Sde2 is an intron‐specific pre‐mRNA splicing regulator activated by ubiquitin‐like processing

The expression of intron‐containing genes in eukaryotes requires generation of protein‐coding messenger RNAs (mRNAs) via RNA splicing, whereby the spliceosome removes non‐coding introns from pre‐mRNAs and joins exons. Spliceosomes must ensure accurate removal of highly diverse introns. We show that Sde2 is a ubiquitin‐fold‐containing splicing regulator that supports splicing of selected pre‐mRNAs in an intron‐specific manner in Schizosaccharomyces pombe. Both fission yeast and human Sde2 are translated as inactive precursor proteins harbouring the ubiquitin‐fold domain linked through an invariant GGKGG motif to a C‐terminal domain (referred to as Sde2‐C). Precursor processing after the first di‐glycine motif by the ubiquitin‐specific proteases Ubp5 and Ubp15 generates a short‐lived activated Sde2‐C fragment with an N‐terminal lysine residue, which subsequently gets incorporated into spliceosomes. Absence of Sde2 or defects in Sde2 activation both result in inefficient excision of selected introns from a subset of pre‐mRNAs. Sde2 facilitates spliceosomal association of Cactin/Cay1, with a functional link between Sde2 and Cactin further supported by genetic interactions and pre‐mRNA splicing assays. These findings suggest that ubiquitin‐like processing of Sde2 into a short‐lived activated form may function as a checkpoint to ensure proper splicing of certain pre‐mRNAs in fission yeast.     

Phosphorylation of Argonaute proteins affects mRNA binding and is essential for microRNA‐guided gene silencing in vivo

Argonaute proteins associate with microRNAs and are key components of gene silencing pathways. With such a pivotal role, these proteins represent ideal targets for regulatory post‐translational modifications. Using quantitative mass spectrometry, we find that a C‐terminal serine/threonine cluster is phosphorylated at five different residues in human and Caenorhabditis elegans. In human, hyper‐phosphorylation does not affect microRNA binding, localization, or cleavage activity of Ago2. However, mRNA binding is strongly affected. Strikingly, on Ago2 mutants that cannot bind microRNAs or mRNAs, the cluster remains unphosphorylated indicating a role at late stages of gene silencing. In C. elegans, the phosphorylation of the conserved cluster of ALG‐1 is essential for microRNA function in vivo. Furthermore, a single point mutation within the cluster is sufficient to phenocopy the loss of its complete phosphorylation. Interestingly, this mutant retains its capacity to produce and bind microRNAs and represses expression when artificially tethered to an mRNA. Altogether, our data suggest that the phosphorylation state of the serine/threonine cluster is important for Argonaute–mRNA interactions.     

Neuronal RNA‐binding protein HuD regulates addiction‐related gene expression and behavior

The neuronal RNA‐binding protein HuD is involved in synaptic plasticity and learning and memory mechanisms. These effects are thought to be due to HuD‐mediated stabilization and translation of target mRNAs associated with plasticity. To investigate the potential role of HuD in drug addiction, we first used bioinformatics prediction algorithms together with microarray analyses to search for specific genes and functional networks upregulated within the forebrain of HuD overexpressing mice (HuD_OE). When this set was further limited to genes in the knowledgebase of addiction‐related genes database (KARG) that contains predicted HuD‐binding sites in their 3′ untranslated regions (3′UTRs), we found that HuD regulates networks that have been associated with addiction‐like behavior. These genes included Bdnf and Camk2a, 2 previously validated HuD targets. Since addiction is hypothesized to be a disorder stemming from altered gene expression causing aberrant plasticity, we sought to test the role of HuD in cocaine conditioned placed preference (CPP), a model of addiction‐related behaviors. HuD mRNA and protein were upregulated by CPP within the nucleus accumbens of wild‐type C57BL/6J mice. These changes were associated with increased expression of Bdnf and Camk2a mRNA and protein. To test this further, we trained HuD_OE and wild‐type mice in CPP and found that HuD_OE mice showed increased cocaine CPP compared with controls. This was also associated with elevated expression of HuD target mRNAs and proteins, CaMKIIα and BDNF. These findings suggest HuD involvement in addiction‐related behaviors such as cocaine conditioning and seeking, through increased plasticity‐related gene expression.     

