Mechanism to regulate the length of small silencing RNAs

调节小沉默RNA长度的机制

• 批准号: 9980435
• 负责人: Ryuya Fukunaga
• 金额: $ 34.39万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-08 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9980435
• 关键词: Active Sites; Affect; Binding; Biochemistry; Biological; Biological Testing; Biology; C-terminal; Cells; Dicer Enzyme; Disease; Double-Stranded RNA; Drosophila genus; Gene Expression; Genes; Genetic; Goals; Health; Healthcare; Heart Diseases; High-Throughput Nucleotide Sequencing; Human; In Vitro; Lead; Length; Macular degeneration; Malignant Neoplasms; Mammals; Measurement; Mediating; Messenger RNA; MicroRNAs; Modeling; Molecular; Molecular Conformation; Mutation; N-terminal; NCOA6 gene; Pathway interactions; Physiological; Play; Production; Protein Isoforms; Proteins; RNA; RNA Interference; RNA Precursors; Regulation; Reporting; Research; Role; Seeds; Site; Small Interfering RNA; Small RNA; Structure; Substrate Specificity; Testing; Virus; base; crosslink; deafness; design; fly; helicase; human disease; in vivo; innovation; inorganic phosphate; mutant; reconstitution; stem

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.

摘要 ~22-24nt等小分子沉默RNA介导的转录后基因沉默(PTGS) Diller酶产生的microRNAs(MiRNAs)和~21ntsiRNA(SiRNAs)，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 功能。如果Dier选择了错误的切割，可能会产生长度错误的小RNA 前体RNA中的位点，会对细胞造成有害影响。因此，理解分子 迪格尔产生的小分子沉默RNA的长度被确定的机制和 监管具有重要意义，也是本提案的研究目标。 1号果蝇从前miRNAs中产生~22-24个核苷酸的miRNA，而2号果蝇则精确地制造 从病毒和转座子来源的长dsRNA中获得~21nT的siRNA。我们最近报道说， 果蝇和哺乳动物产生的miRNA长度可以通过其结合而改变 果蝇中的伙伴蛋白Loqs-PB(Loqs-PB)和哺乳动物中的TRBP，但不是 由他们的替代合作伙伴Loqs-PA和PACT提供。然而，LOQS-1的分子机制。 PB/TRBP，但不是Loqs-PA/PACT，改变了miRNAs的长度是未知的，我们将致力于更好地 理解它。我们将检验这样的假设，即LOQS-PB/TRBP，而不是LOQS-PA/PACT，结合了 在前miRNA茎中心部分的未配对碱基，并改变它们的构象，以便 更改miRNA长度。 与具有更广泛的长度分布(~22-24个核苷酸)的miRNAs相比，~21个核苷酸的长度 SiRNAs更精确。MiRNAs和siRNAs之间的长度差异对它们的 各自的职能。然而，果蝇DICER-2是如何产生如此高精度的siRNA的 未知。基于我们之前和初步的结果，我们将测试假设 DICER-2的PAZ结构域上的磷酸结合口袋在产生精确的~21nT中起着至关重要的作用 SiRNA。我们还假设DICER-2的C末端dsRNA结合域(DsRBD) 此外，还有助于~21nt siRNA生产的精确度。我们假设 磷酸盐结合口袋和C-末端dsRBD锚定末端一磷酸和 分别是长的dsRNAs，从而使RNAs精确地沿着RNaseIII活性位点排列。 为了实现这些目标，我们将使用在体外生物化学中重组的果蝇遗传学，以及 高通量测序。这项拟议中的研究将揭示 对miRNAs和siRNAs的长度进行了定义和调节，这将促进我们对 序列特异性转录后基因沉默。