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Posttranscriptional Regulation of 14q32 MicroRNAs by the CIRBP and HADHB during Vascular Regeneration after Ischemia
Molecular Therapy - Nucleic Acids ( IF 6.5 ) Pub Date : 2018-12-06 , DOI: 10.1016/j.omtn.2018.11.017
Angela Downie Ruiz Velasco 1 , Sabine M J Welten 2 , Eveline A C Goossens 2 , Paul H A Quax 2 , Juri Rappsilber 3 , Gracjan Michlewski 4 , A Yaël Nossent 5
Affiliation  

After induction of ischemia in mice, 14q32 microRNAs are regulated in three distinct temporal patterns. These expression patterns, as well as basal expression levels, are independent of the microRNA genes’ order in the 14q32 locus. This implies that posttranscriptional processing is a major determinant of 14q32 microRNA expression. Therefore, we hypothesized that RNA binding proteins (RBPs) regulate posttranscriptional processing of 14q32, and we aimed to identify these RBPs. To identify proteins responsible for this posttranscriptional regulation, we used RNA pull-down SILAC mass spectrometry (RP-SMS) on selected precursor microRNAs. We observed differential binding of cold-inducible RBP (CIRBP) and hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit beta (HADHB) to the precursors of late-upregulated miR-329-3p and unaffected miR-495-3p. Immunohistochemical staining confirmed expression of both CIRBP and HADHB in the adductor muscle of mice. Expression of both CIRBP and HADHB was upregulated after hindlimb ischemia in mice. Using RBP immunoprecipitation experiments, we showed specific binding of CIRBP to pre-miR-329 but not to pri-miR-329. Finally, using CRISPR/Cas9, we generated HADHB−/− 3T3 cells, which display reduced expression of miR-329 and miR-495 but not their precursors. These data suggest a novel role for CIRBP and HADHB in posttranscriptional regulation of 14q32 microRNAs.



中文翻译:

缺血后血管再生过程中 CIRBP 和 HADHB 对 14q32 MicroRNAs 的转录后调控

在小鼠中诱导缺血后,14q32 microRNAs 以三种不同的时间模式进行调节。这些表达模式以及基础表达水平与 14q32 基因座中 microRNA 基因的顺序无关。这意味着转录后加工是 14q32 microRNA 表达的主要决定因素。因此,我们假设 RNA 结合蛋白 (RBP) 调节 14q32 的转录后加工,我们旨在识别这些 RBP。为了鉴定负责这种转录后调控的蛋白质,我们在选定的前体 microRNA 上使用了 RNA 下拉 SILAC 质谱 (RP-SMS)。我们观察到冷诱导 RBP (CIRBP) 和羟酰基辅酶 A 脱氢酶三功能多酶复合物亚基 β (HADHB) 与晚期上调的 miR-329-3p 和未受影响的 miR-495-3p 前体的差异结合。免疫组织化学染色证实了 CIRBP 和 HADHB 在小鼠内收肌中的表达。小鼠后肢缺血后 CIRBP 和 HADHB 的表达均上调。使用 RBP 免疫沉淀实验,我们显示 CIRBP 与 pre-miR-329 特异性结合,但不与 pri-miR-329 特异性结合。最后,使用 CRISPR/Cas9,我们生成了 我们展示了 CIRBP 与 pre-miR-329 的特异性结合,而不是与 pri-miR-329 的特异性结合。最后,使用 CRISPR/Cas9,我们生成了 我们展示了 CIRBP 与 pre-miR-329 的特异性结合,而不是与 pri-miR-329 的特异性结合。最后,使用 CRISPR/Cas9,我们生成了HADHB -/- 3T3 细胞,其表现出 miR-329 和 miR-495 的表达降低,但它们的前体没有。这些数据表明 CIRBP 和 HADHB 在 14q32 microRNA 的转录后调控中具有新的作用。

更新日期:2018-12-06
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