The Minimal Active Human SVA Retrotransposon Requires Only the 5′-Hexamer and Alu-Like Domains
The Minimal Active Human SVA Retrotransposon Requires Only the 5′-Hexamer and Alu-Like Domains
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DOI：
10.1128/mcb.00860-12
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发表时间：
2012-11-01
影响因子：
5.3
通讯作者：
Kazazian, Haig H., Jr.
中科院分区：
文献类型：
--
作者：
Hancks, Dustin C.;Mandal, Prabhat K.;Kazazian, Haig H., Jr.
由逆转录酶（RT）介导的基于rna的复制，一种称为逆转录的过程，正在人类中进行，是个体间甚至可能是个体内基因组变异的重要来源。长散布元件1 (LINE-1或L1) ORF2蛋白是在顺式和其他RNA中动员其自身RNA所需的RT活性的基因组来源，例如反式中的SINE/可变数串联重复(VNTR)/Alu （SVA）元件。SVA元件类似于通过插入突变或异常mRNA剪接导致人类单基因疾病的2kb人类特异性非编码rna。在此，我们在U2OS细胞中使用SVA逆转录转位细胞培养实验，研究了在l1介导的SVA逆转录中重要的SVA结构域。部分和全结构域的缺失表明，在全长SVA的情况下，去除Alu-like或sin - r结构域几乎没有影响，而去除CT六聚体或VNTR结构域可以导致活性降低75%。另外的实验表明，单独的alu -样片段可以在低水平上进行反转位，而CT六聚体的加入可以使活性增强到全长SVA的2倍。这些结果表明，在U2OS细胞中，没有SVA结构域是逆转录转位所必需的，SVA的5'端（六聚体和alu -样结构域）足以实现逆转录转位。
RNA-based duplication mediated by reverse transcriptase (RT), a process termed retrotransposition, is ongoing in humans and is a source of significant inter-and perhaps intraindividual genomic variation. The long interspersed element 1 (LINE-1 or L1) ORF2 protein is the genomic source for RT activity required for mobilization of its own RNA in cis and other RNAs, such as SINE/variable-number tandem-repeat (VNTR)/Alu (SVA) elements, in trans. SVA elements are similar to 2-kb hominid-specific noncoding RNAs that have resulted in single-gene disease in humans through insertional mutagenesis or aberrant mRNA splicing. Here, using an SVA retrotransposition cell culture assay in U2OS cells, we investigated SVA domains important in L1-mediated SVA retrotransposition. Partial-and whole-domain deletions revealed that removal of either the Alu-like or SINE-R domain in the context of a full-length SVA has little to no effect, whereas removal of the CT hexamer or the VNTR domain can result in a 75% decrease in activity. Additional experiments demonstrate that the Alu-like fragment alone can retrotranspose at low levels while the addition of the CT hexamer can enhance activity as much as 2-fold compared to that of the full-length SVA. These results suggest that no SVA domain is essential for retrotransposition in U2OS cells and that the 5' end of SVA (hexamer and Alu-like domain) is sufficient for retrotransposition.