title: Specificity of recognition of mRNA 5' cap by human nuclear cap-binding complex.
abstract: The heterodimeric nuclear cap-binding complex (CBC) binds to the mono-methylated 5' cap of eukaryotic RNA polymerase II transcripts such as mRNA and U snRNA. The binding is important for nuclear maturation of mRNAs and possibly in the first round of translation and nonsense-mediated decay. It is also essential for nuclear export of U snRNAs in metazoans. We report characterization by fluorescence spectroscopy of the recognition of 5' capped RNA by human CBC. The association constants (K(as)) for 17 mono- and dinucleotide cap analogs as well as for the oligomer m7GpppA(m2') pU(m2')pA(m2') cover the range from 1.8 x 10(6) M(-1) to 2.3 x 10(8) M(-1). Higher affinity for CBC is observed for the dinucleotide compared with mononucleotide analogs, especially for those containing a purine nucleoside next to m7G. The mRNA tetramer associates with CBC as tightly as the dinucleotide analogs. Replacement of Tyr138 by alanine in the CBP20 subunit of CBC reduces the cap affinity except for the mononucleotide analogs, consistent with the crystallographic observation of the second base stacking on this residue. Our spectroscopic studies showed that contrary to the other known cap-binding proteins, the first two nucleotides of a capped-RNA are indispensable for its specific recognition by CBC. Differences in the cap binding of CBC compared with the eukaryotic translation initiation factor 4E (eIF4E) are analyzed and discussed regarding replacement of CBC by eIF4E.
authors.affiliation: Department of Biophysics, Institute of Experimental Physics, Warsaw University, 93 Zwirki & Wigury St., 02-089 Warszawa, Poland.
authors.forename: Remigiusz
authors.initials: R
authors.lastname: Worch
authors.forename: Anna
authors.initials: A
authors.lastname: Niedzwiecka
authors.forename: Janusz
authors.initials: J
authors.lastname: Stepinski
authors.forename: Catherine
authors.initials: C
authors.lastname: Mazza
authors.forename: Marzena
authors.initials: M
authors.lastname: Jankowska-Anyszka
authors.forename: Edward
authors.initials: E
authors.lastname: Darzynkiewicz
authors.forename: Stephen
authors.initials: S
authors.lastname: Cusack
authors.forename: Ryszard
authors.initials: R
authors.lastname: Stolarski
mesh_terms: D001665:Binding Sites; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D039502:Nuclear Cap-Binding Protein Complex / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D012315:RNA Caps / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D017186:Titrimetry
chemical_list: D039502:Nuclear Cap-Binding Protein Complex; D012315:RNA Caps; D012333:RNA, Messenger