Evidence for cooperative tandem binding of hnRNP C RRMs in mRNA processing

• Published in: RNA (Cambridge) 2015-11, Vol.21 (11), p.1931-1942
• Main Authors: Cieniková, Zuzana, Jayne, Sandrine, Damberger, Fred Franz, Allain, Frédéric Hai-Trieu, Maris, Christophe
• Format: Article
• Language: English
• Subjects: Binding Sites - genetics; Heterogeneous-Nuclear Ribonucleoprotein Group C - metabolism; Heterogeneous-Nuclear Ribonucleoproteins - genetics; Humans; Protein Binding - genetics; RNA Recognition Motif Proteins - metabolism; RNA, Messenger - genetics; RNA, Messenger - metabolism; Transcriptome - genetics; Uridine - genetics; Uridine - metabolism
• ISSN: 1355-8382; 1469-9001
• DOI: 10.1261/rna.052373.115

The human hnRNP C is a ubiquitous cellular protein involved in mRNA maturation. Recently, we have shown that this protein specifically recognizes uridine (U) pentamers through its single RNA recognition motif (RRM). However, a large fraction of natural RNA targets of hnRNP C consists of much longer contiguous uridine stretches. To understand how these extended sites are recognized, we studied the binding of the RRM to U-tracts of 8-11 bases. In vivo investigation of internal translation activation of unr (upstream of N-ras) mRNA indicates that the conservation of the entire hnRNP C binding site, UC(U)8, is required for hnRNP C-dependent IRES activation. The assays further suggest a synergistic interplay between hnRNP C monomers, dependent on the protein's ability to oligomerize. In vitro spectroscopic and thermodynamic analyses show that isolated RRMs bind to (U)11 oligomers as dimers. Structural modeling of a ternary double-RRM/RNA complex indicates additionally that two RRM copies can be accommodated on the canonical sequence UC(U)8. The proposed tandem RRM binding is in very good agreement with the transcriptome-wide recognition of extended U-tracts by full-length hnRNP C, which displays a cross-linking pattern consistent with a positively cooperative RRM dimer binding model.