Cell‐specific changes in expression of mRNAs encoding splice variants of Aplysia cell adhesion molecule accompany long‐term synaptic plasticity

Aplysia neurons express several splice variants of apCAM, a member of the Ig superfamily of cell adhesion molecules. The major transmembrane isoform is endocytosed in sensory neurons (SNs) during the early phases of long‐term facilitation (LTF) of SN synapses evoked by serotonin (5‐HT) or in the motor neuron L7 during the early phases of long‐term depression (LTD) of SN synapses evoked by Phe‐Met‐Arg‐Phe‐amide (FMRFa). We used single cell RT‐PCR to evaluate whether expression of mRNAs encoding for different apCAM isoforms in SNs and L7 is regulated during LTF produced by 5‐HT, and LTD produced by FMRFa. Single SNs and L7s express mRNAs encoding for all major isoforms, but the proportion of each isoform expressed differs for the two cells. SN expresses more mRNA encoding for GPI‐linked isoforms, while L7 expresses more mRNA encoding for the major transmembrane isoform. The neuromodulators produced significant changes in the proportional levels of mRNAs encoding for specific apCAM isoforms during the first 4 h after treatments without affecting overall levels of apCAM mRNA. 5‐HT evoked changes that exaggerated cell‐specific differences in isoform expression. FMRFa evoked changes that reduced cell‐specific differences in isoform expression. The effects of the neuromodulators on apCAM mRNA expression were not detected when cells were cultured alone or when SNs were cocultured with another motor cell that failed to induce synapse formation (L11). The results suggest that rapid cell‐specific regulation of splice variant expression may contribute to different forms of long‐term synaptic plasticity. © 2000 John Wiley & Sons, Inc. J Neurobiol 45: 152–161, 2000     

Development and optimization of a cell‐based assay for the selection of synthetic compounds that potentiate bone morphogenetic protein‐2 activity

The requirement of large amounts of the recombinant human bone morphogenetic protein‐2 (BMP‐2) produces a huge translational barrier for its routine clinical use due to high cost. This leads to an urgent need to develop alternative methods to lower costs and/or increase efficacies for using BMP‐2. In this study, we describe the development and optimization of a cell‐based assay that is sensitive, reproducible, and reliable in identifying reagents that potentiate the effects of BMP‐2 in inducing transdifferentiation of C2C12 myoblasts into the osteoblastic phenotype. The assay is based on a BMP‐responsive Smad1‐driven luciferase reporter gene. LIM mineralization protein‐1 (LMP‐1) is a novel intracellular LIM domain protein that has been shown by our group to enhance cellular responsiveness to BMP‐2. Our previous report elucidated that the binding of LMP‐1 with the WW2 domain in Smad ubiquitin regulatory factor‐1 (Smurf1) rescues the osteogenic Smads from degradation. Here, using the optimized cell‐based assay, we first evaluated the activity of the recombinantly prepared proteins, LMP‐1, and its mutant (LMP‐1ΔSmurf1) that lacks the Smurf1‐WW2 domain‐binding motif. Both the wild type and the mutant proteins were engineered to contain an 11‐amino acid HIV‐TAT protein derived membrane transduction domain to aid the cellular delivery of recombinant proteins. The cell‐based reporter assay confirmed that LMP‐1 potentiates the BMP‐induced stimulation of C2C12 cells towards the osteoblastic phenotype. The potentiating effect of LMP‐1 was significantly reduced when a specific‐motif known to interact with Smurf1 was mutated. We validated the results obtained in the reporter assay by also monitoring the expression of mRNA for osteocalcin and alkaline phosphatase (ALP) which is widely accepted osteoblast differentiation marker genes. Finally, we provide further confirmation of our results by measuring the activity of alkaline phosphatase in support of the accuracy and reliability of our cell‐based assay. Direct delivery of synthesized protein can be limited by high cost, instability or inadequate post‐translational modifications. Thus, there would be a clear benefit for a low cost, cell penetrable chemical compound. We successfully used our gene expression‐based assay to choose an active compound from a select group of compounds that were identified by computational screenings as the most likely candidates for mimicking the function of LMP‐1. Among them, we selected SVAK‐3, a compound that showed a dose‐dependent potentiation of BMP‐2 activity in inducing osteoblastic differentiation of C2C12 cells. We show that either the full length LMP‐1 protein or its potential mimetic compound consistently exhibit similar potentiation of BMP‐2 activity even when multiple markers of the osteoblastic phenotype were parallely monitored. Published in 2009 by John Wiley & Sons, Ltd.     

