Abstract
The S1 genome segments of avian and Nelson Bay reovirus encode tricistronic mRNAs containing three sequential partially overlapping open reading frames (ORFs). The translation start site of the 3'-proximal ORF encoding the sigmaC protein lies downstream of two ORFs encoding the unrelated p10 and p17 proteins and more than 600 nucleotides distal from the 5'-end of the mRNA. It is unclear how translation of this remarkable tricistronic mRNA is regulated. We now show that the p10 and p17 ORFs are coordinately expressed by leaky scanning. Translation initiation events at these 5'-proximal ORFs, however, have little to no effect on translation of the 3'-proximal sigmaC ORF. Northern blotting, insertion of upstream stop codons or optimized translation start sites, 5'-truncation analysis, and poliovirus 2A protease-mediated cleavage of eIF4G indicated sigmaC translation derives from a full-length tricistronic mRNA using a mechanism that is eIF4G-dependent but leaky scanning- and translation reinitiation-independent. Further analysis of artificial bicistronic mRNAs failed to provide any evidence that sigmaC translation derives from an internal ribosome entry site. Additional features of the S1 mRNA and the mechanism of sigmaC translation also differ from current models of ribosomal shunting. Translation of the tricistronic reovirus S1 mRNA, therefore, is dependent both on leaky scanning and on a novel scanning-independent mechanism that allows translation initiation complexes to efficiently bypass two functional upstream ORFs.
Highlights
Creation of the 43 S and 48 S preinitiation complexes
Efficient C Translation Initiation Occurs from a Full-length Tricistronic S1 Transcript—Cryptic splice sites or promoters or mRNA cleavage upstream of an internal start codon can potentially confound translation analysis of polycistronic mRNAs [7, 22]. To exclude such trivial explanations for C translation initiation, S1-specific transcripts generated in cells transfected with either the pARV-S1 or pNBV-S1 expression constructs were detected by Northern blotting
C translation derives from a full-length, tricistronic S1 mRNA, not from a truncated mRNA containing the C start site as the 5Ј-proximal AUG codon
Summary
Creation of the 43 S and 48 S preinitiation complexes. Translation initiation generally occurs at the 5Ј-proximal AUG start codon after scanning of the 5Ј-untranslated region (UTR) by the preinitiation complex and recruitment of the 60 S subunit. Further analyses revealed that a novel eIF4G-dependent, scanning-independent mechanism provides efficient ribosome access to the internal C start codon in a manner that is relatively unaffected by translation initiation events occurring at either of the two functional upstream ORFs. Cells and Antibodies—The quail fibroblast cell line, QM5, was maintained as previously described [20] and used for all transfection studies.
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