An evolutionarily conserved tryptophan cage promotes folding of the extended RNA recognition motif in the hnRNPR-like protein family

Abstract

The heterogeneous nuclear ribonucleoprotein (hnRNP) R-like family is a class of RNA binding proteins in the hnRNP superfamily that has diverse functions in RNA processing. Here, we present the 1.90 [A] X-ray crystal structure and solution NMR studies of the first RNA recognition motif (RRM) of human hnRNPR. We find that this domain adopts an extended RRM (eRRM1) featuring a canonical RRM with a structured N-terminal extension (Next) motif that docks against the RRM and extends the {beta}-sheet surface. The adjoining loop positions the Next for docking to the RRM and forms a tryptophan cage motif, which has only been reported previously in a synthetic peptide. Using a combination of mutagenesis, solution NMR spectroscopy, and thermal denaturation studies we evaluate the importance of residues in the Next-RRM interface and adjoining loop on eRRM folding and conformational dynamics. We find that these sites are essential for protein solubility, thermal stability, and conformational ordering. Consistent with their importance, mutations in the Next-RRM interface and loop are associated with several cancers in a survey of somatic mutations in cancer studies. Sequence and structure comparison of the human hnRNPR eRRM1 to experimentally verified and predicted hnRNPR-like proteins reveal conserved features in the eRRM. Significance statementThis study provides the first structural and thermodynamic analysis of the hnRNPR extended RNA recognition motif (eRRM1). Here an N-terminal extension docks alongside a canonical RRM, stabilized by an intervening loop containing a tryptophan cage motif. Highly conserved among hnRNPR-like proteins, this motif has only been previously observed in a synthetic peptide. Substitutions that disrupt observed interactions reduce thermal stability and are associated with cancers. These findings significantly advance our understanding of atypical RRM folding.