Formation of circular polyribosomes on eukaryotic mRNA without cap-structure and poly(A)-tail: a cryo electron tomography study

Abstract

The polyribosomes newly formed on recombinant GFP-encoding mRNAs in a wheat germ cell-free translation system were analyzed using cryo-electron tomography, with sub-tomogram averaging of polysomal ribosomes and reconstruction of 3D structures of individual polyribosomes. The achieved level of resolution in the reconstructed polyribosomes allowed deducing the mRNA path by connecting adjacent exit and entry sites at the ribosomes inside each polyribosome. In this way, the circularity of a significant fraction (about 50%) of translating polyribosomes was proved in the case of the capped poly(A)-tailed mRNA, in agreement with the existing paradigm of the circularization via interaction of cap-bound initiation factor eIF4F with poly(A)-binding protein. However, translation of the capped mRNA construct without poly(A) tail, but with unspecific 3'-UTR derived from non-coding plasmid sequence, also led to the formation of circular polyribosomes in similar proportion (40%). Moreover, the polyribosomes formed on the uncapped non-polyadenylated mRNA with non-synergistic 5'- and 3'-UTRs proved to be circular as well, and appeared in the same proportion as in the previous cases. Thus, the formation of circular polyribosomes was found to be virtually independent of the presence of cap structure and poly(A) tail in mRNA, in contrast to the longstanding paradigm in the field.

Figure 1.

Ribosomal model obtained by sub-tomogram averaging. The head of the 40S subunit is shown in red, the body of the 40S subunit in yellow, the P1/P2 stalk of the 60S subunit in pink and the 60S subunit in blue.

Figure 2.

Cryo-ET analysis of polyribosomes formed in the wheat germ cell-free system with capped polyadenylated mRNA (Cap-5′UTR_βGlobin-scGFP-3′UTR_(N)40-(A)100) after 15 min of translation. (A) Fragment of reconstructed tomogram. Polyribosome models were obtained by fitting the averaged ribosome model as described in text and the Materials and Methods section (monoribosomes are not shown). Arrows point to polyribosomes with confirmed circular mRNA configuration, either ring-shape or double-row polyribosomes. (B) Left: tomographic slices of individual polyribosomes; middle: 3D subtomogram reconstructions of the same polyribosomes. Color indications are the same as in Figure 1. Right: deduced mRNA path within polyribosomes. Orientation of the mRNA chain in each ribosome is symbolized by an arrowhead directed from the mRNA entry to mRNA exit sites of the ribosome. The deduced path of the mRNA chain through the ribosome is shown by a line. The asterisk points to putative 3′-5′ junction.

Figure 3.

Cryo-ET analysis of polyribosomes formed in the wheat germ cell-free system with capped mRNA without polyA (Cap-5′UTR_βGlobin-scGFP-3′UTR_(N)180) after 15 min of translation. (A, B): see the legend in Figure 2.

Figure 4.

Cryo-ET analysis of polyribosomes formed in the wheat germ cell-free system with uncapped non-polyadenylated mRNA (5′UTR_Obe-GFP-3′UTR_TMV) after 15 min of translation. (A, B): see the legend in Figure 2.