Redundant Structural Motifs in a Unique Retroviral Posttranscriptional Control Element Mediate a Novel Mechanism of Translational Enhancement PDF Download

Author: Tiffiney Marie Roberts
Publisher:
ISBN:
Category : RNA
Languages : en
Pages :

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Book Description
Abstract: Retroviruses achieve translation of their unspliced genome-length RNA despite a long and highly structured 52 untranslated region and lack of intron removal. By contrast typical cellular pre-mRNAs either undergo complete splicing and nuclear export or become degraded in the nucleus. Retroviral pre-mRNA interacts with viral or cellular proteins that alter typical posttranscriptional gene expression. Complex retroviruses like human immunodeficiency virus (HIV) encode a specialized regulatory protein whereas simple retroviruses like spleen necrosis virus (SNV) rely solely on cellular posttranscriptional modulators. Our lab identified a novel posttranscriptional control element in SNV RU5 RNA that facilitates expression of unspliced HIV-1 gag reporter RNA. This dissertation research identified that SNV RU5 functions, at least in part, by enhancing translational utilization of unspliced RNA; characterized primary sequence and secondary structure motifs necessary for SNV RU5 activity; and identified a cellular protein that interacts with SNV RU5 and conveys activity. RNA and protein quantitation determined that SNV RU5 enhances translation of nonviral luciferase RNA 8- to 10-fold but does not function as an internal ribosomal entry site. Detailed ribosomal profile analysis determined that SNV RU5 enhances translation initiation by increasing association of the RNA with multiple ribosomes by at least 3.5-fold as compared to a deletion mutant. Deletion and point mutagenesis defined two functionally redundant and synergistic regions necessary for activity. Enzymatic mapping determined that the regions are stable stem-loop structures and that both secondary structure and primary sequence motifs are necessary for activity. The loss-of-function mutations did not effect steady-state level or cytoplasmic accumulation of RU5-gag RNA but eliminated translational utilization. To identify cellular proteins that interact with SNV RU5, four techniques were utilized. First, overexpression assays evaluated three selected cellular proteins for stimulation of SNV RU5 activity. Second, RNA electromobility shift assays verified that binding is recapitulated in vitro. Third, UV-crosslinking experiments determined the relative sizes of SNV RU5-interactive proteins. Finally, affinity chromatography isolated a protein identified as RNA helicase A (RHA), which binds wildtype but not antisense SNV RU5 RNA and increases SNV RU5 activity in overexpression assays. RHA is co-localized with polysomes and correlates with increased translational utilization by SNV RU5.