title: Noncanonical registers and base pairs in human 5' splice-site selection.
abstract: Accurate recognition of splice sites is essential for pre-messenger RNA splicing. Mammalian 5' splice sites are mainly recognized by canonical base-pairing to the 5' end of U1 small nuclear RNA, yet we described multiple noncanonical base-pairing registers by shifting base-pair positions or allowing one-nucleotide bulges. By systematic mutational and suppressor U1 analyses, we prove three registers involving asymmetric loops and show that two-nucleotide bulges but not longer can form in this context. Importantly, we established that a noncanonical uridine-pseudouridine interaction in the 5' splice site/U1 helix contributes to the recognition of certain 5' splice sites. Thermal melting experiments support the formation of noncanonical registers and uridine-pseudouridine interactions. Overall, we experimentally validated or discarded the majority of predicted noncanonical registers, to derive a list of 5' splice sites using such alternative mechanisms that is much different from the original. This study allows not only the mechanistic understanding of the recognition of a wide diversity of mammalian 5' splice sites, but also the future development of better splice-site scoring methods that reliably predict the effects of disease-causing mutations at these sequences.
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 637551, Singapore.
authors.forename: Jiazi
authors.initials: J
authors.lastname: Tan
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 637551, Singapore.
authors.forename: Jia Xin Jessie
authors.initials: JX
authors.lastname: Ho
authors.affiliation: Division of Chemistry & Biological Chemistry, School of Physical and Mathematical Sciences, Nanyang Technological University, 637371, Singapore.
authors.forename: Zhensheng
authors.initials: Z
authors.lastname: Zhong
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 637551, Singapore.
authors.forename: Shufang
authors.initials: S
authors.lastname: Luo
authors.affiliation: Division of Chemistry & Biological Chemistry, School of Physical and Mathematical Sciences, Nanyang Technological University, 637371, Singapore.
authors.forename: Gang
authors.initials: G
authors.lastname: Chen
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 637551, Singapore xroca@ntu.edu.sg.
authors.forename: Xavier
authors.initials: X
authors.lastname: Roca
mesh_terms: D020029:Base Pairing; D057809:HEK293 Cells; D006801:Humans; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D011560:Pseudouridine / Q000737:chemistry; D022821:RNA Splice Sites; D012342:RNA, Small Nuclear / Q000737:chemistry / Q000378:metabolism; D014529:Uridine / Q000737:chemistry
chemical_list: D022821:RNA Splice Sites; D012342:RNA, Small Nuclear; C405483:U1 small nuclear RNA; D011560:Pseudouridine; D014529:Uridine