Mir-146a wild-type 3' sequence identity is dispensable for proper innate immune function in vivo (RNA-Seq)

A long-prevailing model has held that the “seed” region (nucleotides 2-8) of a microRNA is typically sufficient to mediate target recognition and repression. However, numerous recent studies, both within the context of defining miRNA/target pairs by direct physical association and by directly assessing this model in vivo in C. elegans have brought this model into question. To test the importance of miRNA 3' pairing in vivo, in a mammalian system, we engineered a mutant murine mir-146a allele in which the 5' half of the mature microRNA retains the sequence of the wild-type mir-146a but the 3ʹ half has been altered to be anti-complementary to the wild-type miR-146a sequence. Mice homozygous or hemizygous for this mutant allele are phenotypically indistinguishable from wild-type controls and do not recapitulate any of the immunopathology previously described for mir-146a-null mice. Our results strongly support the conclusion that 3ʹ pairing is dispensable in the context of the function of a key mammalian microRNA. Gene expression profiling of either stimulated (1 ng/ml LPS for 16 hours) or resting bone marrow derived macrophages (BMDMs) from miR-146 wild-type (WT), knock-out (KO) and non-seed swap (3'F) mice, in triplicate.

长期以来占据主流的模型认为，微小RNA（microRNA）的“种子区”（核苷酸2至8位）通常足以介导靶标识别与基因沉默。然而，近期多项研究——无论是通过直接物理结合来界定miRNA/靶标配对，还是在秀丽隐杆线虫（C. elegans）体内直接验证该模型——均对这一模型提出了质疑。为在哺乳动物系统体内验证miRNA 3'端配对的重要性，我们构建了突变型小鼠mir-146a等位基因：成熟微小RNA的5'端保留野生型mir-146a的序列，而3'端被改造为与野生型miR-146a序列反向互补。携带该突变等位基因的纯合子与杂合子小鼠，其表型与野生型对照组无显著差异，且未重现此前报道的mir-146a敲除小鼠的免疫病理表型。本研究结果有力支持了如下结论：在关键哺乳动物微小RNA的功能语境中，3'端配对并非必需。本数据集包含来自miR-146野生型（WT）、敲除型（KO）以及非种子区互换（3'F）小鼠的骨髓衍生巨噬细胞（BMDMs）的基因表达谱，这些细胞分别经1 ng/ml脂多糖（LPS）刺激16小时，或处于静息状态，每组设置三次生物学重复。