Nateka L. Jackson

The ELAV RNA-stability factor HuR binds the 5′-untranslated region of the human IGF-IR transcript and differentially represses cap-dependent and IRES-mediated translation

The type I insulin-like growth factor receptor (IGF-IR) is an integral component in the control of cell proliferation, differentiation and apoptosis. The IGF-IR mRNA contains an extraordinarily long (1038 nt) 5′-untranslated region (5′-UTR), and we have characterized a diverse series of proteins interacting with this RNA sequence which may provide for intricate regulation of IGF-IR gene expression at the translational level. Here, we report the purification and identification of one of these IGF-IR 5′-UTR-binding proteins as HuR, using a novel RNA crosslinking/RNase elution strategy. Because HuR has been predominantly characterized as a 3′-UTR-binding protein, enhancing mRNA stability and generally increasing gene expression, we sought to determine whether HuR might serve a different function in the context of its binding the IGF-IR 5′-UTR. We found that HuR consistently repressed translation initiation through the IGF-IR 5′-UTR. The inhibition of translation by HuR was concentration dependent, and could be reversed in trans by addition of a fragment of the IGF-IR 5′-UTR containing the HuR binding sites as a specific competitor, or abrogated by deletion of the third RNA recognition motif of HuR. We determined that HuR repressed translation initiation through the IGF-IR 5′-UTR in cells as well, and that siRNA knockdown of HuR markedly increased IGF-IR protein levels. Interestingly, we also found that HuR potently inhibited IGF-IR translation mediated through internal ribosome entry. Kinetic assays were performed to investigate the mechanism of translation repression by HuR and the dynamic interplay between HuR and the translation apparatus. We found that HuR, occupying a cap-distal position, significantly delayed translation initiation mediated by cap-dependent scanning, but was eventually displaced from its binding site, directly or indirectly, as a consequence of ribosomal scanning. However, HuR perpetually blocked the activity of the IGF-IR IRES, apparently arresting the IRES-associated translation pre-initiation complex in an inactive state. This function of HuR as a 5′-UTR-binding protein and dual-purpose translation repressor may be critical for the precise regulation of IGF-IR expression essential to normal cellular homeostasis.

Alterations in RNA-binding activities of IRES-regulatory proteins as a mechanism for physiological variability and pathological dysregulation of IGF-IR translational control in human breast tumor cells

The type I insulin‐like growth factor receptor (IGF‐IR) is integrally involved in the control of cellular proliferation and survival. An internal ribosomal entry site (IRES) within the 1,038 nucleotide 5′‐untranslated region of the human IGF‐IR mRNA helps to provide the tight control of IGF‐IR expression necessary for maintenance of normal cellular and tissue homeostasis. The IRES maps to a discrete sequence of 85 nucleotides positioned just upstream of the IGF‐IR initiation codon, allowing the ribosome to bypass the highly structured regions of the 5′‐UTR as well as the upstream open reading frame. The authenticity of the IGF‐IR IRES has been confirmed by its sensitivity to deletion of the promoter from a bicistronic reporter construct, and its resistance in a monocistronic reporter construct to co‐expression of a viral 2A protease. We previously characterized HuR as a potent repressor of IGF‐IR translation. Here we demonstrate that hnRNP C competes with HuR for binding the IGF‐IR 5′‐UTR and enhances IRES‐mediated translation initiation in a concentration‐dependent manner. We observed changes in binding of hnRNP C versus HuR to the IGF‐IR 5′‐UTR in response to physiological alterations in cellular environment or proliferative status. Furthermore, we have found distinct alterations in the pattern of protein binding to the IGF‐IR 5′‐UTR in human breast tumor cells in which IGF‐IR IRES activity and relative translational efficiency are aberrantly increased. These results suggest that dysregulation of the IGF‐IR IRES through changes in the activities of RNA‐binding translation‐regulatory proteins could be responsible for IGF‐IR overexpression in a proportion of human breast tumors. J. Cell. Physiol. 217: 172–183, 2008.

