Exploring the role of RRM domains and conserved aromatic residues in RGG motif  of eIF4G-binding translation repressor protein Sbp1

Abstract

Background: RNA binding proteins play crucial role in determining if a given mRNA will be translated, stored, or degraded. Sbp1 is an RGG-motif containing protein that is implicated in affecting mRNA decapping and translation. Sbp1 represses translation by binding eIF4G1 through its RGG-motif and activates decapping when overexpressed. In this report, we have assessed the genetic interaction of Sbp1 with decapping activators such as Dhh1, Pat1, and Scd6. We have further analyzed the importance of different domains and specific conserved residues of Sbp1 in its ability to cause over-expression mediated growth defect. Method: Sequence alignment was performed to identify conserved aromatic residues to be mutated. Using site-directed mutagenesis several point mutations and domain deletions were created in Sbp1 expressed under a galactose-inducible promoter. The mutants were tested for their ability to cause growth defect upon over-expression. The ability of Sbp1 to affect over-expression mediated growth defect of other decapping activators was tested using growth assay. Live cell imaging was done to study localization of Sbp1 and its RRM-deletion mutants to RNA granules upon glucose starvation. Results: Mutation of several aromatic residues in the RGG-motif and that of the phosphorylation sites in the RRM domain of Sbp1 did not affect the growth defect phenotype. Deletion of another eIF4G1-binding RGG-motif protein Scd6 does not affect the ability of Sbp1 to cause growth defect. Moreover, absence of Sbp1 did not affect the growth defect phenotypes observed upon overexpression of decapping activators Dhh1 and Pat1. Strikingly deletion of both the RRM domains (RRM1 and RRM2) and not the RNP motifs within them compromised the growth defect phenotype. Sbp1 mutant lacking both RRM1 and RRM2 was highly defective in localizing to RNA granules. Conclusion: This study identifies an important role of RRM domains independent of the RNP motif in Sbp1 function.

Influence of RRM, RGG and Potential Phosphorylated Sites in Cold-Inducible Protein RBM3 on its Subcellular Localization and Neuroprotective Effects

“…The RRM is widely presented in eukaryotes and composed of two a-helices and four β-sheets. Bhatter et al [31] found that deletion of RRM domains compromised the ability of Sbp1 to induce growth defects in yeast by preventing localization of Sbp1 to RNA granules upon glucose starvation. Eulalio et al [32] showed that the RRM in GW182 protein contributes to miRNA-mediated gene silencing through protein-RNA interaction.…”

Low complexity RGG-motif sequence is required for Processing body (P-body) disassembly

“…Complementation experiments presented in Fig. 4 were performed using endogenously tagged Edc3-mCherry-WT, and Edc3-mCherry Δsbp1 strains transformed with CEN plasmid (pRS315) expressing wild-type Sbp1-GFP or its mutants (Sbp1ΔRGG-GFP, Sbp1ΔRRM2-GFP, and Sbp1 ΔRRM1ΔRRM2-GFP) 59 .…”