Identification of mRNAs bound and regulated by human LIN28 proteins and molecular requirements for RNA recognition
Identification of mRNAs bound and regulated by human LIN28 proteins and molecular requirements for RNA recognition
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DOI：
10.1261/rna.036491.112
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发表时间：
2013-05-01
期刊：
影响因子：
4.5
通讯作者：
Tuschl, Thomas
中科院分区：
文献类型：
--
作者：
Hafner, Markus;Max, Klaas E. A.;Tuschl, Thomas
人类Lin28a和Lin28b是在动物体内保守的RNA结合蛋白，在发育和干细胞重编程过程中起着重要作用。我们在HEK293细胞中使用了可光激活的核糖核苷增强的交联和免疫沉淀(PAR-CLIP)，并在类似于3‘UTR和CDS的9500个位点上发现了一组大致重叠的类似3000个mRNAs。在体外和体内，LIN28优先结合含有富含尿苷的元件和一个或多个侧翼鸟苷的单链RNA，当嵌入到预测的二级结构中时，似乎能够破坏碱基配对来访问这些元件。在HEK293细胞中，LIN28蛋白结合温和稳定了靶mRNAs并增加了蛋白丰度。最主要的目标是它自己的mRNAs以及其他限制性商业惯例和细胞周期调节因子的mRNAs。LIN28蛋白水平的变化也对一些但不是所有let-7miRNA家族成员的丰度进行了负调控，表明let-7前体与LIN28蛋白具有序列特异性结合，并与细胞质miRNA生物发生因子竞争。
Human LIN28A and LIN28B are RNA-binding proteins (RBPs) conserved in animals with important roles during development and stem cell reprogramming. We used Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) in HEK293 cells and identified a largely overlapping set of similar to 3000 mRNAs at similar to 9500 sites located in the 3' UTR and CDS. In vitro and in vivo, LIN28 preferentially bound single-stranded RNA containing a uridine-rich element and one or more flanking guanosines and appeared to be able to disrupt base-pairing to access these elements when embedded in predicted secondary structure. In HEK293 cells, LIN28 protein binding mildly stabilized target mRNAs and increased protein abundance. The top targets were its own mRNAs and those of other RBPs and cell cycle regulators. Alteration of LIN28 protein levels also negatively regulated the abundance of some but not all let-7 miRNA family members, indicating sequence-specific binding of let-7 precursors to LIN28 proteins and competition with cytoplasmic miRNA biogenesis factors.