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调控多种转录后过程，包括mRNA剪接、mRNA稳定性及翻译效率，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。交联免疫沉淀结合高通量测序（Crosslinking immunoprecipitation coupled with 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RNA结合蛋白的靶序列，从而解析疾病发病机制的分子基础。然而，由于实验复杂度高、效率低下且耗时较长，且需从极少量RNA中构建测序文库，HITS-CLIP实验方案仍有待进一步优化。本研究通过优化溴脱氧尿苷交联免疫沉淀（BrdU-CLIP）流程，建立了一种更高效、快速且可重复的CLIP实验方法。该方法可使预扩增CLIP测序文库的产量提升10倍，同时因降低了文库扩增所需的PCR循环数，有效降低了CLIP标签测序读段的重复率；此外，文库产量的离散度显著降低，实验周期缩短了2天。利用该优化后的方法，我们验证了核定位RNA结合蛋白异质性核核糖核蛋白U（HNRNPU）对白介素6（IL-6）表达的调控作用：该蛋白可直接结合海拉细胞中IL-6 mRNA的3'非翻译区（3'-UTR），且该相互作用仅在细胞质组分中被检测到，提示细胞质定位的HNRNPU可参与调控mRNA稳定性。该优化方法可实现靶基因的精准鉴定，为研究核质穿梭型RNA结合蛋白的蛋白-RNA互作图谱提供了有效技术手段。