Search

International audience

TRanscription and EXport (TREX) is a conserved multisubunit complex essential for embryogenesis, organogenesis and cellular differentiation throughout life. By linking transcription, mRNA processing and export together, it exerts a physiologically vital role in the gene expression pathway. In addition, this complex prevents DNA damage and regulates the cell cycle by ensuring optimal gene expression. As the extent of TREX activity in viral infections, amyotrophic lateral sclerosis and cancer emerges, the need for a greater understanding of TREX function becomes evident. A complete elucidation of the composition, function and interactions of the complex will provide the framework for understanding the molecular basis for a variety of diseases. This review details the known composition of TREX, how it is regulated and its cellular functions with an emphasis on mammalian systems.

Cancer testis antigens (CTAs) represented a poorly characterized group of proteins whose expression is normally restricted to testis but are frequently up-regulated in cancer cells. Here we show that one CTA, Luzp4, is an mRNA export adaptor. It associates with the TREX mRNA export complex subunit Uap56 and harbours a Uap56 binding motif, conserved in other mRNA export adaptors. Luzp4 binds the principal mRNA export receptor Nxf1, enhances its RNA binding activity and complements Alyref knockdown in vivo. Whilst Luzp4 is up-regulated in a range of tumours, it appears preferentially expressed in melanoma cells where it is required for growth.

andida albicans hyphae grow in a highly polarized fashion from their tips. This polarized growth requires the continuous delivery of secretory vesicles to the tip region. Vesicle delivery depends on Sec2p, the Guanine Exchange Factor (GEF) for the Rab GTPase Sec4p. GTP bound Sec4p is required for the transit of secretory vesicles from the trans-Golgi to sites of polarized growth. We previously showed that phosphorylation of Sec2p at residue S584 was necessary for Sec2p to support hyphal, but not yeast growth. Here we show that on secretory vesicles SEC2 mRNA is physically associated with Sec2p. Moreover, we show that the phosphorylation of S584 allows SEC2 mRNA to dissociate from Sec2p and we speculate that this is necessary for Sec2p function and/or translation. During hyphal extension, the growing tip may be separated from the nucleus by up to 15 μm. Transport of SEC2 mRNA on secretory vesicles to the tip localizes SEC2 translation to tip allowing a sufficient accumulation of this key protein at the site of polarized growth.

GGGGCC repeat expansions of C9orf72 represent the most common genetic variant of amyotrophic lateral sclerosis and\ud frontotemporal degeneration, but the mechanism of pathogenesis is unclear. Recent reports have suggested that the transcribed\ud repeat might form toxic RNA foci that sequester various RNA processing proteins. Consensus as to the identity of the binding\ud partners is missing and whole neuronal proteome investigation is needed. Using RNA fluorescence in situ hybridization we first\ud identified nuclear and cytoplasmic RNA foci in peripheral and central nervous system biosamples from patients with amyotrophic\ud lateral sclerosis with a repeat expansion of C9orf72 (C9orf72 + ), but not from those patients without a repeat expansion\ud of C9orf72 (C9orf72) or control subjects. Moreover, in the cases examined, the distribution of foci-positive neurons correlated\ud with the clinical phenotype (t-test P5 0.05). As expected, RNA foci are ablated by RNase treatment. Interestingly, we identified\ud foci in fibroblasts from an asymptomatic C9orf72 + carrier. We next performed pulldown assays, with GGGGCC5, in conjunction\ud with mass spectrometry analysis, to identify candidate binding partners of the GGGGCC repeat expansion. Proteins containing\ud RNA recognition motifs and involved in splicing, messenger RNA nuclear export and/or translation were significantly enriched.\ud Immunohistochemistry in central nervous system tissue from C9orf72 + patients with amyotrophic lateral sclerosis demonstrated\ud co-localization of RNA foci with SRSF2, hnRNP H1/F, ALYREF and hnRNP A1 in cerebellar granule cells and with SRSF2, hnRNP\ud H1/F and ALYREF in motor neurons, the primary target of pathology in amyotrophic lateral sclerosis. Direct binding of proteins\ud to GGGGCC repeat RNA was confirmed in vitro by ultraviolet-crosslinking assays. Co-localization was only detected in a small\ud proportion of RNA foci, suggesting dynamic sequestration rather than irreversible binding. Additional immunohistochemistry\ud demonstrated that neurons with and without RNA foci were equally likely to show nuclear depletion of TDP-43 (2 P = 0.75) or\ud poly-GA dipeptide repeat protein inclusions (2 P = 0.46). Our findings suggest two non-exclusive pathogenic mechanisms: (i)\ud functional depletion of RNA-processing proteins resulting in disruption of messenger RNA splicing; and (ii) licensing of expanded\ud C9orf72 pre-messenger RNA for nuclear export by inappropriate association with messenger RNA export adaptor\ud protein(s) leading to cytoplasmic repeat associated non-ATG translation and formation of potentially toxic dipeptide repeat\ud protein.\ud d

