hnRNPA is a novel regulator of a mitochondrial microprotein expression. hnRNPA is a novel regulator of a mitochondrial microprotein expression

Mitochondrial-derived peptides (MDPs) are microproteins that are encoded by short open reading frames within the mitochondrial genome. Several MDPs have been identified in recent years; however, the transcriptional and translational regulation of MDPs remain poorly understood. Here we discovered an RNA binding protein of an MDP called humanin. By conducting RNA pull-down assays from mitochondrial extracts, we confidently pinpointed three humanin RNA binding partners that regulate translation: heterogeneous nuclear ribonucleoproteins A1 (hnRNPA1), hnRNPA2, and hnRNPA3. We also identified a novel post-translational modification in the form of succinylation in hnRNPA1 at lysine residue K-106. Succinylation of hnRNPA1 plays an important role in mitochondrial translocation and binding affinity of hnRNPA1 to humanin mRNA. Furthermore, hnRNPA1 and humanin are highly co-expressed in prostate cancer. We provide the first evidence of mRNA binding proteins modulating the expression of a mitochondrial microprotein. This study articulates important new roles of ribonucleoproteins in modulating microprotein expression in mitochondria. Overall design: Examine RNAs bound to hnRNPA1 in cytosol, mitochondria, and total cell lysates by using RIPseq. Examine differential gene expression by hnRNPA1 knockdown using RNA sequencing

线粒体衍生肽（Mitochondrial-derived peptides, MDPs）是一类由线粒体基因组内短开放阅读框编码的微蛋白。近年来已有多种MDPs被鉴定，但目前对MDPs的转录与翻译调控机制仍不甚明晰。本研究发现了一种名为人类素（humanin）的MDP的RNA结合蛋白。通过对线粒体提取物开展RNA下拉实验，我们可靠地鉴定出3种可调控翻译过程的人类素RNA结合伴侣：异质性核核糖核蛋白A1（heterogeneous nuclear ribonucleoproteins A1, hnRNPA1）、hnRNPA2及hnRNPA3。我们还在hnRNPA1的赖氨酸残基K-106位点发现了一种新型琥珀酰化翻译后修饰形式。hnRNPA1的琥珀酰化在其线粒体易位及与人类素mRNA的结合亲和力中发挥重要作用。此外，hnRNPA1与人类素在前列腺癌组织中呈现高度共表达。本研究首次证实了mRNA结合蛋白可调控线粒体微蛋白的表达，阐明了核糖核蛋白在调控线粒体微蛋白表达中的重要新功能。实验整体设计：通过RNA免疫沉淀测序（RNA Immunoprecipitation Sequencing, RIPseq）检测细胞质、线粒体及全细胞裂解液中与hnRNPA1结合的RNA；通过RNA测序分析hnRNPA1敲低后的差异基因表达。