Bhatter Nupur, Iyyappan Rajan, Mohanan Gayatri, Rajyaguru Purusharth I
Department of Biochemistry, Indian Institute of Science, CV Raman Road, Bangalore, 560012, India.
Institute of Animal Physiology and Genetics, Libechov, Czech Republic.
Wellcome Open Res. 2021 Sep 17;3:102. doi: 10.12688/wellcomeopenres.14709.3. eCollection 2018.
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. 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. 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. This study identifies an important role of RRM domains independent of the RNP motif in Sbp1 function.
RNA结合蛋白在决定特定mRNA是会被翻译、储存还是降解方面发挥着关键作用。Sbp1是一种含有RGG基序的蛋白质，与影响mRNA脱帽和翻译有关。Sbp1通过其RGG基序与eIF4G1结合来抑制翻译，并在过表达时激活脱帽。在本报告中，我们评估了Sbp1与脱帽激活剂如Dhh1、Pat1和Scd6的遗传相互作用。我们进一步分析了Sbp1不同结构域和特定保守残基在其导致过表达介导的生长缺陷能力中的重要性。进行序列比对以鉴定要突变的保守芳香族残基。使用定点诱变在半乳糖诱导型启动子下表达的Sbp1中产生了几个点突变和结构域缺失。测试突变体过表达时导致生长缺陷的能力。使用生长试验测试Sbp1影响其他脱帽激活剂过表达介导的生长缺陷的能力。进行活细胞成像以研究葡萄糖饥饿时Sbp1及其RRM缺失突变体在RNA颗粒中的定位。Sbp1的RGG基序中几个芳香族残基以及RRM结构域中磷酸化位点的突变不影响生长缺陷表型。另一种与eIF4G1结合的RGG基序蛋白Scd6的缺失不影响Sbp1导致生长缺陷的能力。此外，Sbp1的缺失不影响脱帽激活剂Dhh1和Pat1过表达时观察到的生长缺陷表型。引人注目的是，两个RRM结构域（RRM1和RRM2）而非其中的RNP基序的缺失损害了生长缺陷表型。缺乏RRM1和RRM2的Sbp1突变体在定位到RNA颗粒方面存在高度缺陷。 这项研究确定了RRM结构域在Sbp1功能中独立于RNP基序的重要作用。
