Loss of CPSF6 causes developmental disease via bimodal changes in polyadenylation site usage and protein expression [Human]

Most pre-messenger RNA (pre-mRNA) undergo extensive processing to create distinct transcripts from the same gene. One of these processes, alternative polyadenylation, involves over twenty proteins to bind and cleave the pre-mRNA at poly(A) sites that can lie within the 3' UTR, introns, or exons; this can modulate protein function, but the effect of choosing a site internal to the gene vs. within the 3' UTR remains unclear. Here we show that reduced expression of CPSF6, one of the proteins involved in site selection, derails development in both humans and zebrafish by causing a bidirectional shift in poly(A) site usage. CPSF6 insufficiency favors the use of intronic poly(A) sites in neuronal genes, reducing mRNA and protein abundance, but promotes 3' UTR site usage in cardiovascular and skeletal genes, upregulating mRNA and protein.These data thus provide a long-sought link between APA and gene expression and shows that poly(A) site selection influences development. Overall design: Comparative analysis of alternative polyadenylation using polyA-click seq (PAC-seq) on CPSF6 patient derived fibroblasts carrying CPSF6 heterozygous deletion or CPSF6 de novo missense mutation (D535V), compared to fibrobrasts from healthy donors (ATCC CRL-2097, CRL-2091, CRL-2076)

绝大多数前信使RNA（pre-mRNA）需经历广泛的加工过程，才能从同一基因产生不同的转录本。可变聚腺苷酸化（alternative polyadenylation, APA）是这类加工过程之一，该过程需二十余种蛋白质结合并在前信使RNA的poly(A)位点处进行切割，这类位点可位于3'非翻译区（3' UTR）、内含子或外显子中；可变聚腺苷酸化可调控蛋白质功能，但选择基因内部位点与3' UTR内位点所产生的效应仍不明确。
本研究证实，作为poly(A)位点选择相关蛋白之一的CPSF6，其表达水平降低会通过引发poly(A)位点使用的双向偏移，破坏人类与斑马鱼的正常发育。CPSF6功能不全倾向于使神经元基因优先使用内含子区域的poly(A)位点，从而降低其mRNA与蛋白质的丰度；而在心血管与骨骼基因中则会促进3' UTR区域poly(A)位点的使用，进而上调mRNA与蛋白质的表达水平。上述数据为可变聚腺苷酸化与基因表达之间长期以来备受追寻的关联提供了直接证据，并证实poly(A)位点选择会对发育过程产生影响。
整体实验设计：采用polyA点击测序（polyA-click seq, PAC-seq）技术，对携带CPSF6杂合缺失或新发错义突变（D535V）的患者来源成纤维细胞进行可变聚腺苷酸化比较分析，并以健康供体成纤维细胞（ATCC CRL-2097、CRL-2091、CRL-2076）作为对照。