Transcriptome-wide identification of LARP4B binding sites in HEK293 cells. Homo sapiens

mRNAs are key molecules in gene expression and subject to diverse regulatory events. Regulation is accomplished by distinct sets of trans-acting factors that interact with mRNAs and form defined mRNA-protein complexes (mRNPs). The resulting “mRNP code” determines the fate of any given mRNA and thus controlling gene expression at the post-transcriptional level. The La-related protein 4B (LARP4B) belongs to an evolutionarily conserved family of RNA binding proteins characterized by the presence of a La-module implicated in direct RNA binding. Biochemical experiments have shown previously direct interactions of LARP4B with factors of the translation machinery. This finding along with the observation of an association with actively translating ribosomes suggested that LARP4B is a factor contributing to the mRNP code. To gain insight into the function of LARP4B in vivo we tested its mRNA association at the transcriptome level and its impact on the proteome. PAR-CLIP analyses allowed us to identify the in vivo RNA targets of LARP4B. We show that LARP4B binds to a distinct set of cellular mRNAs by contacting their 3´UTRs. Biocomputational analysis combined with in vitro binding assays identified the LARP4B binding motif on mRNA targets. The reduction of cellular LARP4B levels leads to a marked destabilization of its mRNA targets and consequently their reduced translation. Our data identify LARP4B as a component of the mRNP code that influences the expression of its mRNA targets by affecting their stability. Overall design: PAR-CLIP experiments for LARP4B in HEK293cells in two biological replicates

信使RNA（mRNA）是基因表达中的关键分子，且受到多种调控事件的作用。此类调控由多类不同的反式作用因子完成，这些因子可与mRNA结合并形成特定的mRNA-蛋白质复合物（mRNPs）。由此形成的"mRNP编码"可决定任意特定mRNA的命运，从而在转录后水平调控基因表达。La相关蛋白4B（LARP4B）属于一类进化保守的RNA结合蛋白家族，该家族以存在参与直接RNA结合的La结构域（La-module）为特征。既往生化实验已证实，LARP4B可与翻译机器的多种因子发生直接相互作用。这一发现，结合其与活跃翻译核糖体存在关联的观测结果，表明LARP4B是参与构建mRNP编码的调控因子之一。为深入解析LARP4B在体内的功能，我们在转录组层面检测了其与mRNA的结合情况，并分析了其对蛋白质组的影响。光辅助交联免疫沉淀（PAR-CLIP）实验使我们得以鉴定LARP4B在体内的RNA靶标。我们证实，LARP4B可通过结合细胞mRNA的3'非翻译区（3'UTR），与一类特定的细胞mRNA相结合。结合生物计算分析与体外结合实验，我们鉴定出了mRNA靶标上的LARP4B结合基序。细胞内LARP4B水平的降低会导致其mRNA靶标显著不稳定，进而使这些靶标的翻译效率下降。本研究数据证实，LARP4B是mRNP编码的组成成分之一，可通过影响其mRNA靶标的稳定性来调控这些靶标的表达。实验整体设计：在HEK293细胞中针对LARP4B开展PAR-CLIP实验，设置两个生物学重复。