Analysis of the nucleocytoplasmic shuttling RNA-binding protein HNRNPU using optimized HITS-CLIP method

RNA-binding proteins (RBPs) control many types of post-transcriptional regulation, including mRNA splicing, mRNA stability, and translational efficiency, by directly binding to their target RNAs and their mutation and dysfunction are often associated with several human neurological diseases and tumorigenesis. Crosslinking immunoprecipitation (CLIP), coupled with high-throughput sequencing (HITS-CLIP), is a powerful technique for investigating the molecular mechanisms underlying disease pathogenesis by comprehensive identification of RBP target sequences at the transcriptome level. However, HITS-CLIP protocol is still required for some optimization due to experimental complication, low efficiency and time-consuming, whose library has to be generated from very small amounts of RNAs. Here we improved a more efficient, rapid, and reproducible CLIP method by optimizing BrdU-CLIP. Our protocol produced a 10-fold greater yield of pre-amplified CLIP library, which resulted in a low duplicate rate of CLIP-tag reads because the number of PCR cycles required for library amplification was reduced. Variance of the yields was also reduced, and the experimental period was shortened by 2 days. Using this, we validated IL-6 expression by a nuclear RBP, HNRNPU, which directly binds the 3’-UTR of IL-6 mRNA in HeLa cells. Importantly, this interaction was only observed in the cytoplasmic fraction, suggesting a role of cytoplasmic HNRNPU in mRNA stability control. This optimized method enables us to accurately identify target genes and provides a snapshot of the protein-RNA interactions of nucleocytoplasmic shuttling RBPs.

RNA结合蛋白（RNA-binding proteins, RBPs）可通过直接结合靶RNA，调控包括mRNA剪接、mRNA稳定性及翻译效率在内的多种转录后调控过程；其突变与功能异常常与多种人类神经系统疾病及肿瘤发生密切相关。交联免疫沉淀（crosslinking immunoprecipitation, CLIP）结合高通量测序（high-throughput sequencing, HITS-CLIP）是一项强大的研究技术，可在转录组水平全面鉴定RBP的靶序列，从而解析疾病发病的分子机制。然而，由于实验操作复杂、效率低下且耗时较长，HITS-CLIP的实验流程仍需进一步优化，且其文库构建需从极少量RNA中完成。本研究通过优化BrdU-CLIP技术，改良得到一种更高效、快速且可重复的CLIP实验方法。该方案使预扩增CLIP文库的产出量提升10倍，且因减少了文库扩增所需的PCR循环数，显著降低了CLIP标签读段的重复率。同时，实验产出的变异度大幅降低，实验周期缩短了2天。利用该优化方法，我们验证了核定位RNA结合蛋白异质性核核糖核蛋白U（heterogeneous nuclear ribonucleoprotein U, HNRNPU）对白细胞介素6（IL-6）基因的表达调控作用：HNRNPU可直接结合HeLa细胞中IL-6 mRNA的3’-UTR，且该相互作用仅在细胞质组分中被检测到，提示细胞质HNRNPU在mRNA稳定性调控中发挥功能。该优化后的方法可帮助我们精准鉴定靶基因，并为核质穿梭型RBPs的蛋白-RNA互作提供了快照式表征。