Applications and limitations of regulatory RNA elements in synthetic biology and biotechnology

This is the peer reviewed version of this article. It may be used for non-commercial purposed in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions.Synthetic biology requires the design and implementation of novel enzymes, genetic circuits or even entire cells, which can be controlled by the user. RNA‐based regulatory elements have many important functional properties in this regard, such as their modular nature and their ability to respond to specific external stimuli. These properties have led to the widespread exploration of their use as gene regulation devices in synthetic biology. In this review, we focus on two major types of RNA elements: riboswitches and RNA thermometers (RNATs). We describe their general structure and function, before discussing their potential uses in synthetic biology (e.g. in the production of biofuels and biodegradable plastics). We also discuss their limitations, and novel strategies to implement RNA‐based regulatory devices in biotechnological applications. We close with a description of some common model organisms used in synthetic biology, with a focus on the current applications and limitations of RNA‐based regulation.Ye

Eukaryotic initiation factor 5B (eIF5B) regulates temozolomide-mediated apoptosis in brain tumor stem cells (BTSCs)

Permission to archive accepted author manuscriptGlioblastoma multiforme (GBM) is among the deadliest cancers, owing in part to complex inter- and intra-tumor heterogeneity and the presence of a population of stem-like cells called brain tumor stem cells (BTSCs/BTICs). These cancer stem cells survive treatment and confer resistance to the current therapies—namely, radiation and the chemotherapeutic, temozolomide (TMZ). TMZ induces cell death by alkylating DNA, and BTSCs resist this mechanism via a robust DNA damage response. Hence, recent studies aimed to sensitize BTSCs to TMZ using combination therapy, such as inhibition of DNA repair machinery. We have previously demonstrated in established GBM cell lines that eukaryotic initiation factor 5B (eIF5B) promotes the translation of pro-survival and anti-apoptotic proteins. Consequently, silencing eIF5B sensitizes these cells to TRAIL-induced apoptosis. However, established cell lines do not always recapitulate the features of human glioma. Therefore, we investigated this mechanism in patient-derived BTSCs. We show that silencing eIF5B leads to increased TMZ sensitivity in two BTSC lines, BT25 and BT48. Depletion of eIF5B decreases levels of anti-apoptotic proteins in BT48 and sensitizes these cells to TMZ-induced activation of caspase-3, cleavage of PARP, and apoptosis. We suggest that eIF5B represents a rational target to sensitize GBM tumors to the current standard-of-care.Ye

IRES-mediated translation of cellular messenger RNA operates in eIF2α- independent manner during stress

Physiological and pathophysiological stress attenuates global translation via phosphorylation of eIF2α. This in turn leads to the reprogramming of gene expression that is required for adaptive stress response. One class of cellular messenger RNAs whose translation was reported to be insensitive to eIF2α phosphorylation-mediated repression of translation is that harboring an Internal Ribosome Entry Site (IRES). IRES-mediated translation of several apoptosis-regulating genes increases in response to hypoxia, serum deprivation or gamma irradiation and promotes tumor cell survival and chemoresistance. However, the molecular mechanism that allows IRES-mediated translation to continue in an eIF2α-independent manner is not known. Here we have used the X-chromosome linked Inhibitor of Apoptosis, XIAP, IRES to address this question. Using toeprinting assay, western blot analysis and polysomal profiling we show that the XIAP IRES supports cap-independent translation when eIF2α is phosphorylated both in vitro and in vivo. During normal growth condition eIF2α-dependent translation on the IRES is preferred. However, IRES-mediated translation switches to eIF5B-dependent mode when eIF2α is phosphorylated as a consequence of cellular stress

IRES-mediated translation of cellular messenger RNA operates in eIF2α- independent manner during stress

Physiological and pathophysiological stress attenuates global translation via phosphorylation of eIF2α. This in turn leads to the reprogramming of gene expression that is required for adaptive stress response. One class of cellular messenger RNAs whose translation was reported to be insensitive to eIF2α phosphorylation-mediated repression of translation is that harboring an Internal Ribosome Entry Site (IRES). IRES-mediated translation of several apoptosis-regulating genes increases in response to hypoxia, serum deprivation or gamma irradiation and promotes tumor cell survival and chemoresistance. However, the molecular mechanism that allows IRES-mediated translation to continue in an eIF2α-independent manner is not known. Here we have used the X-chromosome linked Inhibitor of Apoptosis, XIAP, IRES to address this question. Using toeprinting assay, western blot analysis and polysomal profiling we show that the XIAP IRES supports cap-independent translation when eIF2α is phosphorylated both in vitro and in vivo. During normal growth condition eIF2α-dependent translation on the IRES is preferred. However, IRES-mediated translation switches to eIF5B-dependent mode when eIF2α is phosphorylated as a consequence of cellular stress

