Department of Genetics, The Hebrew University of Jerusalem, Jerusalem Israel.
Translational and Functional Genomics Branch, National Human Genome Research Institute, NIH, Bethesda, MD USA.
RNA Biol. 2022;19(1):333-352. doi: 10.1080/15476286.2021.2020455. Epub 2021 Dec 31.
Latent 5' splice sites, not normally used, are highly abundant in human introns, but are activated under stress and in cancer, generating thousands of nonsense mRNAs. A previously proposed mechanism to suppress latent splicing was shown to be independent of NMD, with a pivotal role for initiator-tRNA independent of protein translation. To further elucidate this mechanism, we searched for nuclear proteins directly bound to initiator-tRNA. Starting with UV-crosslinking, we identified nucleolin (NCL) interacting directly and specifically with initiator-tRNA in the nucleus, but not in the cytoplasm. Next, we show the association of ini-tRNA and NCL with pre-mRNA. We further show that recovery of suppression of latent splicing by initiator-tRNA complementation is NCL dependent. Finally, upon nucleolin knockdown we show activation of latent splicing in hundreds of coding transcripts having important cellular functions. We thus propose nucleolin, a component of the endogenous spliceosome, through its direct binding to initiator-tRNA and its effect on latent splicing, as the first protein of a nuclear quality control mechanism regulating splice site selection to protect cells from latent splicing that can generate defective mRNAs.
潜伏的 5' 剪接位点，通常不被使用，在人类内含子中高度丰富，但在应激和癌症中被激活，产生数千种无意义的 mRNA。先前提出的抑制潜伏剪接的机制被证明与 NMD 无关，起始 tRNA 对于蛋白质翻译是独立的，具有关键作用。为了进一步阐明这一机制，我们搜索了直接与起始 tRNA 结合的核蛋白。从 UV 交联开始，我们鉴定了核仁素（NCL）在核内直接特异性地与起始 tRNA 相互作用，但不在细胞质中。接下来，我们展示了 ini-tRNA 和 NCL 与前体 mRNA 的关联。我们进一步表明，起始 tRNA 的补充恢复了对潜伏剪接的抑制，这依赖于 NCL。最后，在核仁素敲低后，我们发现数百个具有重要细胞功能的编码转录物中的潜伏剪接被激活。因此，我们提出核仁素，作为内源性剪接体的一部分，通过其与起始 tRNA 的直接结合及其对潜伏剪接的影响，作为一种核质量控制机制的第一个蛋白，调节剪接位点选择，以保护细胞免受可能产生有缺陷的 mRNA 的潜伏剪接。
