Caliciviral protein-based artificial translational activator for mammalian gene circuits with RNA-only delivery

Abstract

Synthetic RNA-based gene circuits enable sophisticated gene regulation without the risk of insertional mutagenesis. While various RNA binding proteins have been used for translational repression in gene circuits, the direct translational activation of synthetic mRNAs has not been achieved. Here we develop Caliciviral VPg-based Translational activator (CaVT), which activates the translation of synthetic mRNAs without the canonical 5′-cap. The level of translation can be modulated by changing the locations, sequences, and modified nucleosides of CaVT-binding motifs in the target mRNAs, enabling the simultaneous translational activation and repression of different mRNAs with RNA-only delivery. We demonstrate the efficient regulation of apoptosis and genome editing by tuning translation levels with CaVT. In addition, we design programmable CaVT that responds to endogenous microRNAs or small molecules, achieving both cell-state-specific and conditional translational activation from synthetic mRNAs. CaVT will become an important tool in synthetic biology for both biological studies and future therapeutic applications.

Rational Design of Aptamer-Tagged tRNAs

“…Thus, full sequestration of these fully active tRNA molecules may require another tight interaction between an RNA structure and its target protein. Instead of the wildtype MS2 hairpin RNA and the wildtype MS2 coat protein, an MS2 hairpin RNA variant with a C -loop (U to C at the -5 position) [ 39 , 40 ] and the MS2 coat protein V29I variant [ 40 , 41 ] were used to enhance the RNA aptamer-coat protein interaction. To make a single-chain MS2 coat protein [ 42 ] together with an Mk PSTK-C domain, two V29I domains were fused with a long linker containing a SUMO domain, a 6xHis tag, and an Mk PSTK-C domain, in this order ( Figure 4 B).…”

Protein-Based Systems for Translational Regulation of Synthetic mRNAs in Mammalian Cells

“…Thus, microbial RBPs are primarily used in mRNA-based mammalian synthetic biology. The representative microbial RBPs used to regulate synthetic mRNAs are coat proteins derived from the bacteriophages MS2 (MS2CP) [2][3][4][5][6][10][11][12][13][14][15][16][17][18][19][20][21] and PP7 (PP7CP) [6,14,21], the archaeal ribosomal protein L7Ae [2][3][4]6,7,11,12,[21][22][23][24][25], and the tetracycline-responsive repressor protein (TetR) from Escherichia coli [23,26]. Among these, TetR is unique in its ability to conditionally dissociate from the target RNA motif by doxycycline addition.…”

“…Since caliciviral RNAs lack the 5' cap, caliciviruses use VPg to recruit ribosomes to their RNAs via a VPg-eIF4F interaction [38][39][40]. We showed that the fusion protein of MS2CP and the feline caliciviral VPg, named caliciviral VPg-based translational activator (CaVT), can activate the translation of A-capped mRNAs containing the MS2CP-target motif in their 5' UTRs [5,20] (Figure 2, the 3rd row).…”

“…As some therapeutic genes may cause adverse effects when they are expressed in inappropriate contexts, context-dependent transgene regulation is helpful to cope with low adverse effects and high therapeutic efficacy. For example, cell-selective expression of pro-apoptotic genes [2][3][4][5][6] may enable cancer cell elimination without damaging healthy cells. Similarly, cardiomyocyte-selective expression of proliferation-promoting genes can be helpful to cope with myocardial regeneration without elevating fibrosis and immune response [7].…”

Programmable mammalian translational modulators by CRISPR-associated proteins

“…small molecules) is useful for fine-tuning gene expressions. Recent efforts have developed drug-responsive translational regulators through RBP engineering 40,41 . However, it has remained a challenge to develop various translational regulatory systems, because doing so requires additional characterization and further engineering of the individual modulators.…”