The metazoan TREX complex is recruited to mRNA during nuclear RNA processing and functions in exporting mRNA to the cytoplasm. Nxf1 is an mRNA export receptor, which binds processed mRNA and transports it through the nuclear pore complex. At present, the relationship between TREX and Nxf1 is not understood. Here we show that Nxf1 uses an intramolecular interaction to inhibit its own RNA-binding activity. When the TREX subunits Aly and Thoc5 make contact with Nxf1, Nxf1 is driven into an open conformation, exposing its RNA-binding domain, allowing RNA binding. Moreover, the combined knockdown of Aly and Thoc5 markedly reduces the amount of Nxf1 bound to mRNA in vivo and also causes a severe mRNA export block. Together, our data indicate that TREX provides a license for mRNA export by driving Nxf1 into a conformation capable of binding mRNA.

SRSF2 is a prototypical SR protein which plays important roles in the alternative splicing of pre-mRNA. It has been shown to be involved in regulatory pathways for maintaining genomic stability and play important roles in regulating key receptors in the heart. We report here the solution structure of the RNA recognition motifs (RRM) domain of free human SRSF2 (residues 9–101). Compared with other members of the SR protein family, SRSF2 structure has a longer L3 loop region. The conserved aromatic residue in the RNP2 motif is absent in SRSF2. Calorimetric titration shows that the RNA sequence 5′AGCAGAGUA3′ binds SRSF2 with a Kd of 61 ± 1 nM and a 1:1 stoichiometry. NMR and mutagenesis experiments reveal that for SFSF2, the canonical β1 and β3 interactions are themselves not sufficient for effective RNA binding; the additional loop L3 is crucial for RNA complex formation. A comparison is made between the structures of SRSF2–RNA complex with other known RNA complexes of SR proteins. We conclude that interactions involving the L3 loop, N- and C-termini of the RRM domain are collectively important for determining selectivity between the protein and RNA.

The herpesvirus proteins HSV-1 ICP27 and HVS ORF57 promote viral mRNA export by utilizing the cellular mRNA export machinery. This function is triggered by binding to proteins of the transcription-export (TREX) complex, in particular to REF/Aly which directs viral mRNA to the TAP/NFX1 pathway and, subsequently, to the nuclear pore for export to the cytoplasm. Here we have determined the structure of the REF-ICP27 interaction interface at atomic-resolution and provided a detailed comparison of the binding interfaces between ICP27, ORF57 and REF using solution-state NMR. Despite the absence of any obvious sequence similarity, both viral proteins bind on the same site of the folded RRM domain of REF, via short but specific recognition sites. The regions of ICP27 and ORF57 involved in binding by REF have been mapped as residues 104–112 and 103–120, respectively. We have identified the pattern of residues critical for REF/Aly recognition, common to both ICP27 and ORF57. The importance of the key amino acid residues within these binding sites was confirmed by site-directed mutagenesis. The functional significance of the ORF57-REF/Aly interaction was also probed using an ex vivo cytoplasmic viral mRNA accumulation assay and this revealed that mutants that reduce the protein-protein interaction dramatically decrease the ability of ORF57 to mediate the nuclear export of intronless viral mRNA. Together these data precisely map amino acid residues responsible for the direct interactions between viral adaptors and cellular REF/Aly and provide the first molecular details of how herpes viruses access the cellular mRNA export pathway.

