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

The heterogeneous nuclear ribonucleoprotein (hnRNP) R-like family is a class of RNA binding proteins in the hnRNP superfamily with diverse functions in RNA processing. Here, we present the 1.90 Å 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 (N) motif that docks against the RRM and extends the β-sheet surface. The adjoining loop is structured and forms a tryptophan cage motif to position the N motif for docking to the RRM. Combining mutagenesis, solution NMR spectroscopy, and thermal denaturation studies, we evaluate the importance of residues in the N-RRM interface and adjoining loop on eRRM folding and conformational dynamics. We find that these sites are essential for protein solubility, conformational ordering, and thermal stability. Consistent with their importance, mutations in the N-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 reveals conserved features in the eRRM.