Mechanism to regulate the length of small silencing RNAs

摘要 转录后基因沉默（PTGS）由小的沉默RNA介导，如~22-24 nt 由Dicer酶产生的微小RNA（miRNA）和~21 nt小干扰RNA（siRNA）是 在生物学的各个方面都很重要。制造具有适当长度的小RNA对于它们的生长至关重要。 功能协调发展的长度不正确的小RNA，如果Dicer选择不正确的切割，则可能产生 前体RNA中的位点，可能会对细胞产生有害影响。因此，了解分子 由Dicer产生的小沉默RNA的长度被确定的机制， 监管的意义重大，也是本提案研究的目标。 Drosophila Dicer-1从pre-miRNAs中产生约22-24 nt的miRNAs，而Dicer-2精确地从pre-miRNAs中产生约22-24 nt的miRNAs。 来自病毒和转座子的长dsRNA的~21 nt siRNA。我们最近报道说， 果蝇Dicer-1和哺乳动物Dicer产生的miRNA长度可以通过其结合而改变 伴侣蛋白Loquacious-PB（Loqs-PB）在果蝇和TRBP在哺乳动物中，分别，但没有 他们的替代伙伴Loqs-PA和PACT。然而，Loqs的分子机制- PB/TRBP，而不是Loqs-PA/PACT，改变miRNAs的长度是未知的，我们的目标是更好地 我们将测试Loqs-PB/TRBP，而不是Loqs-PA/PACT，结合 前体miRNA茎中心部分的未配对碱基，并改变它们的构象， 改变miRNA的长度 与具有更宽长度分布（~22-24 nt）的miRNA相反，~21 nt的长度在mRNA中表达。 siRNA更精确。miRNAs和siRNAs之间的长度差异对于它们的表达是重要的。 各自的功能。然而，Drosophila Dicer-2如何能够以如此高的精度产生siRNA， 未知根据我们以前的初步结果，我们将测试假设， Dicer-2的PAZ结构域中的磷酸盐结合口袋对于产生精确的~21 nt起着关键作用 siRNA。我们还假设Dicer-2的C末端dsRNA结合结构域（dsRBD） 另外有助于~21 nt siRNA生产的精确度。我们假设 磷酸盐结合口袋和C末端dsRBD锚定末端单磷酸盐和主体 长dsRNA，从而使RNA沿RNaseIII活性位点沿着精确排列。 为了实现这些目标，我们将使用果蝇遗传学，体外生物化学重组， 高通量测序。这些研究将揭示这些疾病的分子机制。 miRNAs和siRNAs的长度被定义和调节，这将促进我们对miRNAs和siRNAs的理解。 序列特异性转录后基因沉默。

Abstract Post-transcriptional gene silencing (PTGS) mediated by small silencing RNAs such as ~22-24 nt microRNAs (miRNAs) and ~21 nt small interfering RNAs (siRNAs) produced by Dicer enzymes, is important in every aspect of biology. Making small RNAs with an appropriate length is crucial for their functions. Small RNAs with incorrect lengths, which can be produced if Dicer selects incorrect cleavage sites in precursor RNAs, can cause detrimental effects in cells. Thus, understanding the molecular mechanisms by which the length of small silencing RNAs produced by Dicer are determined and regulated is significant and is the goal of research in this proposal. Drosophila Dicer-1 makes ~22-24 nt miRNAs from pre-miRNAs while Dicer-2 precisely produces ~21 nt siRNAs from long dsRNAs derived from virus and transposon. We recently reported that the miRNA length produced by Drosophila Dicer-1 and mammalian Dicer can be changed by its binding partner proteins Loquacious-PB (Loqs-PB) in Drosophila and TRBP in mammals, respectively, but not by their alternative partners Loqs-PA and PACT. However, the molecular mechanism by which Loqs- PB/TRBP, but not Loqs-PA/PACT, changes the length of miRNAs is unknown, and we will aim to better understand it. We will test the hypothesis that Loqs-PB/TRBP, but not Loqs-PA/PACT, binds the unpaired bases at the central part of the pre-miRNA stem and changes their conformation in order to change the miRNA length. In contrast to miRNAs that have a broader length distribution (~22-24 nt), the length of ~21 nt siRNAs is more precise. The difference in the length between miRNAs and siRNAs is important for their respective functions. However, how Drosophila Dicer-2 can produce siRNAs with such high precision is unknown. Based on our previous and preliminary results, we will test the hypothesis that the phosphate-binding pocket in the PAZ domain of Dicer-2 plays crucial roles to produce precise ~21 nt siRNAs. We also hypothesize that the C-terminal dsRNA-binding domain (dsRBD) of Dicer-2 additionally contributes to the precision of the ~21 nt siRNA production. We hypothesize that the phosphate-binding pocket and C-terminal dsRBD anchors the terminal monophosphate and body of long dsRNAs, respectively, thereby aligning the RNAs precisely along the RNaseIII active site. To achieve these aims, we will use Drosophila genetics, reconstituted in vitro biochemistry, and high-throughput sequencing. The proposed studies will reveal the molecular mechanisms by which the length of miRNAs and siRNAs are defined and regulated, which will advance our understanding of sequence-specific post-transcriptional gene silencing.