Functional cloning of BRF1, a regulator of ARE-dependent mRNA turnover

To identify regulators of AU-rich element (ARE)-dependent mRNA turnover we have followed a genetic approach using a mutagenized cell line (slowC) that fails to degrade cytokine mRNA. Accordingly, a GFP reporter construct whose mRNA is under control of the ARE from interleukin-3 gives an increased fluorescence signal in slowC. Here we describe rescue of slowC by a retroviral cDNA library. Flow cytometry allowed us to isolate revertants with reconstituted rapid mRNA decay. The cDNA was identified as butyrate response factor-1 (BRF1), encoding a zinc finger protein homologous to tristetraprolin. Mutant slowC carries frame-shift mutations in both BRF1 alleles, whereas slowB with intermediate decay kinetics is heterozygous. By use of small interfering (si)RNA, independent evidence for an active role of BRF1 in mRNA degradation was obtained. In transiently transfected NIH 3T3 cells, BRF1 accelerated mRNA decay and antagonized the stabilizing effect of PI3-kinase, while mutation of the zinc fingers abolished both function and ARE-binding activity. This approach, which identified BRF1 as an essential regulator of ARE-dependent mRNA decay, should also be applicable to other cis-elements of mRNA turnover.     

3'-end processing of the maize 27 kDa zein mRNA

Cis -regulatory elements involved in the mRNA 3'-end processing of the 27 kDa zein gene have been investigated by deletion and site-directed mutagenesis analyses. In the 3' flanking region of the 27 kDa zein gene, several AATAAA-like sequences and a sequence resembling the mammalian GT-rich sequence are present around the polyadenylation sites. Among the multiple AATAAA-like sequences, the duplicated AATGAA motifs, located 30–40 bp upstream from the polyadenylation sites, have been shown to play roles as polyadenylation signals. Although either of the two AATGAA motifs can function as a polyadenylation signal in chimeric gene constructs, the one proximal to the polyadenylation sites is likely to be the functional polyadenylation signal in the 27 kDa zein gene. Deletion of the downstream GT-rich sequence as well as alteration of the sequence surrounding the poly-adenylation sites has little effect on the mRNA 3'-end processing. However, the sequence elements located upstream from the polyadenylation signals are essential for the mRNA 3'-end processing. Mutations in the AATGAA motifs or the upstream sequences reduced the level of a reporter gene expression. A model depicting the mechanism involved in the 3'-end processing of the 27 kDa zein mRNA is presented.     

A novel function for the U2AF 65 splicing factor in promoting pre-mRNA 3'-end processing

Splicing and 3′-end processing (including cleavage and polyadenylation) of vertebrate pre-mRNAs are tightly coupled events that contribute to the extensive molecular network that coordinates gene expression. Sequences within the terminal intron of genes are essential to stimulate pre-mRNA 3′-end processing, although the factors mediating this effect are unknown. Here, we show that the pyrimidine tract of the last splice acceptor site of the human β-globin gene is necessary to stimulate mRNA 3′-end formation in vivo and binds the U2AF 65 splicing factor. Naturally occurring β-thalassaemia-causing mutations within the pyrimidine tract reduces both U2AF 65 binding and 3′-end cleavage efficiency. Significantly, a fusion protein containing U2AF 65, when tethered upstream of a cleavage/polyadenylation site, increases 3′-end cleavage efficiency in vitro and in vivo. Therefore, we propose that U2AF 65 promotes 3′-end processing, which contributes to 3′-terminal exon definition.     

A novel function for glucocerebrosidase as a regulator of sterylglucoside metabolism

BackgroundSterols are major cell membrane lipids, and in many organisms they are modified with glucose to generate sterylglucosides. Glucosylation dramatically changes the functional properties of sterols. The formation of sterylglucosides from sterols in plants, fungi, and bacteria uses UDP-glucose as a glucose donor. By contrast, sterylglucoside biosynthesis in mammals is catalyzed by the transglucosylation activity of glucocerebrosidases, with glucosylceramide acting as the glucose donor. Recent success in isolation and structural determination of sterylglucosides in the vertebrate central nervous system shows that transglucosylation also occurs in vivo. These analyses also revealed that sterylglucoside aglycons are composed of several cholesterol-related metabolites, including a plant-type sitosteryl.Scope of reviewIn this review, we discuss the biological functions and metabolism of sterylglucosides. We also summarize new findings from studies on the metabolism of vertebrate sterylglucosides and review the circumstances underlying the recent discovery of sterylglucosides in vertebrate brain. Finally, we discuss the role of sterylglucosides in a variety of neurodegenerative disorders such as Gaucher disease and Parkinson’s disease.Major conclusionsThe biological significance of UDP-glucose-independent sterol glucosylation is still unknown, but it is plausible that glucosylation may provide sterols with novel biological functions. Even though sterol glucosylation is a simple reaction, it can dramatically change the physical properties of sterols.General significanceSterylglucosides may play roles in various physiological processes and in the pathogenesis of different diseases. Arriving at a better understanding of them at the organ and cellular level may open up new approaches to developing therapeutics for a variety of diseases. This article is part of a Special Issue entitled Neuro-glycoscience, edited by Kenji Kadomatsu and Hiroshi Kitagawa.     

