The fate of messenger RNA in cytoplasm plays a crucial role in various cellular processes. However, the mechanisms that decide whether mRNA will be translated, degraded or stored remain unclear. Single stranded nucleic acid binding protein (Sbp1), an Arginine‐Glycine‐Glycine (RGG‐motif) protein, is known to promote transition of mRNA into a repressed state by binding eukaryotic translation initiation factor 4G1 (eIF4G1) and to promote mRNA decapping, perhaps by modulation of Dcp1/2 activity. Sbp1 is known to be methylated on arginine residues in RGG‐motif; however, the functional relevance of this modification in vivo remains unknown. Here, we report that Sbp1 is arginine‐methylated in an hnRNP methyl transferase (Hmt1)‐dependent manner and that methylation is enhanced upon glucose deprivation. Characterization of an arginine‐methylation‐defective (AMD) mutant provided evidence that methylation affects Sbp1 function in vivo. The AMD mutant is compromised in causing growth defect upon overexpression, and the mutant is defective in both localizing to and inducing granule formation. Importantly, the Sbp1‐eIF4G1 interaction is compromised both for the AMD mutant and in the absence of Hmt1. Upon overexpression, wild‐type Sbp1 increases localization of another RGG motif containing protein, Scd6 (suppressor of clathrin deficiency) to granules; however, this property of Sbp1 is compromised in the AMD mutant and in the absence of Hmt1, indicating that Sbp1 repression activity could involve other RGG‐motif translation repressors. Additionally, the AMD mutant fails to increase localization of the decapping activator DEAD box helicase homolog to foci and fails to rescue the decapping defect of a dcp1‐2Δski8 strain, highlighting the role of Sbp1 methylation in decapping. Taken together, these results suggest that arginine methylation modulates Sbp1 role in mRNA fate determination.
P-bodies are conserved mRNP complexes that are implicated in determining mRNA fate by affecting translation and mRNA decay. In this report, we identify RGG-motif containing translation repressor protein Sbp1 as a disassembly factor of P-bodies since disassembly of P-bodies is defective in Δsbp1. RGG-motif is necessary and sufficient to rescue the PB disassembly defect in Δsbp1. Binding studies using purified proteins revealed that Sbp1 physically interacts with Edc3 and Sbp1-Edc3 interaction competes with Edc3-Edc3 interaction. Purified Edc3 forms assemblies, promoted by the presence of RNA and NADH and the addition of purified Sbp1, but not the RGG-deletion mutant, leads to significantly decreased Edc3 assemblies. We further note that the aggregates of human EWSR1 protein, implicated in neurodegeneration, are more persistent in the absence of Sbp1 and overexpression of EWSR1 in Δsbp1 leads to a growth defect. Taken together, our observations suggest a role of Sbp1 in disassembly, which could apply to disease-relevant heterologous protein-aggregates.
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.
RNA granules are conserved mRNP complexes that play an important role in determining mRNA fate by affecting translation repression and mRNA decay. Processing bodies (P-bodies) harbor enzymes responsible for mRNA decay and proteins involved in modulating translation. Although many proteins have been identified to play a role in P-body assembly, a bonafide disassembly factor remains unknown. In this report, we identify RGG-motif translation repressor protein Sbp1 as a disassembly factor of P-bodies. Disassembly of Edc3 granules but not the Pab1 granules (a conserved stress granule marker) that arise upon sodium azide and glucose deprivation stress are defective in Δsbp1. Disassembly of other P-body proteins such as Dhh1 and Scd6 is also defective in Δsbp1. Complementation experiments suggest that the wild type Sbp1 but not an RGG-motif deletion mutant rescues the Edc3 granule disassembly defect in Δsbp1. We observe that purified Edc3 forms assemblies, which is promoted by the presence of RNA and NADH. Strikingly, addition of purified Sbp1 leads to significantly decreased Edc3 assemblies. Although low complexity sequences have been in general implicated in assembly, our results reveal the role of RGG-motif (a low-complexity sequence) in the disassembly of P-bodies. Keywords: RNA granules, granule assembly and disassembly, Sbp1, Edc3, Processing bodies, Stress granules, liquid-liquid phase separation, liquid droplet, RGG-motif
Background: Mechanisms of mRNA fate decisions play an important role in determining if a given mRNA will be translated, stored or degraded upon arrival to cytoplasm. Sbp1 is an important 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 translation repression activity. Method: Sequence alignment was performed to identify conserved aromatic residues to be mutated. Using site-directed mutagenesis several point mutations and domain deletions was 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 RNP motif in Sbp1 repression activity.
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