Evidence for cooperative tandem binding of hnRNP C RRMs in mRNA processing
Evidence for cooperative tandem binding of hnRNP C RRMs in mRNA processing
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DOI：
10.1261/rna.052373.115
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发表时间：
2015-11-01
期刊：
影响因子：
4.5
通讯作者：
Maris, Christophe
中科院分区：
文献类型：
--
作者：
Cienikova, Zuzana;Jayne, Sandrine;Maris, Christophe
人hnRNP C是参与mRNA成熟的普遍存在的细胞蛋白。最近，我们已经表明，这种蛋白质特异性地识别尿苷（U）五聚体通过其单一的RNA识别基序（RRM）。然而，hnRNP C的大部分天然RNA靶标由长得多的连续尿苷段组成。为了了解这些延伸的位点是如何被识别的，我们研究了RRM与8-11个碱基的U-束的结合。对unr（N-ras上游）mRNA内部翻译激活的体内研究表明，hnRNP C依赖性IRES激活需要整个hnRNP C结合位点UC（U）（8）的保守。该测定进一步表明hnRNP C单体之间的协同相互作用，取决于蛋白质的寡聚化能力。体外光谱和热力学分析表明，分离的RRM与（U）（11）寡聚体结合为二聚体。三元双RRM/RNA复合物的结构建模还表明，两个RRM拷贝可以容纳在规范序列UC（U）上（8）。建议的串联RRM结合与全长hnRNP C的扩展U-tracts的转录组范围内的识别非常一致，其显示出与正合作RRM二聚体结合模型一致的交联模式。
The human hnRNP C is a ubiquitous cellular protein involved in mRNA maturation. Recently, we have shown that this protein specifically recognizes uridine (U) pentamers through its single RNA recognition motif (RRM). However, a large fraction of natural RNA targets of hnRNP C consists of much longer contiguous uridine stretches. To understand how these extended sites are recognized, we studied the binding of the RRM to U-tracts of 8-11 bases. In vivo investigation of internal translation activation of unr (upstream of N-ras) mRNA indicates that the conservation of the entire hnRNP C binding site, UC(U)(8), is required for hnRNP C-dependent IRES activation. The assays further suggest a synergistic interplay between hnRNP C monomers, dependent on the protein's ability to oligomerize. In vitro spectroscopic and thermodynamic analyses show that isolated RRMs bind to (U)(11) oligomers as dimers. Structural modeling of a ternary double-RRM/RNA complex indicates additionally that two RRM copies can be accommodated on the canonical sequence UC(U)(8). The proposed tandem RRM binding is in very good agreement with the transcriptome-wide recognition of extended U-tracts by full-length hnRNP C, which displays a cross-linking pattern consistent with a positively cooperative RRM dimer binding model.