An internal ribosomal entry site mediates redox-sensitive translation of Nrf2

Nrf2 plays pivotal roles in coordinating the antioxidant response and maintaining redox homeostasis. Nrf2 expression is exquisitely regulated; Nrf2 expression is suppressed under unstressed conditions but strikingly induced under oxidative stress. Previous studies showed that stress-induced Nrf2 up-regulation results from both the inhibition of Nrf2 degradation and enhanced Nrf2 translation. In the present study, we elucidate the mechanism underlying translational control of Nrf2. An internal ribosomal entry site (IRES) was identified within the 5′ untranslated region of human Nrf2 mRNA. The IRESNrf2 contains a highly conserved 18S rRNA binding site (RBS) that is required for internal initiation. This IRESNrf2 also contains a hairpin structured inhibitory element (IE) located upstream of the RBS. Deletion of this IE remarkably enhanced translation. Significantly, treatment of cells with hydrogen peroxide (H2O2) and phyto-oxidant sulforaphane further stimulated IRESNrf2-mediated translation initiation despite the attenuation of global protein synthesis. Polyribosomal profile assay confirmed that endogenous Nrf2 mRNAs were recruited into polysomal fractions under oxidative stress conditions. Collectively, these data demonstrate that Nrf2 translation is suppressed under normal conditions and specifically enhanced upon oxidant exposure by internal initiation, and provide a mechanistic explanation for translational control of Nrf2 by oxidative stress

Eukaryotic Initiation factor 5B (eIF5B) provides glioblastoma multiforme with resistance towards apoptotic agents

Eukaryotic initiation factors (eIFs) are proteins involved in the general process of mRNA translation. However, under conditions of physiological stress, alternative methods of translation initiation are often activated and relied upon to continue translation of a subset of mRNAs. Specific eIFs such as eIF5B have been shown to use cap-independent mechanisms to promote the production of anti-apoptotic proteins, such as X-linked inhibitor of apoptosis protein (XIAP). In the present study, we have explored if eIF5B provides resistance to glioblastoma multiforme (GBM) cells towards apoptotic agents. Using siRNA, we depleted eIF5B from various GBM cells and treated them with agents that promote apoptosis. In order to activate intrinsic apoptosis pathway we treated these cells with genotoxic agents such as doxorubicin (DXR), and temozolomide (TMZ). Moreover, we treated GBM cells with tumor necrosis factor alpha (TNFα), and/or TNF-related apoptosis-inducing ligand (TRAIL) to activate extrinsic apoptosis pathway. The depletion of eIF5B did not sensitize GBM cells to DXR or TMZ. However, further viability assays showed that knockdown of eIF5B sensitized U343 cells to TRAIL or TRAIL + TNFα. Further, the combination of TRAIL + SMAC- mimetic compounds (SMC) showed statistically significant decreases in cell viability when eIF5B was depleted. So far, six different glioblastoma cell lines have  been assessed for this effect, and further studies are underway to observe the effects of eIF5B depletion on cell viability of various cancer and non-cancer cells. Our preliminary data suggest that eIF5B enhances cell survival of GBM cells and could represent a potentially important therapeutic target. *Indicates presente

The role of eukaryotic initiation factor 5B (eIF5B) in cell cycle regulation

Gene expression is critically regulated at the transcriptional, translational, and post-translational levels. Dysregulation and altered mechanisms of translational control have significant consequences in processes including cell growth, cell death, cell proliferation, and maturation. Initiation is the rate- limiting step of translation, which is inhibited under physiological stress conditions. During the integrated stress response, the α subunit of eukaryotic initiation factor 2 (eIF2) is phosphorylated. This phosphorylation attenuates general translation, by preventing the reformation of the necessary ternary complex. Despite the attenuation of general translation, certain mRNAs containing upstream open reading frames (uORFs) are favorably translated during this condition. uORFs are mRNA elements with a start codon in the 5’ UTR that is out-of-frame with the main coding sequence. Bioinformatic studies have shown 49% of human transcripts to contain uORFs. Preliminary data suggests that the depletion of eIF5B parallels the effects of phosphorylation of eIF2α. eIF5B depletion has been observed to inhibit global translation, while enhancing the translation of certain uORF-containing mRNAs. The Thakor lab has shown the protein p27 to be upregulated when eIF5B is knocked down in U343 (human glioblastoma multiforme) cells. As p27 is a cyclin-dependent kinase inhibitor (CKI), which controls cell progression from the G1 to S phase, this suggests a significant role for eIF5B in the cell cycle. However, eIF5B depletion has been observed to not have significant effects on the cyclin-dependent kinase inhibitor: p21. Further, PI staining and flow cytometric analysis have demonstrated that eIF5B does not have any significant effects on the cell cycle. This data suggests that eIF5B has a significant role in the regulation of p27, but does not contribute to cell cycle regulation. *Indicates presente