Laboratory of Molecular Genetics, Wadsworth Center, Albany, NY, 12201, USA.
Mob DNA. 2012 Aug 2;3(1):12. doi: 10.1186/1759-8753-3-12.
Long-terminal repeat (LTR) retrotransposons have complex modes of mobility involving reverse transcription of their RNA genomes in cytoplasmic virus-like particles (VLPs) and integration of the cDNA copies into the host genome. The limited coding capacity of retrotransposons necessitates an extensive reliance on host co-factors; however, it has been challenging to identify co-factors that are required for endogenous retrotransposon mobility because retrotransposition is such a rare event.
To circumvent the low frequency of Ty1 LTR-retrotransposon mobility in Saccharomyces cerevisiae, we used iterative synthetic genetic array (SGA) analysis to isolate host mutations that reduce retrotransposition. Query strains that harbor a chromosomal Ty1his3AI reporter element and either the rtt101Δ or med1Δ mutation, both of which confer a hypertransposition phenotype, were mated to 4,847 haploid ORF deletion strains. Retrotransposition was measured in the double mutant progeny, and a set of 275 ORF deletions that suppress the hypertransposition phenotypes of both rtt101Δ and med1Δ were identified. The corresponding set of 275 retrotransposition host factors (RHFs) includes 45 previously identified Ty1 or Ty3 co-factors. More than half of the RHF genes have statistically robust human homologs (E < 1 x 10-10). The level of unintegrated Ty1 cDNA in 181 rhfΔ single mutants was altered <2-fold, suggesting that the corresponding co-factors stimulate retrotransposition at a step after cDNA synthesis. However, deletion of 43 RHF genes, including specific ribosomal protein and ribosome biogenesis genes and RNA degradation, modification and transport genes resulted in low Ty1 cDNA levels. The level of Ty1 Gag but not RNA was reduced in ribosome biogenesis mutants bud21Δ, hcr1Δ, loc1Δ, and puf6Δ.
Ty1 retrotransposition is dependent on multiple co-factors acting at different steps in the replication cycle. Human orthologs of these RHFs are potential, or in a few cases, presumptive HIV-1 co-factors in human cells. RHF genes whose absence results in decreased Ty1 cDNA include characterized RNA metabolism and modification genes, consistent with their having roles in early steps in retrotransposition such as expression, nuclear export, translation, localization, or packaging of Ty1 RNA. Our results suggest that Bud21, Hcr1, Loc1, and Puf6 promote efficient synthesis or stability of Ty1 Gag.
长末端重复（LTR）逆转录转座子具有复杂的移动模式，涉及它们的 RNA 基因组在细胞质病毒样颗粒（VLPs）中的逆转录和 cDNA 拷贝整合到宿主基因组中。逆转录转座子的有限编码能力需要高度依赖宿主辅助因子；然而，由于逆转座事件非常罕见，因此很难鉴定出需要的辅助因子。
为了规避酿酒酵母中 Ty1 LTR-逆转录转座子移动的低频率，我们使用迭代合成遗传阵列（SGA）分析来分离降低逆转座的宿主突变。携带染色体 Ty1his3AI 报告元件的查询菌株和 rtt101Δ 或 med1Δ 突变，这两种突变都赋予高转座表型，与 4847 个单倍体 ORF 缺失菌株交配。在双突变体后代中测量逆转座，鉴定出一组 275 个 ORF 缺失，可抑制 rtt101Δ 和 med1Δ 的高转座表型。相应的 275 个逆转座宿主因子（RHF）集包括 45 个先前鉴定的 Ty1 或 Ty3 辅助因子。超过一半的 RHF 基因在统计学上有可靠的人类同源物（E < 1 x 10-10）。在 181 个 rhfΔ 单突变体中，未整合的 Ty1 cDNA 的水平改变了 <2 倍，这表明相应的辅助因子在 cDNA 合成后刺激逆转座。然而，删除 43 个 RHF 基因，包括特定的核糖体蛋白和核糖体生物发生基因以及 RNA 降解、修饰和运输基因，导致 Ty1 cDNA 水平降低。核糖体生物发生突变体 bud21Δ、hcr1Δ、loc1Δ 和 puf6Δ 中 Ty1 Gag 的水平降低，但 RNA 水平没有降低。
Ty1 逆转座依赖于多个辅助因子，在复制周期的不同步骤中发挥作用。这些 RHF 的人类同源物是人类细胞中潜在的，或者在少数情况下，推定的 HIV-1 辅助因子。缺乏导致 Ty1 cDNA 减少的 RHF 基因包括特征性的 RNA 代谢和修饰基因，这与它们在 Ty1 RNA 的表达、核输出、翻译、定位或包装等早期步骤中发挥作用一致。我们的结果表明，Bud21、Hcr1、Loc1 和 Puf6 促进 Ty1 Gag 的有效合成或稳定性。
