Fragile X Mental Retardation Protein: Self-Regulation and miRNA Pathway Involvement

Fragile X syndrome, the most common form of inherited mental impairment in humans, affects 1 of 4000 males and 1 of 8000 females. It is caused by the absence of the fragile X mental retardation protein (FMRP), resulting from a CGG trinucleotide repeat expansion in the 5\u27-untranslated region (UTR) of the fragile x mental retardation-1 (FMR1) gene, and subsequent translational silencing of FMRP. FMRP, a proposed translational regulator of neuronal messenger RNA (mRNA) targets, has three RNA binding domains: two K-homology domains (KH1 and KH2) and one arginine-glycine-glycine (RGG) box domain. FMRP RGG box has been shown to bind with high affinity to G-quadruplex forming mRNAs. G-quadruplexes are formed by stacked G-quartets bonded by Hoogsteen base pairing and stabilized by monocations. FMRP undergoes alternative splicing, including the alternative splice site at exon 15, giving rise to FMRP minor isoforms, truncated within close proximity of the RGG box domain. The binding of FMRP to a proposed G quadruplex structure in the coding region of its own mRNA named FBS has been proposed to affect mRNA splicing events for FMRP minor isoforms. In this study we used biophysical methods to directly demonstrate the folding of FMR1 FBS into two specific G-quadruplexes and analyze its binding by the FMRP isoforms. Additionally, we analyzed the binding of an FMRP mutant in which Ser500 was replaced with Asp500 (ISOP), mimicking FMRP phosphorylation. We showed that the minor splice isoforms bind more tightly to the FBS mRNA, suggesting a negative feedback loop of FMRP binding to its mRNA to regulate alternate splicing. FMRP associates directly with the Ago1 protein, a key component in the microRNA (miRNA) pathway. Interestingly, one of the FMRP mRNA targets, the microtubule associated protein 1B (MAP1B) mRNA, has a G-quadruplex structure in its 5\u27-UTR shown to be bound by the FMRP RGG box, and a potential binding site for the miRNA let-7b in its 3\u27-UTR. In this study we investigated the binding of the let-7b miRNA to this sequence within MAP1B mRNA by using biophysical methods. Dr. Yue Feng at Emory University confirmed the translation regulation of let7b miRNA on MAP1B mRNA

As time flies by: Investigating cardiac aging in the short-lived Drosophila model.

Aging is associated with a decline in heart function across the tissue, cellular, and molecular levels. The risk of cardiovascular disease grows significantly over time, and as developed countries continue to see an increase in lifespan, the cost of cardiovascular healthcare for the elderly will undoubtedly rise. The molecular basis for cardiac function deterioration with age is multifaceted and not entirely clear, and there is a limit to what investigations can be performed on human subjects or mammalian models. Drosophila melanogaster has emerged as a useful model organism for studying aging in a short timeframe, benefitting from a suite of molecular and genetic tools and displaying highly conserved traits of cardiac senescence. Here, we discuss recent advances in our understanding of cardiac aging and how the fruit fly has aided in these developments

Expression patterns of cardiac aging in Drosophila.

Aging causes cardiac dysfunction, often leading to heart failure and death. The molecular basis of age-associated changes in cardiac structure and function is largely unknown. The fruit fly, Drosophila melanogaster, is well-suited to investigate the genetics of cardiac aging. Flies age rapidly over the course of weeks, benefit from many tools to easily manipulate their genome, and their heart has significant genetic and phenotypic similarities to the human heart. Here, we performed a cardiac-specific gene expression study on aging Drosophila and carried out a comparative meta-analysis with published rodent data. Pathway level transcriptome comparisons suggest that age-related, extra-cellular matrix remodeling and alterations in mitochondrial metabolism, protein handling, and contractile functions are conserved between Drosophila and rodent hearts. However, expression of only a few individual genes similarly changed over time between and even within species. We also examined gene expression in single fly hearts and found significant variability as has been reported in rodents. We propose that individuals may arrive at similar cardiac aging phenotypes via dissimilar transcriptional changes, including those in transcription factors and micro-RNAs. Finally, our data suggest the transcription factor Odd-skipped, which is essential for normal heart development, is also a crucial regulator of cardiac aging

Pseudo-acetylation of K326 and K328 of actin disrupts Drosophila melanogaster indirect flight muscle structure and performance

In striated muscle tropomyosin (Tm) extends along the length of F-actin-containing thin filaments. Its location governs access of myosin binding sites on actin and, hence, force production. Intermolecular electrostatic associations are believed to mediate critical interactions between the proteins. For example, actin residues K326, K328 and R147 were predicted to establish contacts with E181 of Tm. Moreover, K328 also potentially forms direct interactions with E286 of myosin when the motor is strongly bound. Recently, LC-MS/MS analysis of the cardiac acetyl-lysine proteome revealed K326 and K328 of actin were acetylated, a post-translational modification (PTM) that masks the residues’ inherent positive charges. Here, we tested the hypothesis that by removing the vital actin charges at residues 326 and 328, the PTM would perturb Tm positioning and/or strong myosin binding as manifested by altered skeletal muscle function and structure in the Drosophila melanogaster model system. Transgenic flies were created that permit tissue-specific expression of K326Q, K328Q, or K326Q/K328Q acetyl-mimetic actin and of wild-type actin via the UAS-GAL4 bipartite expression system. Compared to wild-type actin, muscle-restricted expression of mutant actin had a dose-dependent effect on flight ability. Moreover, excessive K328Q and K326Q/K328Q actin overexpression induced indirect flight muscle degeneration, a phenotype consistent with hypercontraction observed in other Drosophila myofibrillar mutants. Based on F-actin-Tm and F-actin-Tm-myosin models and on our physiological data, we conclude that acetylating K326 and K328 of actin alters electrostatic associations with Tm and/or myosin and thereby augments contractile properties. Our findings highlight the utility of Drosophila as a model that permits efficient targeted design and assessment of molecular and tissue-specific responses to muscle protein modifications, in vivo