The Guanine-rich RNA sequence binding factor 1 (GRSF1) is a member of the heterogeneous nuclear ribonucleoprotein F/H family and has been implicated in RNA processing, RNA transport and translational regulation. Amino acid alignments and homology modeling suggested the existence of three distinct RNA-binding domains and two auxiliary domains. Unfortunately, little is known about the molecular details of GRSF1/RNA interactions. To explore the RNA-binding mechanisms we first expressed full-length human GRSF1 and several truncation mutants, which include the three separated qRRM domains in E. coli, purified the recombinant proteins and quantified their RNA-binding affinity by RNA electrophoretic mobility shift assays. The expression levels varied between 1 and 10mg purified protein per L bacterial liquid culture and for full-length human GRSF1 a binding constant (KD-value) of 0.5μM was determined. In addition, our mechanistic experiments with different truncation mutants allowed the following conclusions: i) Deletion of either of the three RNA-binding domains impaired the RNA-binding affinity suggesting that the simultaneous presence of the three domains is essential for high-affinity RNA-binding. ii) Deletion of the Ala-rich auxiliary domain did hardly affect RNA-binding. Thus, this structural subunit may not be involved in RNA interaction. iii) Deletion of the acidic auxiliary domain improved the RNA-binding suggesting a regulatory role for this structural motif. iv) The isolated RNA-binding domains did not exhibit sizeable RNA-binding affinities. Taken together these data suggest that a cooperative interaction of the three qRRMs is required for high affinity RNA-binding.
Keywords: Electrophoretic mobility shift assays; Protein expression; Protein purification; RNA metabolism; qRRMs.
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