THE AVIAN REOVIRUS TRICISTRONIC S1 mRNA: NEW INSIGHTS INTO CONTROL OF TRANSLATION INITIATION

Abstract

The S1 genome segment of avian reovirus is functionally tricistronic, encoding three independent protein products (named p10, p17 and ?C) from three sequential, partially overlapping open reading frames (ORFs). The dogma of translation initiation, the cap-dependent scanning model, suggests that ribosomes would normally only translate the 5?-proximal ORF. Four alternate mechanisms of translation initiation could account for translation of the downstream ?C ORF; an IRES element, reinitiation, ribosome shunting, and leaky scanning. The objective of my doctoral research was to investigate the translation initiation mechanisms that are operative on the S1 mRNA. Translation of the p10 and p17 ORFs was revealed to be coordinated via standard leaky scanning, while none of the known mechanisms of translation initiation could account for expression of the ?C ORF. Further investigation determined that two alternate cap-dependent mechanisms contribute to translation initiation at the ?C AUG codon. The first mechanism involves a modified version of enhanced leaky scanning. Although insertion of upstream elements known to impede scanning ribosomal subunits dramatically inhibited translation of the downstream ORF in the context of other mRNAs, the same elements only marginally reduced ?C translation. Specific features of the S1 mRNA therefore function to promote leaky scanning and translation of the ?C ORF. The inability to eliminate ?C expression beyond a threshold retention level of ~20-30%, despite the presence of eight upstream start codons that should eliminate leaky scanning, strongly suggests that ribosomes must also utilize a scanning-independent means to access the internal ?C start site. This mechanism for ?C translation initiation, which I termed ribosome handoff, allows ribosomes to bypass upstream elements, and requires a sequence-dependent translation enhancer element present within S1 nucleotides 366-392 that may function to mediate handoff via complementarity with 18S ribosomal RNA. Translation initiation at the ?C start site is therefore made possible by two alternative mechanisms, enhanced leaky scanning and ribosome handoff from the 5?-cap. The novelty of these two mechanisms highlights the complexity of the translation initiation process and the potential heterogeneity of cellular ribosomes, which raises the possibility that internal initiation may be far more common than currently appreciated.