A genome‐scale analysis of mRNAs targeting to plant mitochondria: upstream AUGs in 5' untranslated regions reduce mitochondrial association

Intracellular sorting of mRNAs is an essential process for regulating gene expression and protein localization. Most mitochondrial proteins are nuclear‐encoded and imported into the mitochondria through post‐translational or co‐translational processes. In the latter case, mRNAs are found to be enriched in the vicinity of mitochondria. A genome‐scale analysis of mRNAs associated with mitochondria has been performed to determine plant cytosolic mRNAs targeted to the mitochondrial surface. Many messengers encoding mitochondrial proteins were found associated with mitochondria. These mRNAs correspond to particular functions and complexes, such as respiration or mitoribosomes, which indicates a coordinated control of mRNA localization within metabolic pathways. In addition, upstream AUGs in 5' untranslated regions (UTRs), which modulate the translation efficiency of downstream sequences, were found to negatively affect the association of mRNAs with mitochondria. A mutational approach coupled with in vivo mRNA visualization confirmed this observation. Moreover, this technique allowed the identification of 3'‐UTRs as another essential element for mRNA localization at the mitochondrial surface. Therefore, this work offers new insights into the mechanism, function and regulation of the association of cytosolic mRNAs with plant mitochondria.     

Two new miR‐16 targets: caprin‐1 and HMGA1, proteins implicated in cell proliferation

Background information. miRNAs (microRNAs) are a class of non‐coding RNAs that inhibit gene expression by binding to recognition elements, mainly in the 3′ UTR (untranslated region) of mRNA. A single miRNA can target several hundred mRNAs, leading to a complex metabolic network. miR‐16 (miRNA‐16), located on chromosome 13q14, is involved in cell proliferation and apoptosis regulation; it may interfere with either oncogenic or tumour suppressor pathways, and is implicated in leukaemogenesis. These data prompted us to search for and validate novel targets of miR‐16. Results. In the present study, by using a combined bioinformatics and molecular approach, we identified two novel putative targets of miR‐16, caprin‐1 (cytoplasmic activation/proliferation‐associated protein‐1) and HMGA1 (high‐mobility group A1), and we also studied cyclin E which had been previously recognized as an miR‐16 target by bioinformatics database. Using luciferase activity assays, we demonstrated that miR‐16 interacts with the 3′ UTR of the three target mRNAs. We showed that miR‐16, in MCF‐7 and HeLa cell lines, down‐regulates the expression of caprin‐1, HMGA1a, HMGA1b and cyclin E at the protein level, and of cyclin E, HMGA1a and HMGA1b at the mRNA levels. Conclusions. Taken together, our data demonstrated that miR‐16 can negatively regulate two new targets, HMGA1 and caprin‐1, which are involved in cell proliferation. In addition, we also showed that the inhibition of cyclin E expression was due, at least in part, to a decrease in its mRNA stability.