PhD student, Universidad de Concepción
Our aim is to understand better how translational control can affect cell viability during stress
Our cells require an acute control over which proteins are being synthesized and how much of it is bing made. This phenomenon is critical for maintaining any cell function, mediating the adequate cell response to changes in our tissues (the glucose availability or temperature) and even fluctuations in the environment, as the UV light exposure.
A common response to stressful conditions is the inhibition of protein synthesis, due to the high energy expend required to maintain it. When this happens, the molecules from which genetic information is translated into proteins (mRNAs) can be assembled into stable structures until the stressful conditions are gone.
One subset of these structures is called Stress Granules (SGs). SGs are believed to protect untranslated mRNAs from degradation and by doing so, cells do not need the de-novo synthesis of those mRNA molecules when stress is gone.
Our work is focused on how SG assembly can be modulated during stress to sustain cell viability, because it could have important repercussions in diseases in which the translational control is altered, like cancer and some mental disorders as schizophrenia.
Abstract: Although tumor protein D52 (TPD) family proteins were first identified nearly 20 years ago, their molecular regulatory mechanisms remain unclear. Therefore, we investigated the post-transcriptional regulation of TPD52 family genes. An RNA immunoprecipitation assay showed the potential binding ability of TPD52 family mRNAs to several RNA-binding proteins, and an RNA degradation assay revealed that TPD52 is subject to more prominent post-transcriptional regulation than TPD53 and 54. We subsequently focused on the 3'-untranslated region (3'-UTR) of TPD52 as a cis -acting element in post-transcriptional gene regulation. Several deletion mutants of the 3'-UTR of TPD52 mRNA were constructed and ligated to the 3' end of a reporter green fluorescence protein gene. RNA degradation assay revealed that a minimal cis -acting region, located in the 78-280 region of the 5'-proximal region of the 3'-UTR, stabilized the reporter mRNA. Biotin pull-down and RNA immunoprecipitation assays revealed specific binding of the region to T-cell intracellular antigen 1 (TIA-1) and TIA-1-related protein (TIAR). Knockdown of TIA-1/TIAR decreased not only the expression, but also the stability of TPD52 mRNA; it also decreased the expression and stability of the reporter gene ligated to the 3' end of the 78-280 fragment. Stimulation of TGF-b and EGF decreased the binding ability of these factors, resulted in decreased mRNA stability. These results indicate that the 78-280 fragment and TIA-1/TIAR concordantly contribute to mRNA stability as a cis -acting element and trans -acting factor(s), respectively. Thus, we herein report the specific interactions between these elements in the post-transcriptional regulation of the TPD52 gene.
Pub.: 17 Mar '17, Pinned: 30 Aug '17
Abstract: Mammalian stress granules (SGs) contain stalled translation preinitiation complexes that are assembled into discrete granules by specific RNA-binding proteins such as G3BP. We now show that cells lacking both G3BP1 and G3BP2 cannot form SGs in response to eukaryotic initiation factor 2α phosphorylation or eIF4A inhibition, but are still SG-competent when challenged with severe heat or osmotic stress. Rescue experiments using G3BP1 mutants show that phosphomimetic G3BP1-S149E fails to rescue SG formation, whereas G3BP1-F33W, a mutant unable to bind G3BP partner proteins Caprin1 or USP10, rescues SG formation. Caprin1/USP10 binding to G3BP is mutually exclusive: Caprin binding promotes, but USP10 binding inhibits, SG formation. G3BP interacts with 40S ribosomal subunits through its RGG motif, which is also required for G3BP-mediated SG formation. We propose that G3BP mediates the condensation of SGs by shifting between two different states that are controlled by the phosphorylation of S149 and by binding to Caprin1 or USP10.
Pub.: 30 Mar '16, Pinned: 30 Aug '17
Abstract: Caprin-1 plays roles in many important biological processes, including cellular proliferation, innate immune response, stress response and synaptic plasticity. Caprin-1 has been implicated in several human diseases, including osteosarcoma, breast cancer, viral infection, hearing loss and neurodegenerative disorders. The functions of Caprin-1 depend on its molecular-interaction network. Direct interactions have been established between Caprin-1 and the fragile X mental retardation protein (FMRP), Ras GAP-activating protein-binding protein 1 (G3BP1) and the Japanese encephalitis virus (JEV) core protein. Here, crystal structures of a fragment (residues 132–251) of Caprin-1, which adopts a novel all-α-helical fold and mediates homodimerization through a substantial interface, are reported. Homodimerization creates a large and highly negatively charged concave surface suggestive of a protein-binding groove. The FMRP-interacting sequence motif forms an integral α-helix in the dimeric Caprin-1 structure in such a way that the binding of FMRP would not disrupt the homodimerization of Caprin-1. Based on insights from the structures and existing biochemical data, the existence of an evolutionarily conserved ribonucleoprotein (RNP) complex consisting of Caprin-1, FMRP and G3BP1 is proposed. The JEV core protein may bind Caprin-1 at the negatively charged putative protein-binding groove and an adjacent E-rich sequence to hijack the RNP complex.
Pub.: 25 May '16, Pinned: 30 Aug '17
Abstract: Angiogenin (ANG) is a secreted ribonuclease that cleaves tRNA to initiate a stress-response program in mammalian cells. Here we show that ANG inhibits protein synthesis and promotes arsenite- and pateamine A-induced assembly of stress granules (SGs). These effects are abrogated in cells transfected with the ANG inhibitor RNH1. Transfection of natural or synthetic 5'- but not 3'-tRNA fragments (tRNA-derived stress-induced RNAs; tiRNAs) induces the phospho-eukaryotic translation initiation factor 2alpha-independent assembly of SGs. Natural 5'-tiRNAs but not 3'-tiRNAs are capped with a 5'-monophosphate that is required for optimal SG assembly. These findings reveal that SG assembly is a component of the ANG- and tiRNA-induced stress response program.
Pub.: 05 Feb '10, Pinned: 30 Aug '17
Abstract: Protein synthesis stands at the last stage of the central dogma of molecular biology, providing a final regulatory layer for gene expression. Reacting to environmental cues and internal signals, the translation machinery can quickly tune the translatome from a pre-existing pool of RNAs, before the transcriptome changes. Although the translation reaction itself has been known since the 1950s, the quantitative or even qualitative measurement of its efficacy in cells has posed experimental and analytic hurdles. In this review, we outline the array of state-of-the-art methods that have emerged to tackle the hidden aspects of translational control.
Pub.: 02 Jun '17, Pinned: 30 Aug '17
Abstract: Translational control of gene expression is an important regulator of growth, homeostasis and aging in Drosophila The ability to measure changes in protein synthesis in response to genetic and environmental cues is therefore important in studying these processes. Here we describe a simple and cost effective approach to assay protein synthesis in Drosophila larval cells and tissues. The method is based on the incorporation of puromycin into nascent peptide chains. Using an ex vivo approach, we label newly synthesized peptides in larvae with puromycin and then measure levels of new protein synthesis using an anti-puromycin antibody. We show that this method can detect changes in protein synthesis in specific cells and tissues in the larvae, either by immunostaining or western blotting. We find that the assay reliably detects changes in protein synthesis induced by two known stimulators of mRNA translation - the nutrient/TORC1 kinase pathway and the transcription factor dMyc. We also use the assay to describe how protein synthesis changes through larval development and in response to two environmental stressors - hypoxia and heat-shock. We propose that this puromycin-labelling assay is a simple, but robust method to detect protein synthesis changes at the levels of cells, tissues or whole body in Drosophila.
Pub.: 24 Jun '17, Pinned: 30 Aug '17