科研动态｜Mol Cell：吴炜祥课题组揭示mRNA修饰m6Am的首个阅读器蛋白

m⁶Am是一种丰富的mRNA修饰，位于转录的第一个核苷酸上，已被证明影响多种疾病。然而，对于m⁶Am的分子功能，学术界仍存在多种相互矛盾的模型，包括mRNA稳定性、mRNA不稳定性、翻译增强以及翻译抑制等。m⁶Am作用机制尚不明确，很大程度上是因为尚未发现能够特异性结合m⁶Am并介导其功能的“阅读器蛋白”。

m⁶Am is an abundant mRNA modification located on the first transcribed nucleotide and has been shown to impact multiple diseases[1,2]. However, multiple studies have reported various conflicting models regarding m6Am’s molecular function, ranging from mRNA stabilization, mRNA destabilization, translation enhancement to translation inhibition. That the mechanistic nature of m⁶Am’s role remains unclear is largely due to the lack of a known m⁶Am ‘reader’ that can bind specifically to m⁶Am to mediate its function.

为了解决这一争议，作者使用定量蛋白质组学筛选出优先结合m⁶Am修饰的蛋白质，最终鉴定出转录终止因子PCF11。值得注意的是，PCF11包含多个约13个氨基酸长度的FEGP基序，被称为“FEGP重复序列”，这些序列对于特异性结合m⁶Am是必需的。FEGP重复序列仅在拥有m⁶Am修饰的物种中保守存在。这表明，PCF11的FEGP基序可能与mRNA的m⁶Am修饰共同进化，使得PCF11能够识别并结合mRNA上的m⁶Am。

To address this controversy, the authors used quantitative proteomics to screen for proteins that preferentially bind to the m⁶Am modification. This yielded the transcriptional terminator PCF11. Notably, PCF11 contains multiple ~13mer FEGP-containing motifs, termed “FEGP-repeats”, which are necessary for specific binding to m⁶Am. These FEGP-repeats are conserved only in species possessing m⁶Am. This suggests that the PCF11 FEGP-repeats domain may have co-evolved with the emergence of mRNA m⁶Am modification to enable PCF11 to recognize and bind m⁶Am on mRNA.

接下来，作者探索了PCF11的已知功能，以更好地理解m⁶Am的分子功能。PCF11的一个已知功能是促进转录提前终止。通过对转录提前终止本和成熟转录终止本的定量分析，作者发现m⁶Am抑制了PCF11介导的转录提前终止。此外，m⁶Am对转录提前终止的抑制作用反过来促进了全长转录以及m⁶Am基因的高表达。值得注意的是，m⁶Am的功能在PCF11低表达的情况下更为显著。这一发现表明，当PCF11低表达时，m⁶Am能够更有效地抑制PCF11的功能。

Next, the authors explored known functions of PCF11 to better understand m⁶Am’s molecular function. One known PCF11 function is to promote premature transcription termination[3]. By quantifying premature and mature transcripts, the authors showed that m⁶Am suppresses premature transcription termination mediated by PCF11. Furthermore, m⁶Am suppression of premature termination can, in turn, promote full-length transcription and greater expression of m⁶Am genes, and this effect is enhanced when PCF11 is depleted. This suggests that m⁶Am is better able to suppress PCF11 when PCF11 is limiting.

最后，作者重点研究了MYCN高表达的小儿神经母细胞瘤。ATRA（全反式维甲酸）常用于神经母细胞瘤患者的维持治疗，但该疗法的作用机制尚不明确，因此深入研究ATRA的作用机制可以揭示为何某些患者会对维甲酸疗法产生耐药性。由于ATRA可以降低PCF11的水平，作者推测此时增强的m⁶Am功能可能在维甲酸治疗中发挥作用。确实，作者发现，m⁶Am的耗竭会导致ATRA处理的神经母细胞瘤继续表现出肿瘤干细胞样特性，从而使细胞对ATRA产生耐药性。该研究为理解基于维甲酸的神经母细胞瘤治疗的耐药性提供了一条新的思路。

