Calabrese J Mauro, Sharp Phillip A
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02144, USA.
RNA. 2006 Dec;12(12):2092-102. doi: 10.1261/rna.224606. Epub 2006 Oct 24.
Studies of mammalian RNA interference (RNAi) have focused largely on the actions of microRNAs; however, in other organisms, endogenous short-interfering RNAs (siRNAs) are involved in silencing processes. To date, similar molecules have been difficult to characterize in mammalian cells. P19 is a plant suppressor of RNA silencing that binds with high affinity to siRNAs. Here, the short RNAs bound by P19 in mouse embryonic stem (ES) cells have been characterized. We show that P19 selectively immunoprecipitates endogenous short RNAs from ES cells. Cloning of immunoprecipitated RNA reveals a strong selection for short RNAs that are exact matches to ribosomal RNA (rRNA), with particular short rRNA species highly enriched in P19 immunoprecipitates. Complementary strands to the enriched rRNAs were not cloned, which was surprising because P19 was previously thought to bind only siRNAs. We show that P19 binds tightly to a noncanonical dsRNA substrate comprised of a short RNA annealed to a much longer partner, such that the double-stranded region between the two is 19 base pairs long. Binding to similar endogenous species might explain the association of P19 with short rRNAs in ES cells. Finally, we show that the P19-enriched rRNAs are not involved in canonical RNAi, as they exist in the absence of Dicer and do not function as post-transcriptional gene silencers. Our results support the previous observation that endogenous siRNAs are not abundant molecules in mouse ES cells.
对哺乳动物RNA干扰（RNAi）的研究主要集中在微小RNA的作用上；然而，在其他生物体中，内源性短干扰RNA（siRNA）参与了沉默过程。迄今为止，类似的分子在哺乳动物细胞中很难被鉴定。P19是一种RNA沉默的植物抑制因子，它能与siRNA高亲和力结合。在此，已对小鼠胚胎干细胞（ES）中与P19结合的短RNA进行了鉴定。我们发现P19能从ES细胞中选择性地免疫沉淀内源性短RNA。对免疫沉淀RNA的克隆显示，对与核糖体RNA（rRNA）完全匹配的短RNA有强烈的选择，特定的短rRNA种类在P19免疫沉淀中高度富集。未克隆到富集rRNA的互补链，这很令人惊讶，因为P19以前被认为只结合siRNA。我们发现P19紧密结合一种非经典的双链RNA底物，该底物由一条短RNA与一条长得多的互补链退火形成，使得两者之间的双链区域为19个碱基对长。与类似内源性种类的结合可能解释了P19在ES细胞中与短rRNA的关联。最后，我们发现P19富集的rRNA不参与经典的RNAi，因为它们在没有Dicer的情况下存在，并且不作为转录后基因沉默子发挥作用。我们的结果支持了之前的观察，即内源性siRNA在小鼠ES细胞中不是丰富的分子。
