A stable hairpin preceded by a short open reading frame promotes nonlinear ribosome migration on a synthetic mRNA leader

Abstract

The regulation of cauliflower mosaic virus (CaMV) pregenomic 35S RNA translation occurs via nonlinear ribosome migration (ribosome shunt) and is mediated by an elongated hairpin structure in the leader. The replacement of the viral leader by a series of short, low-energy stems in either orientation supports efficient ribosomal shunting, showing that the stem per se, and not its sequence, is recognized by the translation machinery. The requirement for cis-acting sequences from the unstructured terminal regions of the viral leader was analyzed: the 59-terminal polypyrimidine stretch and the short upstream open reading frame (uORF) A stimulate translation, whereas the 39-flanking region seems not to be essential. Based on these results, an artificial leader was designed with a stable stem flanked by unstructured sequences derived from parts of the 59-and 39-proximal regions of the CaMV 35S RNA leader. This artificial leader is shunt-competent in translation assays in vivo and in vitro, indicating that a low-energy stem, broadly used as a device to successfully interfere with ribosome scanning, can efficiently support translation, if preceded by a short uORF. The synthetic 140-nt leader can functionally replace the CaMV 35S RNA 600-nt leader, thus implicating the universal role that nonlinear ribosome scanning could play in translation initiation in eukaryotes.

Upstream Open Reading Frames as Regulators of mRNA Translation

“…However, a GUG codon appears to initiate a uORF in the Fli-1 mRNA (56), illustrating the need to remain cognizant of the potential for non-AUG codons to serve as uORF initiation sites. cis-acting elements such as internal ribosomal entry sites or sequences that promote ribosomal shunting may enable ribosomes to circumvent the uAUG codon altogether and thus evade the regulatory effects of the uORF (21,47,68).…”

Continuous and Discontinuous Ribosome Scanning on the Cauliflower Mosaic Virus 35 S RNA Leader Is Controlled by Short Open Reading Frames

“…sORF A and stem section 1 of the CaMV RNA leader can be exchanged for other natural or synthetic sORFs and stable energy-rich hairpin (Kozak stem), respectively, without greatly affecting the efficiency of shunting (22,26,27,35). Note, however, that in the special case of sORFs whose products repress the downstream termination process shunting is promoted with only a low efficiency (26).…”

Mechanism of ribosome shunting in Rice tungro bacilliform pararetrovirus

“…At the molecular level, amechanism of shunting has been investigated most extensivelyfor CaMV (Fü tterer et al1990(Fü tterer et al , 1993D ominguez et al 1998;Hemmings-Mieszczak et al 1998Hemmings-Mieszczak and Hohn 1999;Pooggin et al 1998Pooggin et al ,2 000, 2001. Ac urrent model of the CaMVs hunting states that 40S ribosomal subunits with acomplement of initiation factors (hereafter, scanningr ibosomes) engage the CaMV pregenomic 35S RNA at the capped 5 9 -end, scan for ashort distance of 60 nt to encounterafirstAUG,the startcodonof the59 -proximalshort ORF (sORF A), and amajority of the scanning ribosomes assemble at thisAUG into complete 80S ribosomes to initiate translation.…”