Coupling of Gab1 to C-Met, Grb2, and Shp2 Mediates Biological Responses

Gab1 is a substrate of the receptor tyrosine kinase c-Met and involved in c-Met–specific branching morphogenesis. It associates directly with c-Met via the c-Met–binding domain, which is not related to known phosphotyrosine-binding domains. In addition, Gab1 is engaged in a constitutive complex with the adaptor protein Grb2. We have now mapped the c-Met and Grb2 interaction sites using reverse yeast two-hybrid technology. The c-Met–binding site is localized to a 13–amino acid region unique to Gab1. Insertion of this site into the Gab1-related protein p97/Gab2 was sufficient to confer c-Met–binding activity. Association with Grb2 was mapped to two sites: a classical SH3-binding site (PXXP) and a novel Grb2 SH3 consensus-binding motif (PX(V/I)(D/N)RXXKP). To detect phosphorylation-dependent interactions of Gab1 with downstream substrates, we developed a modified yeast two-hybrid assay and identified PI(3)K, Shc, Shp2, and CRKL as interaction partners of Gab1. In a trk-met-Gab1–specific branching morphogenesis assay, association of Gab1 with Shp2, but not PI(3)K, CRKL, or Shc was essential to induce a biological response in MDCK cells. Overexpression of a Gab1 mutant deficient in Shp2 interaction could also block HGF/SF-induced activation of the MAPK pathway, suggesting that Shp2 is critical for c-Met/Gab1-specific signaling

The uORF-containing thrombopoietin mRNA escapes nonsense-mediated decay (NMD)

Platelet production is induced by the cytokine thrombopoietin (TPO). It is physiologically critical that TPO expression is tightly regulated, because lack of TPO causes life-threatening thrombocytopenia while an excess of TPO results in thrombocytosis. The plasma concentration of TPO is controlled by a negative feedback loop involving receptor-mediated uptake of TPO by platelets. Furthermore, TPO biosynthesis is limited by upstream open reading frames (uORFs) that curtail the translation of the TPO mRNA. uORFs are suggested to activate RNA degradation by nonsense-mediated decay (NMD) in a number of physiological transcripts. Here, we determine whether NMD affects TPO expression. We show that reporter mRNAs bearing the seventh TPO uORF escape NMD. Importantly, endogenously expressed TPO mRNA from HuH7 cells is unaffected by abrogation of NMD by RNAi. Thus, regulation of TPO expression is independent of NMD, implying that mRNAs bearing uORFs cannot generally be considered to represent NMD targets

Global variations in diabetes mellitus based on fasting glucose and haemogloblin A1c

Fasting plasma glucose (FPG) and haemoglobin A1c (HbA1c) are both used to diagnose diabetes, but may identify different people as having diabetes. We used data from 117 population-based studies and quantified, in different world regions, the prevalence of diagnosed diabetes, and whether those who were previously undiagnosed and detected as having diabetes in survey screening had elevated FPG, HbA1c, or both. We developed prediction equations for estimating the probability that a person without previously diagnosed diabetes, and at a specific level of FPG, had elevated HbA1c, and vice versa. The age-standardised proportion of diabetes that was previously undiagnosed, and detected in survey screening, ranged from 30% in the high-income western region to 66% in south Asia. Among those with screen-detected diabetes with either test, the agestandardised proportion who had elevated levels of both FPG and HbA1c was 29-39% across regions; the remainder had discordant elevation of FPG or HbA1c. In most low- and middle-income regions, isolated elevated HbA1c more common than isolated elevated FPG. In these regions, the use of FPG alone may delay diabetes diagnosis and underestimate diabetes prevalence. Our prediction equations help allocate finite resources for measuring HbA1c to reduce the global gap in diabetes diagnosis and surveillance.peer-reviewe

Heterogeneous contributions of change in population distribution of body mass index to change in obesity and underweight NCD Risk Factor Collaboration (NCD-RisC)

From 1985 to 2016, the prevalence of underweight decreased, and that of obesity and severe obesity increased, in most regions, with significant variation in the magnitude of these changes across regions. We investigated how much change in mean body mass index (BMI) explains changes in the prevalence of underweight, obesity, and severe obesity in different regions using data from 2896 population-based studies with 187 million participants. Changes in the prevalence of underweight and total obesity, and to a lesser extent severe obesity, are largely driven by shifts in the distribution of BMI, with smaller contributions from changes in the shape of the distribution. In East and Southeast Asia and sub-Saharan Africa, the underweight tail of the BMI distribution was left behind as the distribution shifted. There is a need for policies that address all forms of malnutrition by making healthy foods accessible and affordable, while restricting unhealthy foods through fiscal and regulatory restrictions

