Mechanisms and biomedical implications of -1 programmed ribosome frameshifting on viral and bacterial mRNAs

Abstract

Some proteins are expressed as a result of a ribosome frameshifting event that is facilitated by a slippery site and downstream secondary structure elements in the mRNA. This review summarizes recent progress in understanding mechanisms of -1 frameshifting in several viral genes, including IBV 1a/1b, HIV-1 gag-pol, and SFV 6K, and in Escherichia coli dnaX. The exact frameshifting route depends on the availability of aminoacyl-tRNAs: the ribosome normally slips into the -1-frame during tRNA translocation, but can also frameshift during decoding at condition when aminoacyl-tRNA is in limited supply. Different frameshifting routes and additional slippery sites allow viruses to maintain a constant production of their key proteins. The emerging idea that tRNA pools are important for frameshifting provides new direction for developing antiviral therapies.

Keywords: RNA; tRNA; frameshifting; protein synthesis; recoding; regulation; ribosome; translation.

Figure 1

Sequence and structure of mRNA frameshifting motifs. (A) From left to right, schematics of frameshifting motifs of IBV 1a/1b, dnaX of E. coli, gag‐pol of HIV‐1, and 6K of SFV. Slippery sites (SS) are indicated and highlighted in green. The regulatory downstream mRNA element is a pseudoknot (PK) or a stem‐loop (SL), as indicated. pSS2 in HIV‐1 stands for the second putative SS. (B) Chemical probing of the mRNA secondary structure element in the SFV 6K mRNA. In vitro transcribed mRNA was treated with dimethyl sulfate (DMS; A‐ and C‐specific), 1‐cyclohexyl‐3‐(2‐morpholinoethyl) carbodiimide metho‐p‐toluenesulfonate (CMCT; U‐specific and low reactivity toward G) and β‐ethoxy‐α‐ketobutyraldehyde (kethoxal KE; G‐specific, and analyzed base modifications by primer extensions 109. (–) indicates untreated mRNA. Positions of reverse transcription (RT) stops due to modification were visualized on a sequencing gel using fluorescence primer complementary to positions 109–129 nucleotides of the mRNA (60–80 nucleotides downstream the SS). wt mRNA has the native sequence; test mRNA has been optimized for translation in E. coli (see lower panel; SD is Shine–Dalgarno sequence, AUG is the start codon, G61 is mutated to C to remove a potential initiation codon); IC is the initiation complex of test mRNA with 70S ribosomes. C, U, A, G are sequencing lanes. Numbered nucleotides to the left refer to the nucleotides in the SFV mRNA starting from the slippery site as indicated in c. LSL is lower stem‐loop, USL is upper stem‐loop. (C) Secondary structure of SFV 6K mRNA based on bioinformatics prediction 27 and probing results. Modified nucleotides are marked with circles: red for the wt mRNA, blue for the test mRNA and green for the test mRNA in the IC. Sequences in boxes indicate nucleotides forming lower (LSL) and upper (USL) stems. Primer‐binding site for RT is marked with an arrow; triangle on the 5′ of the primer indicates its fluorescence label Atto647N.

Figure 2

Kinetic mechanisms of FFR (upper) and FLR (lower) –1PRF pathways on the gag‐pol mRNA of HIV‐1. FFR results from one‐tRNA slippage with peptidyl‐tRNA^{P he} in the P site (in magenta) when the A site is vacant due to low availability of Leu‐tRNA^{L eu( UAA )}. FLR arises upon frameshifting during translocation of tRNA^{P he} and peptidyl‐Phe‐Leu‐tRNA^{L eu( UAA )} (in green) and is prevalent at excess of Leu‐tRNA^{L eu( UAA )}. After reading the slippery site, translation can continue in the –1‐frame by incorporating Arg at the AGG codon (red) or in 0‐frame by decoding Gly at the GGG codon (blue). The –1‐frame commitment steps on the FFR and FLR routes is marked in red. –1PRF on SFV 6K, IBV 1a/1b, and E. coli dnaX can, in principle, follow the same two routes. The existence of the two‐tRNA route is well‐documented 15, 29, 30, 45, 46, 57. The prevalence of the one‐tRNA route for SFV 6K depends on the concentration of tRNA^{L eu( UAA )} in the infected neuronal cells, which is not known (see text below). The one‐tRNA slippage on IBV 1a/1b could occur before decoding of the first slippery seqence codon UUA by the respective rare tRNA^{L eu( UAA )}, however, the existence of the respective –1‐frame peptide product containing Phe‐Lys, rather than Leu‐Lys, has not been tested and the abundance of tRNA^{L eu( UAA )} in avian host cells is unknown. For dnaX, tRNA^{L ys} that reads the slippery site codons is abundant and the one‐tRNA frameshifting pathway is only elicited by starvation.

Figure 3

Mechanism of –1PRF on the SFV 6K mRNA. Translation was carried out in HiFi buffer at 37 °C as described in 69 for the gag‐pol mRNA; concentrations were Ser‐tRNA^{S er} and Phe‐tRNA^{P he} (with 0.8 μm each) and Lys‐tRNA^{L ys}, Val‐tRNA^{V al}, Ala‐tRNA^{A la} and Thr‐tRNA^{T hr} (0.25 μm each) and IC (0.08 μm) programmed with the 6K mRNA. Translation products were separated by reversed phase high‐performance liquid chromatography 69. 0‐frame products were identified based on the incorporation of [¹⁴C]Val, –1‐frame peptides using [¹⁴C]Ala and [¹⁴C]Thr. The –1PRF efficiency was calculated as a ratio between –1‐frame peptides and the sum of –1‐frame and all 0‐frame products, multiplied by 100%. (A) Schematic of the frameshifting site. The model SFV mRNA containing native SS and SL is optimized for translation in E. coli by introducing a SD sequence and a start codon AUG followed by AAG (Lys) to improve translation efficiency. (B) Effect of Phe‐tRNA^{P he} on FFS peptide formation in the absence of Leu‐tRNA^{L eu( UAA )}. Translation was carried out using tRNAs aminoacylated with M, S, K, F. (C) Dependence of –1PRF on Leu‐tRNA^{L eu( UAA )} concentration. Translation was carried out with M, S, K, F, L, V, A, and T aa‐tRNAs. (D) Effect of Val‐tRNA^{V al} (green circles) and Ser‐tRNA^{S er} (gray circles) concentrations on –1PRF efficiency. Translation was carried out using an equimolar concentrations of Leu‐tRNA^{L eu( UAA )} and aa‐tRNAs as in C. The large excess of Ser‐tRNA is required to ensure efficient translation of the SFV mRNA, which contains three Ser codons read by different tRNA^{S er} isoacceptors in the total tRNA^{S er} used in these experiments.

Figure 4

The role of the pSS2 in maintaining the permissive Gag to Gag‐Pol ratio. Top, in the wt gag‐pol mRNA, –1PRF on the SS1 accounts for most of the Gag‐Pol product and pSS2 is silent. Middle, a compensatory mutation in pSS2 that emerges in response to treatment with inhibitors targeting the viral protease makes pSS2 slippery. The production of Gag and Gag‐Pol is unchanged, but a small fraction of ribosomes slips into –2‐frame, resulting in synthesis of a truncated protein. Bottom, when SS1 is mutated, –1‐frameshifting on pSS2 restores Gag‐Pol production to about 70% of that on the wt sequence.