A novel assay for viral microRNA function identifies a single nucleotide polymorphism that affects Drosha processing
A novel assay for viral microRNA function identifies a single nucleotide polymorphism that affects Drosha processing
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DOI：
10.1128/jvi.02734-05
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发表时间：
2006-06-01
影响因子：
5.4
通讯作者：
Cullen, Bryan R.
中科院分区：
文献类型：
--
作者：
Gottwein, Eva;Cai, Xuezhong;Cullen, Bryan R.
MicroRNAs （miRNAs）是一类类似于22核苷酸的非编码rna，在转录后水平上抑制特定靶基因的表达。最近，从潜伏感染的细胞中克隆出了致病性人类疱疹病毒卡波西肉瘤相关疱疹病毒（KSHV）编码的11个mirna。虽然Northern分析证实了这些mirna的表达，但它们抑制靶基因表达的能力尚未得到证实。我们设计了一种新的miRNA功能测定方法，该方法使用慢病毒指示载体，在Renilla荧光素酶基因的3'非翻译区为每个给定的miRNA携带两个完全互补的靶位。该试验使我们能够证明每个病毒miRNA在细胞与Renilla荧光素酶指示载体和萤火虫荧光素酶对照载体共转导时的活性。在kshv感染的BC-1和bbcl -1细胞中，而不是在未感染的对照细胞中，Renilla荧光素酶的表达选择性地降低了10倍。有趣的是，其中一种病毒mirna （miR-K5）在BC-1细胞中比在bbcl -1细胞中表现出更高的活性。对两种病毒基因组的序列分析显示，miR-K5前体茎环中存在单核苷酸多态性，这抑制了成熟miR-K5在BCBL-1细胞中的表达。我们发现，存在于bbcl -1中的原代miR-K5序列导致体内和体外的Drosha加工减少。这是miRNA前体中自然发生的序列多态性的首次报道，该多态性导致加工减少，从而降低成熟miRNA的表达和功能水平。
MicroRNAs (miRNAs) are a class of similar to 22-nucleotide noncoding RNAs that inhibit the expression of specific target genes at the posttranscriptional level. Recently, 11 miRNAs encoded by the pathogenic human herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) were cloned from latently infected cells. While the expression of these miRNAs has been confirmed by Northern analysis, their ability to inhibit target gene expression has not been demonstrated. We have devised a novel assay for miRNA function that uses lentiviral indicator vectors carrying two perfectly complementary target sites for each given miRNA in the 3' untranslated region of the Renilla luciferase gene. This assay allowed us to demonstrate the activity of each viral miRNA upon cotransduction of cells with the Renilla luciferase indicator vector together with a firefly luciferase control vector. In KSHV-infected BC-1 and BCBL-1 cells, but not uninfected control cells, Renilla luciferase expression was selectively reduced up to 10-fold. Interestingly, one of the viral miRNAs (miR-K5) exhibited much higher activity in BC-1 cells than in BCBL-1 cells. Sequence analysis of both viral genomes revealed a single nucleotide polymorphism in the miR-K5 precursor stem-loop, which inhibits the expression of mature miR-K5 in BCBL-1 cells. We show that the primary miR-K5 sequence present in BCBL-1 results in diminished processing by Drosha both in vivo and in vitro. This is the first report of a naturally occurring sequence polymorphism in an miRNA precursor that results in reduced processing and therefore lower levels of mature miRNA expression and function.