 1000-fold less sensitive to changes in miRNA concentration. Thus, our data indicate that the sensitivity of the MiRAR can be tuned depending on the concentration range of miRNA of interest. In the future, we envision that changes in sensitivity or adaptation to other miRNA species can be achieved by either fusing the 3 -UTR of a miRNA-regulated gene to gfp, or by mutating the binding sites of e.g., miR-122 or let-7 in the existing constructs to another seed sequence.
In summary, we present a versatile reporter system that is adaptable to different miRNAs and is scalable in terms of sensitivity. Changes in miRNA metabolism in response to extracellular stimuli or over the lifetime of a cell can be monitored in a time resolved manner at the single-cell level without further interfering with the cellular environment. This tool will allow unprecedented insight into miRNA metabolism and biology. Future efforts will include the generation of stable cell lines containing MiRARs directed at certain miRNAs, and adaptation of the current constructs to other target miRNAs. Compared to luciferase-based reporters, our GFP-based reporter circumvents use of chemicals (luciferin) as an additional screening step and may provide a useful tool for high-throughput screening of small molecule chemotherapeutics that are effective in altering miRNA content or activity.