The human IGF1R IRES likely operates through a Shine-Dalgarno-like interaction with the G961 loop (E-site) of the 18S rRNA and is kinetically modulated by a naturally-polymorphic polyU loop

IGF1R is a proto‐oncogene with potent mitogenic and antiapoptotic activities, and its expression must be tightly regulated to maintain normal cellular and tissue homeostasis. We previously demonstrated that translation of the human IGF1R mRNA is controlled by an internal ribosome entry site (IRES), and delimited the core functional IRES to a 90‐nucleotide segment of the 5′‐untranslated region positioned immediately upstream of the initiation codon. Here we have analyzed the sequence elements that contribute to the function of the core IRES. The Stem2/Loop2 sequence of the IRES exhibits near‐perfect Watson–Crick complementarity to the G961 loop (helix 23b) of the 18S rRNA, which is positioned within the E‐site on the platform of the 40S ribosomal subunit. Mutations that disrupt this complementarity have a negative impact on regulatory protein binding and dramatically decrease IRES activity, suggesting that the IGF1R IRES may recruit the 40S ribosome by a eukaryotic equivalent of the Shine–Dalgarno (mRNA–rRNA base‐pairing) interaction. The homopolymeric Loop3 sequence of the IRES modulates accessibility and limits the rate of translation initiation mediated through the IRES. Two functionally distinct allelic forms of the Loop3 poly(U)‐tract are prevalent in the human population, and it is conceivable that germ‐line or somatic variations in this sequence could predispose individuals to development of malignancy, or provide a selectable growth advantage for tumor cells. J. Cell. Biochem. 110: 531–544, 2010.

Inborn stress reactivity shapes adult behavioral consequences of early-life maternal separation stress.

Early-life experience strongly impacts neurodevelopment and stress susceptibility in adulthood. Maternal separation (MS), an established model of early-life adversity, has been shown to negatively impact behavioral and endocrine responses to stress in adulthood. However, the impact of MS in rats with heightened inborn stress susceptibility has not been fully explored. To address this issue we conducted MS in Wistar-Kyoto (WKY) rats, an animal model of comorbid depression and anxiety, and Wistar rats, which share a similar genetic background with WKYs. WKY and Wistar pups experienced either 180-min daily MS or 15-min separation (neonatal handling) during the first two postnatal weeks, and were tested for depressive- and anxiety- like behaviors in adulthood. Exposure to early-life MS in WKY rats decreased anxiety- and depressive- like behaviors, leading to increased exploration on the open field test (OFT), enhanced social interaction, and diminished immobility on the forced swim test. MS had an opposite effect in Wistar offspring, leading to enhanced anxiety-like behaviors, such as reduced OFT exploration and decreased social interaction. These findings are consistent with the match/mismatch theory of disease and the predictive adaptive response, which suggests that early life stress exposure can confer adaptive value in later life within certain individuals. Our data supports this theory, showing that early-life MS has positive and perhaps adaptive effects within stress-vulnerable WKY offspring. Future studies will be required to elucidate the neurobiological underpinnings of contrasting behavioral effects of MS on WKY vs. Wistar offspring.

mrtl—A translation/localization regulatory protein encoded within the human c-myc locus and distributed throughout the endoplasmic and nucleoplasmic reticular network

mrtl (myc‐related translation/localization regulatory factor) is a previously uncharacterized protein synthesized from the first open reading frame contained within the human c‐myc P0 transcript, ∼800 nucleotides upstream of the Myc coding sequence. The mrtl protein, 114 amino acids in length, is projected to contain an N‐terminal transmembrane domain and a highly charged C‐terminal interaction domain with homology to numerous RNA‐binding proteins. Using monoclonal antibodies raised against the hydrophilic C‐terminal domain, endogenous mrtl was visualized in human breast tumor cell lines and primary mammary epithelial cells at the nuclear envelope and contiguous endoplasmic/nucleoplasmic reticulum. mrtl colocalizes and coimmunoprecipitates with translation initiation factor eIF2α and the 40S ribosomal protein RACK1, and appears capable of binding specifically to the c‐myc RNA. Inducible ectopic overexpression of wild‐type mrtl interferes with the function of endogenous mrtl, which results in loss of Myc from the nucleus. Furthermore, treatment of cells with a peptide derived from the C‐terminal domain displaces endogenous mrtl and causes a dramatic reduction in total cellular Myc protein levels. Together with our previous work demonstrating complete loss of tumorigenicity in association with ectopic expression of the c‐myc P0 5′‐UTR (containing the mrtl coding sequence), these results suggest that mrtl may serve an important function in regulating Myc translation and localization to the nucleus, perhaps ultimately contributing to the role of the c‐myc locus in oncogenesis. J. Cell. Biochem. 105: 1092–1108, 2008.

Genetic predisposition to high anxiety- and depression-like behavior coincides with diminished DNA methylation in the adult rat amygdala.

