Expansion of GGGGCC repeats in C9orf72 causes familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia, but the underlying mechanism is unclear. Using RNA pulldown and immunohistochemistry in ALS biosamples, Cooper-Knock et al. identify proteins that bind to the repeat expansions. Disrupted RNA splicing and/or nuclear export may underlie C9orf72-ALS pathogenesis.

Increasing a protein concentration in solution to the required level, without causing aggregation and precipitation is often a challenging but important task, especially in the field of structural biology; as little as 20% of nonmembrane proteins have been found to be suitable candidates for structural studies predominantly due to poor protein solubility. We demonstrate here that simultaneous addition of charged amino acids L-Arg and L-Glu at 50 mM to the buffer can dramatically increase the maximum achievable concentration of soluble protein (up to 8.7 times). These amino acids are effective in preventing protein aggregation and precipitation, and they dramatically increase the long-term stability of the sample; additionally, they protect protein samples from proteolytic degradation. Specific protein-protein and protein-RNA interactions are not adversely affected by the presence of these amino acids. These additives are particularly suitable for situations where high protein concentration and long-term stability are required, including solution-state studies of isotopically labeled proteins by NMR.

REF/Yra1p interacts with TAP/Mex67p, and in yeast this interaction leads to displacement of Sub2p from Yra1p (7). TAP heterodimerizes with p15 and binds nucleoporins through central and C-terminal domains (8), directing the mRNP to the nuclear pore and promoting transport to the cytoplasm. On the cytoplasmic side of the nuclear pore, Dbp5p triggers displacement of Mex67p from mRNA. Yra1p binds mRNA early during its nuclear maturation but is no longer bound once it reaches the nuclear periphery (9). Consistent with this finding, analysis of Balbiani ring pre-mRNPs shows that UAP56 and REF accompany the mRNP to the nuclear periphery where UAP56 and then REF dissociate during translocation through the pore (10).Although Yra1p is essential for yeast mRNA export, depletion of REF in higher eukaryotes does not block bulk mRNA export (11), suggesting that other proteins can fulfill this role and that there may be functional redundancy between export adaptors. The shuttling SR proteins 9G8, SRp20, and SF2/ASF directly bind TAP by short arginine-rich peptides (12, 13) and can function as export factors (14). Even in yeast, other proteins can recruit Mex67p to the mRNP, including Yra2p (15) and Npl3p.The fact that TAP binds RNA weakly in vitro led to the idea that export factors such as REF, which bind RNA avidly, bridge the interaction between TAP and mRNA, leading to the term mRNA export adaptors (15). However, both TAP and Mex67p are readily UV-cross-linked to mRNA in vivo (16)(17)(18), suggesting a direct stable interaction at some point during export. Here, we show that mRNA is handed over from export adaptors to TAP and that at least in vitro, export adaptors have the ability to enhance the RNA-binding activity of TAP. ResultsThe RNA-and TAP-Binding Sites on REF Overlap. In Saccharomyces cerevisiae, Mex67p binding to Yra1p triggers displacement of Sub2p (7), so we established whether this is the case for the mammalian orthologs. We examined whether UAP56 coimmunoprecipitated (Co-IP) with REF2-I (REF) in the presence of increasing amounts of TAP. This analysis revealed that TAP triggered dissociation of UAP56 from REF (Fig. 1A, lanes 5 and 6).We analyzed organization of the resulting REF-TAP-RNA ternary complex by examining how REF binds RNA. NMR

SummaryMessenger RNA (mRNA) export adaptors play an important role in the transport of mRNA from the nucleus to the cytoplasm. They couple early mRNA processing events such as 5′ capping and 3′ end formation with loading of the TAP/NXF1 export receptor onto mRNA. The canonical adaptor REF/ALY/Yra1 is recruited to mRNA via UAP56 and subsequently delivers the mRNA to NXF1 [1]. Knockdown of UAP56 [2, 3] and NXF1 [4–7] in higher eukaryotes efficiently blocks mRNA export, whereas knockdown of REF only causes a modest reduction, suggesting the existence of additional adaptors [8–10]. Here we identify a new UAP56-interacting factor, UIF, which functions as an export adaptor, binding NXF1 and delivering mRNA to the nuclear pore. REF and UIF are simultaneously found on the same mRNA molecules, and both proteins are required for efficient export of mRNA. We show that the histone chaperone FACT specifically binds UIF, but not REF, via the SSRP1 subunit, and this interaction is required for recruitment of UIF to mRNA. Together the results indicate that REF and UIF represent key human adaptors for the export of cellular mRNAs via the UAP56-NXF1 pathway.

