Abstract
MicroRNAs have emerged as important markers and regulators of cell identity. Precise measurements of cellular miRNA levels rely traditionally on RNA extraction and thus do not allow to follow miRNA expression dynamics at the level of single cells. Non-invasive miRNA sensors present an ideal solution but they critically depend on the performance of suitable ubiquitous promoters that reliably drive expression both in pluripotent and differentiated cell types. Here we describe the engineering of bidirectional promoters that drive the expression of precise ratiometric fluorescent miRNA sensors in single mouse embryonic stem cells (mESCs) and their differentiated derivatives. These promoters are based on combinations of the widely used CAG, EF1α and PGK promoters as well as the CMV and PGK enhancers. miR-142-3p, which is known to be bimodally expressed in mESCs, served as a model miRNA to gauge the precision of the sensors. The performance of the resulting miRNA sensors was assessed by flow cytometry in single stable transgenic mESCs undergoing self-renewal or differentiation. EF1α promoters arranged back-to-back failed to drive the robustly correlated expression of two transgenes. Back-to-back PGK promoters were shut down during mESC differentiation. However, we found that a back-to-back arrangement of CAG promoters with four CMV enhancers provided both robust expression in mESCs undergoing differentiation and the best signal-to-noise for measurement of miRNA activity in single cells among all the sensors we tested. Such a bidirectional promoter is therefore particularly well suited to study the dynamics of miRNA expression during cell fate transitions at the single cell level.
Highlights
IntroductionMicroRNAs (miRNAs) are a class of small non-coding RNAs that play important roles in the post transcriptional regulation of gene expression [1]. miRNA genes are predominantly transcribed by RNA polymerase II [2] either from their own transcriptional units or being found in introns of their host genes [3,4,5,6,7,8,9,10]
MicroRNAs are a class of small non-coding RNAs that play important roles in the post transcriptional regulation of gene expression [1]. miRNA genes are predominantly transcribed by RNA polymerase II [2] either from their own transcriptional units or being found in introns of their host genes [3,4,5,6,7,8,9,10]
We found that a back-to-back arrangement of CAG promoters with four CMV enhancers provided both robust expression in mouse embryonic stem cells (mESCs) undergoing differentiation and the best signal-to-noise for measurement of miRNA activity in single cells among all the sensors we tested
Summary
MicroRNAs (miRNAs) are a class of small non-coding RNAs that play important roles in the post transcriptional regulation of gene expression [1]. miRNA genes are predominantly transcribed by RNA polymerase II [2] either from their own transcriptional units or being found in introns of their host genes [3,4,5,6,7,8,9,10]. Bidirectional Promoter Engineering for Single Cell MicroRNA Sensors doi:10.1371/journal.pone.0155177.g001. A complex of the RNase Dicer [14] and TRBP [15] cleaves the stem-loop pre-miRNA into 20–25 base pair long double-stranded RNA fragments. The guide strand of miRNA duplex is selectively loaded into the RNA-induced silencing complex (RISC) [16,17,18,19], where it guides RISC to target mRNAs based on sequence complementarity. Partial complementarity is needed for animal miRNAs to bind to their target genes and it renders them far more promiscuous in their target selection with single miRNAs often regulating dozens of transcripts [21]. Pairing of the “seed” region (the nucleotides 2–8 at the 5’-end of the miRNA) to the 3’-UTR of mRNAs is often sufficient for metazoan miRNAs to recognize their targets [22]. Metazoan miRNAs first elicit translational repression of their targets followed by target deadenylation and degradation [23,24,25]
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