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 × 10⁶ M⁻¹ to 2.3 × 10⁸ 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.

Keywords

• CBC
• mRNA 5′
• cap
• molecular recognition
• fluorescence
• binding energy
• CBC, cap-binding complex
• eIF4E, eukaryotic initiation factor 4E
• Gm2′, 2′-O-methylguanosine
• Am2′, 2′-O-methyladenosine
• Um2′, 2′-O-methyluridine
• m6A, N6-methyladenosine
• m7G, 7-methylguanosine
• m22,7 G, N2,7-dimethyl guanosine
• m32,2,7 G, N2,N2,7-trimethylguanosine
• m7GTP, 7-methylguanosine triphosphate and similarly other nucleoside triphosphates
• m22,7 GTP, N2,7-dimethylguanosine triphosphate
• m7GpppG, P1-7-methylguanosine-5′ P3-guanosine-5′ triphosphate, and similarly other dinucleoside triphosphates
• EDTA, ethylenediaminetetraacetic acid
• DTT, dithiothreitol
• HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
• wt, wild type.