Mouse let-7 miRNA populations exhibit RNA editing that is constrained in the 5'-seed/cleavage/anchor regions and stabilize predicted mmu-let-7a: mRNA duplexes
Mouse let-7 miRNA populations exhibit RNA editing that is constrained in the 5'-seed/cleavage/anchor regions and stabilize predicted mmu-let-7a: mRNA duplexes
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DOI：
10.1101/gr.078246.108
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发表时间：
2008-10-01
期刊：
影响因子：
7
通讯作者：
Gunaratne, Preethi H.
中科院分区：
文献类型：
--
作者：
Reid, Jeffrey G.;Nagaraja, Ankur K.;Gunaratne, Preethi H.
对小鼠卵巢、胚胎胰腺（E14.5）和胰岛素分泌β细胞（β TC-3）中表达的数百万< 30-nt rna的大规模平行测序显示，与前体miRNA和小鼠基因组参考序列相比，接近50%的成熟miRNA（主要代表mmu-let-7家族）显示内部插入/缺失和替换。大约12% -20%与mu-let-7种群相关的物种表现出序列差异，这些差异在核苷酸3 -7（5'-种子）和10-15（切割和锚位点）上显著减少。这一观察结果与测序错误不一致，并使我们提出这些变化主要是由miRNA靶mRNA复合物上的转录后rna编辑活性引起的。内部核苷酸修饰在第9个核苷酸位置最丰富。常见的U-to-G第九碱基编辑导致抑制素缺乏小鼠中下调的let-7a靶点的稳定性显著增加（Inha(-/-)）。u -插入（14.8%）多于u -缺失（1.5%），并且存在被切割的中间体，这表明哺乳动物TUTase（末端尿苷基转移酶）介导的dutp依赖的u -插入/ u -缺失周期可能是一种可能的机制。我们推测，mmu-let-7a双凸块的mRNA靶点定向编辑稳定了“松散”的miRNA: mRNA靶点关联和功能，扩大了靶点库和/或增强了mRNA在翻译抑制中的衰变。我们的研究结果还表明，从下一代测序（NGS）技术（“内组学”）生成的数百万序列中，系统地研究给定细胞类型中特定RNA类别的序列变异，可以广泛地用于推断完全未表征RNA的特定部分的功能限制。
Massively parallel sequencing of millions of < 30-nt RNAs expressed in mouse ovary, embryonic pancreas (E14.5), and insulin-secreting beta-cells (beta TC-3) reveals that similar to 50% of the mature miRNAs representing mostly the mmu-let-7 family display internal insertion/deletions and substitutions when compared to precursor miRNA and the mouse genome reference sequences. Approximately, 12% -20% of species associated with mmu-let-7 populations exhibit sequence discrepancies that are dramatically reduced in nucleotides 3 -7 (5'-seed) and 10-15 (cleavage and anchor sites). This observation is inconsistent with sequencing error and leads us to propose that the changes arise predominantly from post-transcriptional RNA-editing activity operating on miRNA: target mRNA complexes. Internal nucleotide modifications are most enriched at the ninth nucleotide position. A common ninth base edit of U-to-G results in a significant increase in stability of down-regulated let-7a targets in inhibin-deficient mice (Inha(-/-)). An excess of U-insertions (14.8%) over U-deletions (1.5%) and the presence of cleaved intermediates suggest that a mammalian TUTase (terminal uridylyl transferase) mediated dUTP-dependent U-insertion/ U-deletion cycle may be a possible mechanism. We speculate that mRNA target site-directed editing of mmu-let-7a duplex-bulges stabilizes " loose" miRNA: mRNA target associations and functions to expand the target repertoire and/or enhance mRNA decay over translational repression. Our results also demonstrate that the systematic study of sequence variation within specific RNA classes in a given cell type from millions of sequences generated by next-generation sequencing (NGS) technologies ("intranomics") can be used broadly to infer functional constraints on specific parts of completely uncharacterized RNAs.