A novel function of Drosophila eIF4A as a negative regulator of Dpp/BMP signalling that mediates SMAD degradation

Signalling by the TGF-beta superfamily member and BMP orthologue Decapentaplegic (Dpp) is crucial for multiple developmental programmes and has to be tightly regulated. Here, we demonstrate that the Drosophila Dpp pathway is negatively regulated by eukaryotic translation initiation factor 4A (eIF4A), which mediates activation-dependent degradation of the Dpp signalling components Mad and Medea. eIF4A mutants exhibit increased Dpp signalling and accumulation of Mad and phospho-Mad. Overexpression of eIF4A decreases Dpp signalling and causes loss of Mad and phospho-Mad. Furthermore, eIF4A physically associates with Mad and Medea, and promotes their degradation following activation of Dpp signalling in a translation-independent manner. Finally, we show that eIF4A acts synergistically with, but independently of, the ubiquitin ligase DSmurf, indicating that a dual system controls SMAD degradation. Thus, in addition to being an obligatory component of the cap-dependent translation initiation complex, eIF4A has a novel function as a specific inhibitor of Dpp signalling that mediates the degradation of SMAD homologues.     

Genome level analysis of rice mRNA 3'-end processing signals and alternative polyadenylation

The position of a poly(A) site of eukaryotic mRNA is determined by sequence signals in pre-mRNA and a group of polyadenylation factors. To reveal rice poly(A) signals at a genome level, we constructed a dataset of 55 742 authenticated poly(A) sites and characterized the poly(A) signals. This resulted in identifying the typical tripartite cis-elements, including FUE, NUE and CE, as previously observed in Arabidopsis. The average size of the 3′-UTR was 289 nucleotides. When mapped to the genome, however, 15% of these poly(A) sites were found to be located in the currently annotated intergenic regions. Moreover, an extensive alternative polyadenylation profile was evident where 50% of the genes analyzed had more than one unique poly(A) site (excluding microheterogeneity sites), and 13% had four or more poly(A) sites. About 4% of the analyzed genes possessed alternative poly(A) sites at their introns, 5′-UTRs, or protein coding regions. The authenticity of these alternative poly(A) sites was partially confirmed using MPSS data. Analysis of nucleotide profile and signal patterns indicated that there may be a different set of poly(A) signals for those poly(A) sites found in the coding regions. Based on the features of rice poly(A) signals, an updated algorithm termed PASS-Rice was designed to predict poly(A) sites.     

Mutant-specific gene expression profiling identifies SRY-related HMG box 11b (SOX11b) as a novel regulator of vascular development in zebrafish

Previous studies have identified two zebrafish mutants, cloche and groom of cloche, which lack the majority of the endothelial lineage at early developmental stages. However, at later stages, these avascular mutant embryos generate rudimentary vessels, indicating that they retain the ability to generate endothelial cells despite this initial lack of endothelial progenitors. To further investigate molecular mechanisms that allow the emergence of the endothelial lineage in these avascular mutant embryos, we analyzed the gene expression profile using microarray analysis on isolated endothelial cells. We find that the expression of the genes characteristic of the mesodermal lineages are substantially elevated in the kdrl ⁺ cells isolated from avascular mutant embryos. Subsequent validation and analyses of the microarray data identifies Sox11b, a zebrafish ortholog of SRY-related HMG box 11 (SOX11), which have not previously implicated in vascular development. We further define the function sox11b during vascular development, and find that Sox11b function is essential for developmental angiogenesis in zebrafish embryos, specifically regulating sprouting angiogenesis. Taken together, our analyses illustrate a complex regulation of endothelial specification and differentiation during vertebrate development.     

Kinetic study of the HIV-1 DNA 3'-end processing

The 3'-processing of viral DNA extremities is the first step in the integration process catalysed by human immunodeficiency virus (HIV)-1 integrase (IN). This reaction is relatively inefficient and processed DNAs are usually detected in vitro under conditions of excess enzyme. Despite such experimental conditions, steady-state Michaelis-Menten formalism is often applied to calculate characteristic equilibrium/kinetic constants of IN. We found that the amount of processed product was not significantly affected under conditions of excess DNA substrate, indicating that IN has a limited turnover for DNA cleavage. Therefore, IN works principally in a single-turnover mode and is intrinsically very slow (single-turnover rate constant = 0.004 min(-1)), suggesting that IN activity is mainly limited at the chemistry step or at a stage that precedes chemistry. Moreover, fluorescence experiments showed that IN-DNA product complexes were very stable over the time-course of the reaction. Binding isotherms of IN to DNA substrate and product also indicate tight binding of IN to the reaction product. Therefore, the slow cleavage rate and limited product release prevent or greatly reduce subsequent turnover. Nevertheless, the time-course of product formation approximates to a straight line for 90 min (apparent initial velocity), but we show that this linear phase is due to the slow single-turnover rate constant and does not indicate steady-state multiple turnover. Finally, our data ruled out the possibility that there were large amounts of inactive proteins or dead-end complexes in the assay. Most of complexes initially formed were active although dramatically slow.     