Finally, the authors focused on pediatric neuroblastoma, characterized by amplified MYCN expression. All-trans-retinoic acid (ATRA) is used in maintenance therapy for neuroblastoma patients but the mechanistic basis for this therapy’s efficacy is unclear so establishing additional insights about ATRA’s mechanism can reveal why certain patients exhibit retinoid therapy resistance[4]. Since ATRA also reduces PCF11 levels, the authors suspected that the enhanced m⁶Am function here could play a role in retinoid therapy. Indeed, the authors found that m⁶Am depletion causes ATRA-treated neuroblastoma to continue exhibiting tumor stem-like properties, effectively making the cells resistant to ATRA. This study thus provides an alternative avenue to understand resistance to retinoid-based neuroblastoma therapy.

综上所述，这项研究不仅首次鉴定了m⁶Am的特异性阅读蛋白及其功能，还在机制层面提出了不同于常规表观转录组学范式的特例：RNA修饰与其阅读器蛋白结合后会激活该蛋白的功能。例如，N⁶-甲基腺苷（m⁶A）的阅读蛋白YTHDF2与m⁶A结合后，会介导m⁶A修饰的mRNA降解。同样，作者最初预期m⁶Am的结合会激活PCF11的功能。然而，他们发现m⁶Am的结合实际上抑制了PCF11的功能，从而发现了RNA修饰和阅读蛋白之间常见关系的一个特例。

In summary, this study not only identified the specific reader protein of m⁶Am and its function for the first time but also proposed an exception to the conventional paradigms of epitranscriptomics at the mechanistic level: Binding of the reader to the RNA modification induces the reader’s function. For instance, binding of the N⁶-methyladenosine (m⁶A) reader YTHDF2 to m⁶A induces YTHDF2 to direct m⁶A-modified mRNA towards RNA degradation[5]. Likewise, the authors initially expected m⁶Am-binding to induce PCF11’s function. Instead, they find m⁶Am-binding suppresses PCF11’s function, thereby identifying an exception to the common RNA modification-Reader relationships. 

深圳湾实验室特聘研究员吴炜祥为该文通讯作者，深圳湾实验室与香港大学联合培养博士生安会会为该文的第一作者。

Dr. Wee Siong Sho Goh, a Principal Investigator at Shenzhen Bay Laboratory, is the corresponding author of the paper, and Huihui An, a joint PhD student of Shenzhen Bay Laboratory and the University of Hong Kong, is the first author of the paper.

参考文献 References

1. Mauer J, Luo X, Blanjoie A, et al. Reversible methylation of m6Am in the 5′ cap controls mRNA stability. Nature. 2017;541(7637):371-375. doi:10.1038/nature21022

2. Relier S, Ripoll J, Guillorit H, et al. FTO-mediated cytoplasmic m6Am demethylation adjusts stem-like properties in colorectal cancer cell. Nat Commun. 2021;12(1):1716. doi:10.1038/s41467-021-21758-4

3. Kamieniarz-Gdula K, Gdula MR, Panser K, et al. Selective Roles of Vertebrate PCF11 in Premature and Full-Length Transcript Termination. Mol Cell. 2019;74(1):158-172.e9. doi:10.1016/j.molcel.2019.01.027

4. Ogorodnikov A, Levin M, Tattikota S, et al. Transcriptome 3′end organization by PCF11 links alternative polyadenylation to formation and neuronal differentiation of neuroblastoma. Nat Commun. 2018;9(1):5331. doi:10.1038/s41467-018-07580-5

5. Zaccara S, Jaffrey SR. A Unified Model for the Function of YTHDF Proteins in Regulating m6A-Modified mRNA. Cell. 2020;181(7):1582-1595.e18. doi:10.1016/j.cell.2020.05.012