A Novel Role for Nucleolin in Splice Site Selection

Latent 59 splice sites are highly abundant in human introns, yet, are apparently not normally used. Splicing at most of these sites would incorporate in-frame stop codons generating nonsense mRNAs. Importantly, under stress and in cancer, splicing at latent sites is activated generating nonsense mRNAs from thousands of genes. Previous studies point to an unresolved RNA quality control mechanism that suppresses latent splicing independently of NMD. They further demonstrated a pivotal role for initiator-tRNA in this mechanism, through its interaction with the AUG codon, independent of its role in protein translation. To further elucidate this mechanism, here we searched for nuclear proteins directly bound to initiator-tRNA in the nucleus. We identified nucleolin (NCL), a multifunctional, abundant, and conserved protein, as a novel regulator of splice site selection. Starting with UV crosslinking, we show that NCL is directly and specifically interacting with initiator-tRNA in the nucleus, but not in the cytoplasm. In support of NCL involvement in this mechanism, we show activation of latent splicing in hundreds of transcripts upon NCL knockdown, disrupting gene transcripts involved in several important cellular pathways and cell metabolism functions (e.g. transcription factors, oncogenes, kinases, splicing factors, translation factors, and genes affecting cell motility, proliferation, and cellular trafficking). We thus propose NCL, a component of the endogenous spliceosome, through its direct interaction with initiator-tRNA and its effect on latent splicing as the first documented protein of a nuclear quality control mechanism that regulates splice site selection to protect cells from latent splicing that would generate defective mRNAs.