Stimulation of −1 programmed ribosomal frameshifting by a metabolite-responsive RNA pseudoknot

Abstract

Specific recognition of metabolites by functional RNA motifs within mRNAs has emerged as a crucial regulatory strategy for feedback control of biochemical reactions. Such riboswitches have been demonstrated to regulate different gene expression processes, including transcriptional termination and translational initiation in prokaryotic cells, as well as splicing in eukaryotic cells. The regulatory process is usually mediated by modulating the accessibility of specific sequence information of the expression platforms via metabolite-induced RNA conformational rearrangement. In eukaryotic systems, viral and the more limited number of cellular decoding À1 programmed ribosomal frameshifting (PRF) are commonly promoted by a 39 mRNA pseudoknot. In addition, such À1 PRF is generally constitutive rather than being regulatory, and usually results in a fixed ratio of products. We report here an RNA pseudoknot capable of stimulating À1 PRF whose efficiency can be tuned in response to the concentration of S-adenosylhomocysteine (SAH), and the improvement of its frameshifting efficiency by RNA engineering. In addition to providing an alternative approach for small-molecule regulation of gene expression in eukaryotic cells, such a metabolite-responsive pseudoknot suggests a plausible mechanism for metabolite-driven translational regulation of gene expression in eukaryotic systems.

Endogenous ribosomal frameshift signals operate as mRNA destabilizing elements through at least two molecular pathways in yeast

“…The EST2 signal is both highly efficient at promoting À1 RF [$55%, see (15)], and is predicted to be quite stable ($À27 to À24 kcal/mol depending on the particular folding solution, see http:// prfdb.umd.edu/). It is important to note however that the software used to predict mRNA pseudoknots can neither identify base triples, which make major contributions to frameshifting (40)(41)(42)(43)(44), let alone calculate their contributions to thermodynamic stability. Regardless, this combination of high frameshifting and thermodynamic stability results in very strong destabilization via NMD ( Figure 2B), and perhaps NGD as well ( Figure 2C).…”

Synergetic regulation of translational reading-frame switch by ligand-responsive RNAs in mammalian cells

“…The discovery of alternative splicing regulation by TPP riboswitches in fungi and plants ( 4 ) suggests that other RNA-mediated gene-expression platforms may provide a better framework for constructing successful eukaryotic riboswitches. Recently, engineered metabolite-responsive RNA pseudo-knots of prokaryotic origin were shown to possess ligand-specific -1 programmed ribosomal frameshifting (-1 PRF) stimulation activity in reticulocyte lysate ( 10 , 11 ). The -1 PRF causes an elongating ribosome to shift a single nucleotide in the 5′-direction of mRNA, leading to a -1 reading-frame switch during decoding.…”

“…Indeed, translational frameshifts have been found to serve as nutrient sensors ( 13 ). However, while the success of converting a ligand-responsive pseudo-knot into a ligand-dependent -1 PRF stimulator suggests that translational reading-frame switch regulation holds promise as an expression platform for engineering mammalian riboswitches, its general application is hampered by the difficulty in finding specific ligand-responsive -1 PRF pseudo-knots ( 10 , 11 ).…”

Structural basis for recognition ofS-adenosylhomocysteine by riboswitches

“…These riboswitches promote expression of the mRNA via an SAH-stabilized conformation that either prevents formation of a rho-independent transcriptional terminator or exposes the Shine-Dalgarno sequence to permit ribosome binding . Intriguingly, the SAH riboswitch has been shown to be capable of regulating gene expression in human cells as an engineered ribosomal frame-shifting pseudoknot (Chou et al 2010).…”