Starega-Roslan Julia, Galka-Marciniak Paulina, Krzyzosiak Wlodzimierz J
Department of Molecular Biomedicine, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, Poznan 61-704, Poland.
Department of Molecular Biomedicine, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, Poznan 61-704, Poland
Nucleic Acids Res. 2015 Dec 15;43(22):10939-51. doi: 10.1093/nar/gkv968. Epub 2015 Sep 30.
The ribonuclease Dicer excises mature miRNAs from a diverse group of precursors (pre-miRNAs), most of which contain various secondary structure motifs in their hairpin stem. In this study, we analyzed Dicer cleavage in hairpin substrates deprived of such motifs. We searched for the factors other than the secondary structure, which may influence the length diversity and heterogeneity of miRNAs. We found that the nucleotide sequence at the Dicer cleavage site influences both of these miRNA characteristics. With regard to cleavage mechanism, we demonstrate that the Dicer RNase IIIA domain that cleaves within the 3' arm of the pre-miRNA is more sensitive to the nucleotide sequence of its substrate than is the RNase IIIB domain. The RNase IIIA domain avoids releasing miRNAs with G nucleotide and prefers to generate miRNAs with a U nucleotide at the 5' end. We also propose that the sequence restrictions at the Dicer cleavage site might be the factor that contributes to the generation of miRNA duplexes with 3' overhangs of atypical lengths. This finding implies that the two RNase III domains forming the single processing center of Dicer may exhibit some degree of flexibility, which allows for the formation of these non-standard 3' overhangs.
核糖核酸酶Dicer从多种前体（前体miRNA）中切除成熟的miRNA，其中大多数在其发夹茎中含有各种二级结构基序。在本研究中，我们分析了缺乏此类基序的发夹底物中的Dicer切割。我们寻找除二级结构之外可能影响miRNA长度多样性和异质性的因素。我们发现Dicer切割位点的核苷酸序列会影响miRNA的这两个特征。关于切割机制，我们证明在pre-miRNA的3'臂内切割的Dicer RNase IIIA结构域比RNase IIIB结构域对其底物的核苷酸序列更敏感。RNase IIIA结构域避免释放带有G核苷酸的miRNA，并且更倾向于生成5'端带有U核苷酸的miRNA。我们还提出，Dicer切割位点的序列限制可能是导致生成具有非典型长度3'突出端的miRNA双链体的因素。这一发现意味着构成Dicer单一加工中心的两个RNase III结构域可能表现出一定程度的灵活性，这使得这些非标准3'突出端得以形成。