HighlightsLow (vs. high) novelty‐reactive rats exhibit distinct amygdalar transcriptomes.DNA methylation patterns differed across the genome of low (vs. high) reactive rats.Low novelty‐reactive rats displayed many hypomethylated genomic sites.Increased dietary methyl donors improved helpless behavior in low reactive rats. ABSTRACT Understanding biological mechanisms that shape vulnerability to emotional dysfunction is critical for elucidating the neurobiology of psychiatric illnesses like anxiety and depression. To elucidate molecular and epigenetic alterations in the brain that contribute to individual differences in emotionality, our laboratory utilized a rodent model of temperamental differences. Rats bred for low response to novelty (Low Responders, LRs) are inhibited in novel situations and display high anxiety, helplessness, and diminished sociability compared to High Novelty Responder (HR) rats. Our current transcriptome profiling experiment identified widespread gene expression differences in the amygdala of adult HR/LR rats; we hypothesize that HR/LR gene expression and downstream behavioral differences stem from distinct epigenetic (specifically DNA methylation) patterning in the HR/LR brain. Although we found similar levels of DNA methyltransferase proteins in the adult HR/LR amygdala, next‐generation sequencing analysis of the methylome revealed 793 differentially methylated genomic sites between the groups. Most of the differentially methylated sites were hypermethylated in HR versus LR, so we next tested the hypothesis that enhancing DNA methylation in LRs would improve their anxiety/depression‐like phenotype. We found that increasing DNA methylation in LRs (via increased dietary methyl donor content) improved their anxiety‐like behavior and decreased their typically high levels of Forced Swim Test (FST) immobility; however, dietary methyl donor depletion exacerbated LRs’ high FST immobility. These data are generally consistent with findings in depressed patients showing that treatment with DNA methylation‐promoting agents improves depressive symptoms, and highlight epigenetic mechanisms that may contribute to individual differences in risk for emotional dysfunction.

Altered metabolic activity in the developing brain of rats predisposed to high versus low depression-like behavior.

Individual differences in human temperament can increase the risk of psychiatric disorders like depression and anxiety. Our laboratory utilized a rat model of temperamental differences to assess neurodevelopmental factors underlying emotional behavior differences. Rats selectively bred for low novelty exploration (Low Responders, LR) display high levels of anxiety- and depression-like behavior compared to High Novelty Responder (HR) rats. Using transcriptome profiling, the present study uncovered vast gene expression differences in the early postnatal HR versus LR limbic brain, including changes in genes involved in cellular metabolism. These data led us to hypothesize that rats prone to high (versus low) anxiety/depression-like behavior exhibit distinct patterns of brain metabolism during the first weeks of life, which may reflect disparate patterns of synaptogenesis and brain circuit development. Thus, in a second experiment we examined activity of cytochrome C oxidase (COX), an enzyme responsible for ATP production and a correlate of metabolic activity, to explore functional energetic differences in the HR/LR early postnatal brain. We found that HR rats display higher COX activity in the amygdala and specific hippocampal subregions compared to LRs during the first 2 weeks of life. Correlational analysis examining COX levels across several brain regions and multiple early postnatal time points suggested desynchronization in the developmental timeline of the limbic HR versus LR brain during the first two postnatal weeks. These early divergent COX activity levels may reflect altered circuitry or synaptic activity in the early postnatal HR/LR brain, which could contribute to the emergence of their distinct behavioral phenotypes.

A paternal methyl donor depleted diet leads to increased anxiety- and depression-like behavior in adult rat offspring

Epigenetic mechanisms such as DNA methylation elicit lasting changes in gene expression and likely mediate gene–environment interactions that shape brain development, behavior, and emotional health. Myriad environmental factors influence DNA methylation, including methyl donor content in the paternal diet, could influence methylation in offspring via changes in the paternal germ line. The present study examines the effects of paternal methyl donor dietary deficiency on offspring’s emotional behaviors, including anxiety, social interaction, and depression-like behavior. We previously found that rats bred to display high levels of anxiety- and depression-like behavior exhibit diminished DNA methylation in the amygdala. We also observed that depleting dietary methyl donor content exacerbated the rats’ already high levels of anxiety- and depression-like behavior. Here we sought to determine whether paternal dietary methyl donor depletion elicits intergenerational effects on first generation (F1) offspring’s behavior (potentially triggering a similar increase in anxiety- and/or depression-like behavior). Thus, adult male rats prone to high anxiety/depression-like behavior, were fed either a methyl donor depleted (DEP) or control (CON) diet for 5 weeks prior to mating. They were paired with females and resultant F1 male offspring were subjected to a behavioral test battery in adulthood. F1-DEP offspring showed a similar behavioral profile to the F0 males, including greater depression-like behavior in the forced swim test (FST) and increased anxiety-like behavior in the open field test (OFT). Future work will interrogate molecular changes in the brains of F1 offspring that mediate these intergenerational effects of paternal methyl donor dietary content on offspring emotional behavior.