Background: Combinatorial RNA interference (co-RNAi) is a valuable tool for highly effective gene suppression of single and multiple-genes targets, and can be used to prevent the escape of mutation-prone transcripts. There are currently three main approaches used to achieve co-RNAi in animal cells; multiple promoter/shRNA cassettes, long hairpin RNAs (lhRNA) and miRNA-embedded shRNAs, however, the relative effectiveness of each is not known. The current study directly compares the ability of each co-RNAi method to deliver pre-validated siRNA molecules to the same gene targets.\ud \ud Results: Double-shRNA expression vectors were generated for each co-RNAi platform and their ability to suppress both single and double-gene reporter targets were compared. The most reliable and effective gene silencing was achieved from the multiple promoter/shRNA approach, as this method induced additive suppression of single-gene targets and equally effective knockdown of double-gene targets. Although both lhRNA and microRNA-embedded strategies provided efficient gene knockdown, suppression levels were inconsistent and activity varied greatly for different siRNAs tested. Furthermore, it appeared that not only the position of siRNAs within these multi-shRNA constructs impacted upon silencing activity, but also local properties of each individual molecule. In addition, it was also found that the insertion of up to five promoter/shRNA cassettes into a single construct did not negatively affect the efficacy of each individual shRNA.\ud \ud Conclusions: By directly comparing the ability of shRNAs delivered from different co-RNA platforms to initiate knockdown of the same gene targets, we found that multiple U6/shRNA cassettes offered the most reliable and predictable suppression of both single and multiple-gene targets. These results highlight some important strengths and pitfalls of the currently used methods for multiple shRNA delivery, and provide valuable insights for the design and application of reliable co-RNAi.

The REF/ALY mRNA export adaptor binds TAP/\ud NXF1 via an arginine-rich region, which overlaps\ud with its RNA-binding domain. When TAP binds a\ud REF:RNA complex, it triggers transfer of the RNA\ud from REF to TAP. Here, we have examined the\ud effects of arginine methylation on the activities of\ud the REF protein in mRNA export. We have mapped\ud the arginine methylation sites of REF using\ud mass spectrometry and find that several arginines\ud within the TAP and RNA binding domains\ud are methylated in vivo. However, arginine methylation\ud has no effect on the REF:TAP interaction.\ud Instead, arginine methylation reduces the\ud RNA-binding activity of REF in vitro and in vivo.\ud The reduced RNA-binding activity of REF in its\ud methylated state is essential for efficient displacement\ud of RNA from REF by TAP in vivo. Therefore,\ud arginine methylation fine-tunes the RNA-binding\ud activity of REF such that the RNA–protein interaction\ud can be readily disrupted by export factors\ud further down the pathway.

This paper provides a detailed overview of developments in transducer materials technology relating to their current and future applications in micro-scale devices. Recent advances in piezoelectric, magnetostrictive and shape-memory alloy systems are discussed and emerging transducer materials such as magnetic nanoparticles, expandable micro-spheres and conductive polymers are introduced. Materials properties, transducer mechanisms and end applications are described and the potential for integration of the materials with ancillary systems components is viewed as an essential consideration. The review concludes with a short discussion of structural polymers that are extending the range of micro-fabrication techniques available to designers and production engineers beyond the limitations of silicon fabrication technology. Original Publication:S.A. Wilson, R.P.-J. Jourdain, Q. Zhang, R.A. Dorey, C.R. Bowen, Magnus Willander, Qamar Ul Wahab, M. Willander, S.M. Al-hilli, O. Nur, E. Quandt, C. Johansson, E. Pagounis, M. Kohl, J. Matovic, B. Samel, der Wijngaart W. van, Edwin Jager, D. Carlsson, Z. Djinovic, M. Wegener, C. Moldovan, E. Abad, M. Wendlandt, C. Rusu and K. Persson, New materials for micro-scale sensors and actuators. An engineering review, 2007, Materials science & engineering. R, Reports, (56), 1-129.http://dx.doi.org/10.1016/j.mser.2007.03.001Copyright: Elsevierhttp://www.elsevier.com/

A new and simple method is described for the determination of the piezoelectric coefficients d(33,f) and e(31,f) for piezoelectric films deposited on substrates using a conventional point-loading 'd(33)' or 'Berlincourt' piezometer. An analytical mathematical model is developed which simulates the dynamical flexure of such films when a ring-supported sample is subject to central loading. Classical plate theory and elastic analysis are used to calculate the stresses in doped lead zirconate titanate (PZT) him for different radii of supporting rings, enabling both piezoelectric coefficients to be determined through a simple modification to the piezometer. The analytical model for the radial stresses has been evaluated in comparison with the results of finite element analysis and has shown a good correlation. The new measurement technique has been applied to both thick films of PZT and thin films of manganese-doped lead zirconate titanate (PMZT) on silicon substrates. The values of d(33,f) and e(31,f) obtained experimentally are found to be similar to these that have been determined by more elaborate methods.