Oppositional Poly(A) Tail Length Regulation by FMRP and CPEB1

Poly(A) tail length is regulated in both the nucleus and cytoplasm. One factor that controls polyadenylation in the cytoplasm is CPEB1, an RNA binding protein that associates with specific mRNA 3'UTR sequences to tether enzymes that add and remove poly(A). Two of these enzymes, the noncanonical poly(A) polymerases Gld2 (TENT2, PAPD4, Wispy) and Gld4 (TENT4B, PAPD5, TRF4, TUT3), interact with CPEB1 to extend poly(A). To identify additional RNA binding proteins that might anchor Gld4 to RNA, we expressed double tagged Gld4 in U87MG cells, which was used for sequential immunoprecipitation and elution followed by mass spectrometry. We identified several RNA binding proteins that co-precipitated with Gld4, among which was FMRP. To assess whether FMRP regulates polyadenylation, we performed TAIL-seq from WT and FMRP-deficient HEK293 cells. Surprisingly, loss of FMRP resulted in an overall increase in poly(A), which was also observed for several specific mRNAs. Conversely, loss of CPEB1 elicited an expected decrease in poly(A), which was examined in cultured neurons. We also examined polyadenylation in wild type (WT) and FMRP-deficient mouse brain cortex by direct RNA Nanopore sequencing, which identified RNAs with both increased and decreased poly(A). Our data show that FMRP has a role in mediating poly(A) tail length, which adds to its repertoire of RNA regulation. Identification of Poly(A) tail length for all transcripts in WT and FMRP Knock-out mice using direct RNA sequencing (PromethION)Poly(A)尾(Poly(A) tail)长度在细胞核与细胞质中均受到调控。其中，调控细胞质多聚腺苷酸化的关键因子之一为CPEB1，这是一类可结合特定mRNA 3'非翻译区(3'UTR)序列的RNA结合蛋白(RNA binding protein)，能够招募负责添加或移除Poly(A)尾的酶类。此类酶中有两种属于非典型Poly(A)聚合酶(Poly(A) polymerase)，分别为Gld2（别名TENT2、PAPD4、Wispy）与Gld4（别名TENT4B、PAPD5、TRF4、TUT3），二者均可与CPEB1相互作用以延伸Poly(A)尾。为鉴定能够将Gld4锚定至RNA的其他RNA结合蛋白，我们在U87MG细胞中表达了带双标签的Gld4，随后通过依次免疫沉淀、洗脱结合质谱分析开展实验。最终筛选到多个与Gld4共沉淀的RNA结合蛋白，其中便包括脆性X智力低下蛋白(FMRP, Fragile X Mental Retardation Protein)。为评估FMRP是否调控多聚腺苷酸化过程，我们分别对野生型(WT, wild type)与FMRP缺陷型HEK293细胞开展尾端测序(TAIL-seq)。令人意外的是，FMRP缺失会导致整体Poly(A)尾长度增加，该现象在数种特异性mRNA中也可观察到。与之相反，CPEB1缺失则会引发预期中的Poly(A)尾长度降低，这一结果在培养的神经元中得到验证。我们还通过直接RNA纳米孔测序技术，对野生型与FMRP缺陷型小鼠大脑皮层的多聚腺苷酸化水平进行了检测，该测序手段鉴定出了Poly(A)尾长度发生上调与下调的RNA分子。本研究数据表明，FMRP在介导Poly(A)尾长度调控中发挥重要作用，进一步拓展了其在RNA调控领域的功能谱系。此外，本研究利用直接RNA测序技术(PromethION平台)，完成了野生型与FMRP敲除(Knock-out, KO)小鼠体内所有转录本的Poly(A)尾长度鉴定。