Tailored enrichment strategy detects low abundant small noncoding RNAs in HIV-1 infected cells.
Tailored enrichment strategy detects low abundant small noncoding RNAs in HIV-1 infected cells.
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DOI：
10.1186/1742-4690-9-27
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发表时间：
2012-03-29
期刊：
影响因子：
3.3
通讯作者：
Fischer M
中科院分区：
文献类型：
--
作者：
Althaus CF;Vongrad V;Niederöst B;Joos B;Di Giallonardo F;Rieder P;Pavlovic J;Trkola A;Günthard HF;Metzner KJ;Fischer M
不同种类的小非编码RNA（sncRNA）是不同类型生物体基因表达的重要调节因子。虽然近年来已经鉴定了数量迅速增加的sncRNA，但低丰度的sncRNA的分离仍然具有挑战性。病毒编码的sncRNA，特别是RNA病毒的sncRNA，可以以非常低的水平表达。HIV-1最好地说明了这一点，其中病毒编码的sncRNA占HIV-1感染细胞中所有sncRNA的约0.1-1.0%或未被检测到。因此，我们应用了一种新的序列靶向富集策略来捕获HIV-1感染的原代CD 4 + T淋巴细胞和巨噬细胞中的HIV-1衍生的sncRNA，其允许低丰度sncRNA的富集超过100倍。从来自五个独立实验的九个不同的sncRNA文库中克隆和测序了892个单独的HIV-1 sncRNA。这些克隆占所产生文库中所有sncRNA克隆的高达90%。216个HIV-1 sncRNA可区分为独特的克隆。然而，它们分布在整个HIV-1基因组中，形成某些簇，并且几乎10%显示反义方向。HIV-1 sncRNA的长度在16至89个核苷酸之间变化，在31至50个核苷酸处有一个意想不到的峰值，因此比细胞microRNA或短干扰RNA（siRNA）长。示例性的HIV-1 sncRNA也在用不同的原代HIV-1分离株感染的细胞中产生，并且可以抑制HIV-1复制。HIV-1感染的细胞产生病毒编码的sncRNA，这可能在HIV-1生命周期中发挥作用。此外，以序列特异性方式富集低丰度sncRNA的巨大能力高度推荐我们的选择策略用于任何类型的研究，其中所寻求的sncRNA的起源或靶序列是已知的。
The various classes of small noncoding RNAs (sncRNAs) are important regulators of gene expression across divergent types of organisms. While a rapidly increasing number of sncRNAs has been identified over recent years, the isolation of sncRNAs of low abundance remains challenging. Virally encoded sncRNAs, particularly those of RNA viruses, can be expressed at very low levels. This is best illustrated by HIV-1 where virus encoded sncRNAs represent approximately 0.1-1.0% of all sncRNAs in HIV-1 infected cells or were found to be undetected. Thus, we applied a novel, sequence targeted enrichment strategy to capture HIV-1 derived sncRNAs in HIV-1 infected primary CD4+ T-lymphocytes and macrophages that allows a greater than 100-fold enrichment of low abundant sncRNAs. Eight hundred and ninety-two individual HIV-1 sncRNAs were cloned and sequenced from nine different sncRNA libraries derived from five independent experiments. These clones represent up to 90% of all sncRNA clones in the generated libraries. Two hundred and sixteen HIV-1 sncRNAs were distinguishable as unique clones. They are spread throughout the HIV-1 genome, however, forming certain clusters, and almost 10% show an antisense orientation. The length of HIV-1 sncRNAs varies between 16 and 89 nucleotides with an unexpected peak at 31 to 50 nucleotides, thus, longer than cellular microRNAs or short-interfering RNAs (siRNAs). Exemplary HIV-1 sncRNAs were also generated in cells infected with different primary HIV-1 isolates and can inhibit HIV-1 replication. HIV-1 infected cells generate virally encoded sncRNAs, which might play a role in the HIV-1 life cycle. Furthermore, the enormous capacity to enrich low abundance sncRNAs in a sequence specific manner highly recommends our selection strategy for any type of investigation where origin or target sequences of the sought-after sncRNAs are known.
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影响因子：
3.3
作者：
Landry S;Halin M;Lefort S;Audet B;Vaquero C;Mesnard JM;Barbeau B
通讯作者：
Barbeau B
影响因子：
5.4
作者：
MICHAEL, NL;VAHEY, MT;REDFIELD, RR
通讯作者：
REDFIELD, RR
影响因子：
56.9
作者：
Kapranov, Philipp;Cheng, Jill;Gingeras, Thomas R.
通讯作者：
Gingeras, Thomas R.
影响因子：
32.4
作者：
Bennasser, Y;Le, SY;Jeang, KT
通讯作者：
Jeang, KT
影响因子：
5.4
作者：
Lin, Jennifer;Cullen, Bryan R.
通讯作者：
Cullen, Bryan R.