Abstract
RNA-binding protein with multiple splicing (designated RBPMS) is a higher vertebrate mRNA-binding protein containing a single RNA recognition motif (RRM). RBPMS has been shown to be involved in mRNA transport, localization and stability, with key roles in axon guidance, smooth muscle plasticity, as well as regulation of cancer cell proliferation and migration. We report on structure-function studies of the RRM domain of RBPMS bound to a CAC-containing single-stranded RNA. These results provide insights into potential topologies of complexes formed by the RBPMS RRM domain and the tandem CAC repeat binding sites as detected by photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation. These studies establish that the RRM domain of RBPMS forms a symmetrical dimer in the free state, with each monomer binding sequence-specifically to all three nucleotides of a CAC segment in the RNA bound state. Structure-guided mutations within the dimerization and RNA-binding interfaces of RBPMS RRM on RNA complex formation resulted in both disruption of dimerization and a decrease in RNA-binding affinity as observed by size exclusion chromatography and isothermal titration calorimetry. As anticipated from biochemical binding studies, over-expression of dimerization or RNA-binding mutants of Flag-HA-tagged RBPMS were no longer able to track with stress granules in HEK293 cells, thereby documenting the deleterious effects of such mutations in vivo.
Highlights
RNA recognition motifs (RRMs) constitute the most abundant RNA-binding domains in higher vertebrates that play diverse roles in post-transcriptional gene expression processes ranging from mRNA and rRNA processing to RNA transport, localization and stability
The structure of the complex reveals the specific recognition of the CAC motif by the RBPMS RRM domain, as well as the dimeric arrangement of the protein that could enable the dimer to bind a pair of tandem CAC elements separated by a spacer of sufficient nucleotide length
The RRM of RBPMS adopts the classical RRM fold composed of a four-stranded antiparallel β-sheet packed against a pair of α-helices (Fig. 1b)
Summary
RNA recognition motifs (RRMs) constitute the most abundant RNA-binding domains in higher vertebrates that play diverse roles in post-transcriptional gene expression processes ranging from mRNA and rRNA processing to RNA transport, localization and stability (reviewed in Clery et al 2008; Gerstberger et al 2014; Lunde et al 2007; Serganov & Patel, 2008). A systematic study of transcriptome-wide mRNA targets of the RRM domain of RBPMS using photoactivatableribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) has identified RNA targets composed of tandem CAC trinucleotide motifs separated by variable spacer segments (Farazi et al 2014). We have investigated the impact of structure-guided dimerization and RNA-binding mutants of RBPMS RRM on RNA-binding affinity and oligomerization, as well as localization of RBPMS to cytoplasmic stress granules under oxidative stress conditions
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