Translation of an uncapped mRNA involves scanning

Area of Science:

• Molecular Biology
• Virology
• Gene Expression

Background:

• Human immunodeficiency virus (HIV) Tat protein is essential for viral replication.
• Tat protein is transcribed from an uncapped, nonpolyadenylated mRNA (VA-Tat RNA) under the control of an RNA polymerase III promoter.
• The presence of an upstream open reading frame (ORF) in VA-Tat RNA suggests potential scanning during translation, even without a cap structure.

Purpose of the Study:

• To investigate the mechanism of translation initiation for uncapped mRNA.
• To provide definitive evidence for ribosomal scanning on uncapped transcripts.
• To explore the role of secondary structures and upstream ORFs in regulating Tat protein synthesis.

Main Methods:

• Transfection of mammalian cells with modified VA-Tat RNA constructs.
• Introduction of secondary structures with varying stabilities into the 5'-untranslated region.
• Mutagenesis to alter the overlap between upstream and downstream ORFs.
• Analysis of Tat protein synthesis via translation.

Main Results:

• Highly stable secondary structures in the 5'-untranslated region significantly inhibited Tat synthesis, confirming mRNA scanning.
• Lower stability structures did not inhibit translation, supporting the scanning model.
• Increasing the overlap between the upstream ORF and the Tat ORF reduced downstream translation.
• Ribosome reinitiation, potentially in a 3' to 5' direction, was implied by overlapping ORF results.
• Similar translation patterns were observed for capped polymerase II transcripts.

Conclusions:

• Uncapped viral mRNA is translated via a scanning mechanism, similar to capped cellular mRNA.
• Ribosomal scanning efficiency is influenced by mRNA secondary structure and upstream ORFs.
• Evidence suggests ribosomes can reinitiate translation, possibly through a 3' to 5' scanning mechanism in overlap regions.