Characterizing the binding mechanism of the splicing factor hnRNP H and RNA G-quadruplex forming sequences

HnRNP-H is a splicing factor that regulates splicing by binding to RNA through unique RNA recognition motifs called quasi-RRMs (q-RRMs). These motifs, which lack conserved aromatic and basic residues that appear in canonical RRMs, recognize poly-guanine (poly-G) RNA sequences that have the potential to fold into stable G-quadruplex secondary structures. G-quadruplexes are constructed of guanine-planar tetrads stacking upon each other when each tetrad is composed of four guanines from four distinct G-repeats that associate through hoogsteen hydrogen bonding. The fold and topology of G-quadruplex structures are dictated by the number of G-repeats and the number of guanines in each repeat, which, in turn, contribute to the stability of the structure. In our study, we aim to characterize the interactions and mechanism of binding between the qRRM fragments of hnRNP-H (qRRM1, qRRM2, and qRRM1-2) and RNA sequences with varying numbers of G-repeats. To date, we examined the interactions between the qRRMs to four repeats of GGGGCC (G4C2x4), which showed high affinity to qRRM1-2 and qRRM1, but surprisingly not to qRRM2. G4C2x4 is heterogeneous in solution since it can fold into various G-quadruplex multimers. Recently we obtained homogeneous populations using gel filtration, which enabled us to grow crystals of qRRM1-2 in complex with G4C2x4. These structures will provide essential data for understanding the unique interactions between hnRNP-H and G-quadruplex-forming sequences.