A novel role for nucleolin in splice site selection

Abstract

Latent 5ʹ 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.

A Quality Control Mechanism of Splice Site Selection Abrogated under Stress and in Cancer

“…Finally, NCL knockdown resulted in activation of latent splicing in hundreds of coding transcripts that have important cellular functions. We thus proposed NCL as the first protein component in a nuclear quality control mechanism that regulates splice site selection, thereby protecting cells from latent splicing that can generate defective mRNAs [ 46 ].…”

“…We propose that, at this stage, NCL can now be added in the model as a protein directly binding ini-tRNA, likely along with additional proteins. This step helps establish a register for the recognition of the reading frame [ 46 ]. The second SOS element involves the cooperative polymerization of protein(s) that bind triplets of nucleotides and in the absence of a PTC it reaches the selected 5′SS; this step is the quality control “confirmation” step of SS selection that triggers the remodeling of the spliceosome to its functional state ( Figure 3 b, left).…”