Abstract
Translation of Giardiavirus (GLV) mRNA is initiated at an internal ribosome entry site (IRES) in the viral transcript. The IRES localizes to a downstream portion of 5′ untranslated region (UTR) and a part of the early downstream coding region of the transcript. Recent studies indicated that the IRES does not require a pre-initiation complex to initiate translation but may directly recruit the small ribosome subunit with the help of a number of trans-activating protein factors. A La autoantigen homologue in the viral host Giardia lamblia, GlLa, was proposed as one of the potential trans-activating factors based on its specific binding to GLV-IRES in vitro. In this study, we further elucidated the functional role of GlLa in GLV-IRES mediated translation in Giardia by knocking down GlLa with antisense morpholino oligo, which resulted in a reduction of GLV-IRES activity by 40%. An over-expression of GlLa in Giardia moderately stimulated GLV-IRES activity by 20%. A yeast inhibitory RNA (IRNA), known to bind mammalian and yeast La autoantigen and inhibit Poliovirus and Hepatitis C virus IRES activities in vitro and in vivo, was also found to bind to GlLa protein in vitro and inhibited GLV-IRES function in vivo. The C-terminal domain of La autoantigen interferes with the dimerization of La and inhibits its function. An over-expression of the C-terminal domain (200–348aa) of GlLa in Giardia showed a dominant-negative effect on GLV-IRES activity, suggesting a potential inhibition of GlLa dimerization. HA tagged GlLa protein was detected mainly in the cytoplasm of Giardia, thus supporting a primary role of GlLa in translation initiation in Giardiavirus.
Highlights
Recognition of the initiation codon by small ribosomal subunit is a key step in translation initiation
The Rluc activity was assayed 5 hrs post-transfection and the outcome showed that the GLVIRES mediated translation of Rluc reporter was inhibited by,40% in the GlLa knockdown cells when compared to the controls (Fig. 1C)
In order to understand the mechanism of translation initiation at an internal ribosome entry site (IRES), it is essential to identify the cellular trans-acting proteins that bind to it and recruit ribosomes to initiate the process
Summary
Recognition of the initiation codon by small ribosomal subunit is a key step in translation initiation. Cap-dependent translation is initiated by the binding of a pre-initiation complex (the 40S ribosomal subunit combined with eIF1, eIF1A, eIF3 and eIF2-GTP-tRNA) to the 59 cap of mRNA through an interaction with eIF4G in the eIF4F complex bound to the cap. This complex initiates a downstream scanning along the mRNA for the initiation codon to begin translation [1]. Numerous capped cellular mRNAs were discovered to contain IRESs in their 59-UTRs and shown to utilize IRES mediated translation initiation when normal cap dependent translation is severely compromised during conditions of cell stress, cell cycle, development and diseases [3]
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