Tandem donor splice sites (5'ss) are unique regions with at least two GU dinucleotides serving as splicing cleavage sites. The Δ3 tandem 5'ss are a specific subclass of 5'ss separated by 3 nucleotides which can affect protein function by inserting/deleting a single amino acid. One 5'ss is typically preferred, yet factors governing particular 5'ss choice are not fully understood. A highly conserved exon 21 of the STAT3 gene was chosen as a model to study Δ3 tandem 5'ss splicing mechanisms. Based on multiple lines of experimental evidence, endogenous U1 snRNA most likely binds only to the upstream 5'ss. However, the downstream 5'ss is used preferentially, and the splice site choice is not dependent on the exact U1 snRNA binding position. Downstream 5'ss usage was sensitive to exact nucleotide composition and dependent on the presence of downstream regulatory region. The downstream 5'ss usage could be best explained by two novel interactions with endogenous U6 snRNA. U6 snRNA enables the downstream 5'ss usage in STAT3 exon 21 by two mechanisms: (i) binding in a novel non-canonical register and (ii) establishing extended Watson-Crick base pairing with the downstream regulatory region. This study suggests that U6:5'ss interaction is more flexible than previously thought.

• MeSH
• Exons * MeSH
• HeLa Cells MeSH
• Humans MeSH
• RNA Splice Sites * MeSH
• RNA, Small Nuclear * metabolism   genetics   MeSH
• Base Sequence MeSH
• RNA Splicing MeSH
• STAT3 Transcription Factor * metabolism   genetics   MeSH
• Protein Binding MeSH
• Binding Sites genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• RNA Splice Sites * MeSH
• RNA, Small Nuclear * MeSH
• STAT3 protein, human MeSH Browser
• STAT3 Transcription Factor * MeSH
• U1 small nuclear RNA MeSH Browser
• U6 small nuclear RNA MeSH Browser