Abstract
In Giardia lamblia, enhanced translation of luciferase mRNA, flanked between the 5'-untranslated region (UTR) and 3 '-end of giardiavirus transcript, requires the presence of the initial 264-nucleotide (nt) viral capsid-coding region. By introducing the transcripts of dicistronic viral constructs into Giardia, we demonstrated that the 264-nt downstream region alone is insufficient to function as an internal ribosome entry site (IRES) without including a portion of the 5 '-UTR as well. Deletion analysis showed that efficient internal initiation requires the last 253 nts (nts 114-367) of the 5 '-UTR in combination with the downstream 264 nts. Specific mutations that disrupted the predicted secondary structural elements in either the 5 '-UTR or the 264-nt capsid-coding region completely abolished the IRES-mediated translation of downstream cistron, suggesting that the IRES activity requires the presence of these structures in both regions. Mutations that abolished translation of the first cistron did not, however, affect the IRES-mediated translation of the second cistron, indicating that this IRES-mediated translation is independent of the translation of the upstream cistron. This is, to our knowledge, the first reported identification of a viral IRES with an estimated size of 517 nts that extends to both sides of the initiation site.
Highlights
The viral transcript is not capped [6]
A background firefly luciferase gene (Fluc) activity (75.5 Ϯ 2.2 relative light units (RLU)/g of protein) was detected in the lysate of Giardia trophozoites transfected with the transcript of this construct, whereas the Renilla luciferase gene (Rluc) activity amounted to 18,487 Ϯ 1210 RLU/g of protein in the same lysate
When both the 5Ј-untranslated region (UTR) and the coding region were placed between the two open reading frames, the Fluc activity in the lysate of transfected cells was increased to 17,489 Ϯ 666 RLU/g of protein, 240-fold of the background, resulting in a Fluc/Rluc ratio of 946 Ϯ 36 ϫ 10Ϫ3 (Fig. 1)
Summary
The viral transcript is not capped [6]. Chimeric mRNA containing a full-length firefly luciferase transcript flanked by the 367-nt GLV 5Ј-UTR and a 2022-nt 3Ј terminus of GLV (ϩ)strand RNA was introduced into GLV-infected Giardia trophozoites via electroporation [4]. In this study we expressed the in vitro transcripts of dicistronic cDNA constructs in Giardia and demonstrated further inclusion of a 253-nt downstream portion of the 5Ј-UTR in the IRES resulting in an overall 517-nt IRES extending to both sides of the initiation codon at an approximately equal distance.
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