a Department of Biochemistry , College of Medicine and Health Sciences, United Arab Emirates University , Al Ain , UAE.
b Université de Strasbourg , CNRS, Architecture et Réactivité de l'ARN , Strasbourg , France.
RNA Biol. 2018;15(8):1047-1059. doi: 10.1080/15476286.2018.1486661. Epub 2018 Jul 5.
Packaging the mouse mammary tumor virus (MMTV) genomic RNA (gRNA) requires the entire 5' untranslated region (UTR) in conjunction with the first 120 nucleotides of the gag gene. This region includes several palindromic (pal) sequence(s) and stable stem loops (SLs). Among these, stem loop 4 (SL4) adopts a bifurcated structure consisting of three stems, two apical loops, and an internal loop. Pal II, located in one of the apical loops, mediates gRNA dimerization, a process intricately linked to packaging. We thus hypothesized that the bifurcated SL4 structure could constitute the major gRNA packaging determinant. To test this hypothesis, the two apical loops and the flanking sequences forming the bifurcated SL4 were individually mutated. These mutations all had deleterious effects on gRNA packaging and propagation. Next, single and compensatory mutants were designed to destabilize then recreate the bifurcated SL4 structure. A structure-function analysis using bioinformatics predictions and RNA chemical probing revealed that mutations that led to the loss of the SL4 bifurcated structure abrogated RNA packaging and propagation, while compensatory mutations that recreated the native SL4 structure restored RNA packaging and propagation to wild type levels. Altogether, our results demonstrate that SL4 constitutes the principal packaging determinant of MMTV gRNA. Our findings further suggest that SL4 acts as a structural switch that can not only differentiate between RNA for translation versus packaging/dimerization, but its location also allows differentiation between spliced and unspliced RNAs during gRNA encapsidation.
包装鼠乳腺肿瘤病毒（MMTV）基因组 RNA（gRNA）需要整个 5'非翻译区（UTR）与 gag 基因的前 120 个核苷酸结合。该区域包含几个回文（pal）序列和稳定的茎环（SL）。其中，茎环 4（SL4）采用分叉结构，由三个茎、两个顶端环和一个内部环组成。位于一个顶端环中的 pal II 介导 gRNA 二聚化，这是一个与包装密切相关的过程。因此，我们假设分叉的 SL4 结构可以构成 gRNA 主要包装决定因素。为了验证这一假设，我们分别突变了两个顶端环和形成分叉 SL4 的侧翼序列。这些突变都对 gRNA 包装和复制产生了有害影响。接下来，设计了单突变和补偿突变来破坏然后重新创建分叉 SL4 结构。使用生物信息学预测和 RNA 化学探测进行的结构功能分析表明，导致 SL4 分叉结构丢失的突变会破坏 RNA 的包装和复制，而重新创建天然 SL4 结构的补偿突变则将 RNA 的包装和复制恢复到野生型水平。总之，我们的结果表明 SL4 是 MMTV gRNA 的主要包装决定因素。我们的研究结果进一步表明，SL4 作为一种结构开关，不仅可以区分用于翻译与包装/二聚化的 RNA，而且其位置还允许在 gRNA 包裹过程中区分剪接和非剪接的 RNA。