The role of the Brr5/Ysh1 C-terminal domain and its homolog Syc1 in mRNA 3'-end processing in Saccharomyces cerevisiae

The cleavage/polyadenylation factor (CPF) of Saccharomyces cerevisiae is thought to provide the catalytic activities of the mRNA 3'-end processing machinery, which include endonucleolytic cleavage at the poly(A) site, followed by synthesis of an adenosine polymer onto the new 3'-end by the CPF subunit Pap1. Because of similarity to other nucleases in the metallo-beta-lactamase family, the Brr5/Ysh1 subunit has been proposed to be the endonuclease. The C-terminal domain of Brr5 lies outside of beta-lactamase homology, and its function has not been elucidated. We show here that this region of Brr5 is necessary for cell viability and mRNA 3'-end processing. It is highly homologous to another CPF subunit, Syc1. Syc1 is not essential, but its removal improves the growth of other processing mutants at restrictive temperatures and restores in vitro processing activity to cleavage/ polyadenylation-defective brr5-1 extract. Our findings suggest that Syc1, by mimicking the essential Brr5 C-terminus, serves as a negative regulator of mRNA 3'-end formation.     

The gut microbiota as a novel regulator of cardiovascular function and disease

The gut microbiome has emerged as a critical regulator of human physiology. Deleterious changes to the composition or number of gut bacteria, commonly referred to as gut dysbiosis, has been linked to the development and progression of numerous diet-related diseases, including cardiovascular disease (CVD). Most CVD risk factors, including aging, obesity, certain dietary patterns, and a sedentary lifestyle, have been shown to induce gut dysbiosis. Dysbiosis is associated with intestinal inflammation and reduced integrity of the gut barrier, which in turn increases circulating levels of bacterial structural components and microbial metabolites that may facilitate the development of CVD. The aim of the current review is to summarize the available data regarding the role of the gut microbiome in regulating CVD function and disease processes. Particular emphasis is placed on nutrition-related alterations in the microbiome, as well as the underlying cellular mechanisms by which the microbiome may alter CVD risk.     

Identification of a novel phosphorylation site in protein phosphatase inhibitor-1 as a negative regulator of cardiac function

Human and experimental heart failure is characterized by increases in type-1 protein phosphatase activity, which may be partially attributed to inactivation of its endogenous regulator, protein phosphatase inhibitor-1. Inhibitor-1 represents a nodal integrator of two major second messenger pathways, adenosine 3',5'-cyclic monophosphate (cAMP) and calcium, which mediate its phosphorylation at threonine 35 and serine 67, respectively. Here, using recombinant inhibitor-1 wild-type and mutated proteins, we identified a novel phosphorylation site in inhibitor-1, threonine 75. This phosphoamino acid was phosphorylated in vitro by protein kinase Calpha independently and to the same extent as serine 67, the previous protein kinase Calpha-identified site. Generation of specific antibodies for the phosphorylated and dephosphorylated threonine 75 revealed that this site is phosphorylated in rat and dog hearts. Adenoviral-mediated expression of the constitutively phosphorylated threonine 75 inhibitor-1 in isolated myocytes was associated with specific stimulation of type-1 protein phosphatase activity and marked inhibition of the sarcoplasmic calcium pump affinity for calcium, resulting in depressed contractility. Thus, phosphorylation of inhibitor-1 at threonine 75 represents a new mechanism of cardiac contractility regulation, partially through the alteration of sarcoplasmic reticulum calcium transport activity.     

Sequence requirements of the bidirectional yeast TRP4 mRNA 3'-end formation signal.

The yeast TRP4 3'-end formation signal functions in both orientations in an in vivo test system. We show here that the TRP4 3'-end formation element consists of two functionally different sequence regions. One region of approximately 70 nucleotides is located in the untranslated region between the translational stop codon and the major poly(A) site. The major poly(A) site is not part of this region and can be deleted without a decrease in TRP4 3'-end formation. 5'and 3'deletions and point mutations within this region affected 3'-end formation similarly in both orientations. In the center of this region the motif TAGT is located on the antisense strand. Point mutations within this motif resulted in a drastic reduce of 3'-end formation activity in both orientations. A second region consists of the 3'-end of the TRP4 open reading frame and is required for 3'-end formation in forward orientation. A single point mutation in a TAGT motif of the TRP4 open reading frame abolished TRP4 mRNA 3'-end formation in forward orientation and had no effect on the reverse orientation.