Exon Junction Complexes: Supervising the Gene Expression Assembly Line

The exon junction complex (EJC) is an RNA-binding protein complex that is assembled and deposited onto mRNAs during splicing. The EJC comprises four core components that bind to not only canonical sites upstream of exon-exon junctions, but also to noncanonical sites at other positions in exons. EJC-associated proteins are recruited by the EJC at different steps of gene expression to execute the multiple functions of the EJC. Recently, new insights have been obtained into how EJCs stimulate pre-mRNA splicing, and mRNA export, translation, and degradation. Furthermore, mutations in EJC core components were shown to result in severe disorders in humans, demonstrating the critical physiological role of the EJC. Hence, the EJC has been identified as an important player in post-transcriptional gene regulation in metazoans

Physiological and pathophysiological role of nonsense-mediated mRNA decay

Nonsense-mediated messenger RNA (mRNA) decay (NMD) is a quality control mechanism that degrades irregular or faulty mRNAs. NMD mainly degrades mRNAs, which contain a premature termination codon (PTC) and therefore encode a truncated protein. Furthermore, NMD alters the expression of different types of cellular mRNAs, the so-called endogenous NMD substrates. In this review, we focus on the impact of NMD on cellular and molecular physiology. We specify key classes of NMD substrates and provide a detailed overview of the physiological function of gene regulation by NMD. We also describe different mechanisms of NMD substrate degradation and how the regulation of the NMD machinery affects cellular physiology. Finally, we outline the physiological functions of central NMD factors

Harnessing short poly(A)-binding protein-interacting peptides for the suppression of nonsense-mediated mRNA decay

Nonsense-mediated mRNA decay (NMD) is a cellular process that eliminates messenger RNA (mRNA) substrates with premature translation termination codons (PTCs). In addition, NMD regulates the expression of a number of physiological mRNAs, for example transcripts containing long 3'UTRs. Current models implicate the interaction between cytoplasmic poly(A)-binding protein (PABPC1) and translation termination in NMD. Accordingly, PABPC1 present within close proximity of a termination codon antagonizes NMD. Here, we use reporter mRNAs with different NMD-inducing 3'UTRs to establish a general NMD-inhibiting property of PABPC1. NMD-inhibition is not limited to PABPC1, but can also be achieved by peptides consisting of the PABP-interacting motif 2 (PAM2) of different proteins when recruited to an NMD-inhibiting position of NMD reporter transcripts. The short PAM2 peptides efficiently suppress NMD activated by a long 3'UTR, an exon-junction complex (EJC) and individual EJC components, and stabilize a PTC-containing beta-globin mRNA. In conclusion, our results establish short PABPC1-recruiting peptides as potent but position-dependent inhibitors of mammalian NMD

Studying the composition of mRNPs in vitro using splicing-competent cell extracts

The correct processing and faithful decoding of mRNAs during gene expression depends on the interaction with RNA-binding proteins (RBPs). The association of RBPs with pre-mRNAs starts during transcription by RNA polymerase II and undergoes constant remodeling during pre-mRNA processing and later steps of genes expression. Recently developed high throughput methods enabled to define RBP binding sites in vivo and to identify a large number of novel RBPs in eukaryotic cells. However, the detailed characterization of RBP-RNA interactions as well as the analysis of functional RNPs is greatly facilitated by well-defined in vitro systems. Here, we describe a versatile method to study the assembly and splicing-dependent remodeling of mRNPs in vitro. This method employs splicing-competent whole cell extracts (WCE) generated from transfected human embryonic kidney (HEK) 293 cells. FLAG-tagged proteins present in the WCE are incorporated into mRNPs in vitro and afterwards used to immunoprecipitate substrate RNAs. We outline the principles of purifying in vitro assembled mRNPs and provide detailed protocols for the preparation and use of whole cell extracts. Alternative purification strategies and RNA substrates are discussed. (C) 2013 Elsevier Inc. All rights reserved