Summary Expression of tumor suppressor p53 is regulated at multiple levels, disruption of which often leads to cancer. We have adopted an approach combining computational systems modeling with experimental validation to elucidate the translation regulatory network that controls p53 expression post DNA damage. The RNA-binding protein HuR activates p53 mRNA translation in response to UVC-induced DNA damage in breast carcinoma cells. p53 and HuR levels show pulsatile change post UV irradiation. The computed model fitted with the observed pulse of p53 and HuR only when hypothetical regulators of synthesis and degradation of HuR were incorporated. miR-125b, a UV-responsive microRNA, was found to represses the translation of HuR mRNA. Furthermore, UV irradiation triggered proteasomal degradation of HuR mediated by an E3-ubiquitin ligase tripartite motif-containing 21 (TRIM21). The integrated action of miR-125b and TRIM21 constitutes an intricate control system that regulates pulsatile expression of HuR and p53 and determines cell viability in response to DNA damage.

Translation control plays a key role in variety of cellular processes. Translation initiation factors augment translation, whereas translation repressor proteins inhibit translation. Different repressors act by distinct mechanisms to accomplish the repression process. Although messenger RNAs (mRNAs) can be repressed at various steps of translation, most repressors have been reported to target the initiation step. We focus on one such translation repressor, an Arginine-Glycine-Glycine (RGG)-motif containing protein Scd6. Using this protein as a model, we present a discourse on the known and possible functions of this repressor, its mechanism of action and its recently reported regulation. We suggest a case for conservation of the mechanism employed by Scd6 along with its regulation in orthologs, and propose that Scd6 family of proteins will be an ideal tool to understand translation control and mRNA fate decision mechanisms across biological systems. This article is categorized under: Translation > Translation Regulation RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes.

It is generally believed that human mature erythrocytes do not possess functional ribosomes, and therefore cannot synthesize proteins. However, the absence of translation is not consistent with the long lifespan of mature erythrocytes. They stay viable and functional for about 115 days in the circulatory system. Here, using highly pure preparation of human mature erythrocytes, we demonstrate the presence of translation by polysome profiling, [35S]methionine labelling and RiboPuromycylation. [35S]methionine labelling revealed that the translation in mature erythrocytes is about 10% of that observed in reticulocytes. We could observe polysomes by transmission electron microscopy in these cells. RNA-seq and quantitative RT-PCR performed on polysome fraction of these cells revealed that HBA (alpha globin) and HBB (beta globin) transcripts are translated. Using luciferase-based reporter assay and mutational studies, we show that the sequence of the 5' untranslated region is crucial for the translation of these transcripts. Furthermore, mature erythrocytes showed reduced expression of globin proteins (alpha and beta) when treated with translation inhibitors. Overall, we provide multiple lines of evidence for translation of globin mRNAs in human mature erythrocytes.

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Running title: RGG-motif proteins suppress clathrin heavy chain deficiency. AbstractClathrin, made up of the heavy-and light-chains, constitutes one of the most abundant protein in vesicles involved in intracellular protein trafficking and endocytosis. YPR129W, which encodes RGGmotif containing translation repressor was identified as a part of multi-gene construct (SCD6) that suppressed clathrin deficiency. However, the contribution of YPR129W alone in suppressing clathrin deficiency has not been documented. In this study we identify YPR129W as a necessary and sufficient gene in a multigene construct SCD6 that suppresses clathrin deficiency. Importantly, we identify cytoplasmic RGG-motif protein encoding gene PSP2 as a novel suppressor of clathrin deficiency. Three other RGG-motif protein encoding genes SBP1, DED1 and GBP2 do not suppress clathrin deficiency. DHH1, a DEAD-box RNA helicase with translation repression activity also fails to rescue clathrin deficiency. α-factor secretion assay suggests that suppression of clathrin deficiency by SCD6 and PSP2 is not mediated by the rescue of the trans-Golgi network (TGN) protein sorting defect observed in the absence of CHC1. Detailed domain analysis of the two suppressors reveals that the RGG-motif of both Scd6 and Psp2 is important for suppressing clathrin deficiency. Additionally, the Lsm domain deletion as well as the arginine to alanine mutation in the arginine methylation defective (AMD) mutant render Scd6 defective in suppressing clathrin deficiency. Overall based on our results using SCD6 and PSP2 proteins, we identify a novel role of RGG-motif in suppressing clathrin deficiency. Since both the suppressors are RNA-binding granule-resident proteins, this study opens an exciting avenue for exploring the connection between clathrin function and cytoplasmic RNA metabolism.