The binding of extracellular ATP to the P2X 7 receptor opens an integral cation-permeable channel; it also leads to membrane blebbing and, in certain immune cells, interleukin-1␤ secretion and eventual death. The latter three effects are unique to the P2X 7 receptor; also unique among P2X receptors is the long intracellular C terminus of the protein. We have shown that the C-terminal domain of the P2X 7 receptor is responsible for the cell blebbing phenotype. A screen for proteins that associate with the C-terminal domain of the P2X 7 receptor and might mediate the blebbing phenotype, identified epithelial membrane protein 2 (EMP-2). The interaction between EMP-2 and P2X 7 was confirmed biochemically by co-immunoprecipitation, co-purification, and glutathione S-transferase pull-down assays, and this interaction was entirely dependent on the C-terminal domain of P2X 7 . The P2X 7 receptor also interacted with the other members of the epithelial membrane protein family (EMP-1, EMP-3, and PMP-22). All four EMPs were found to be expressed in HEK-293 cells and in THP-1 monocytes, which express P2X 7 receptors. Interestingly, the constitutive overexpression of any of the EMPs in HEK-293 cells led to cell blebbing, annexin V binding, and cell death, by a caspase-dependent pathway. These findings suggest that the P2X 7 C-terminal domain associates with EMPs, and this interaction may mediate some aspects of the downstream signaling following P2X 7 receptor activation. P2X 7 receptors belong to a family of ion channels gated by extracellular ATP (1), all with the same predicted topology of two transmembrane domains and intracellular N and C termini (1). The P2X 7 receptor shares 40 -45% amino acid identity with the other P2X proteins, but it is structurally distinct at the C terminus, extending for an additional 100 -200 amino acids (2). P2X 1-6 receptors are widely distributed in both neuronal and non-neuronal cells, whereas P2X 7 receptors are most highly expressed in immune and epithelial cells (3, 4). Brief stimulation (10 -30 s) of the P2X 7 receptor leads to the formation of a channel permeable to large cations, as is also seen for some of the other P2X receptors (2, 5). However, more prolonged activation of the P2X 7 receptor results in extensive membrane blebbing from within seconds to minutes (5), and eventual cell death (5-7), although such responses are not observed with other P2X receptors. We hypothesized that the C terminus of the P2X 7 receptor might engage other cellular proteins to mediate its distinct responses of membrane blebbing and cell death. We tested this hypothesis by using a construct containing the soluble C-terminal domain (Asn 356 -Tyr 595 ) of the rat P2X 7 protein as bait in a yeast two-hybrid screen. Because HEK-293 cells expressing P2X 7 receptors exhibit pronounced blebbing and eventual cell death when stimulated with ATP (5),we sought interacting proteins using a HEK-293 cell library.

The role of herpes simplex virus ICP27 protein in mRNA export is investigated by microinjection into Xenopus laevis oocytes. ICP27 dramatically stimulates the export of intronless viral mRNAs, but has no effect on the export of cellular mRNAs, U snRNAs or tRNA. Use of inhibitors shows, in contrast to previous suggestions, that ICP27 neither shuttles nor exports viral mRNA via the CRM1 pathway. Instead, ICP27‐mediated viral RNA export requires REF and TAP/NXF1, factors involved in cellular mRNA export. ICP27 binds directly to REF and complexes containing ICP27, REF and TAP are found in vitro and in virally infected cells. A mutant ICP27 that does not interact with REF is inactive in viral mRNA export. We propose that ICP27 associates with viral mRNAs and recruits TAP/NXF1 via its interaction with REF proteins, allowing the otherwise inefficiently exported viral mRNAs to access the TAP‐mediated export pathway. This represents a novel mechanism for export of viral mRNAs.

Birds have played a central role in many biological disciplines, particularly ecology, evolution, and behavior. The chicken, as a model vertebrate, also represents an important experimental system for developmental biologists, immunologists, cell biologists, and geneticists. However, genomic resources for the chicken have lagged behind those for other model organisms, with only 1845 nonredundant full-length chicken cDNA sequences currently deposited in the EMBL databank. We describe a large-scale expressed-sequence-tag (EST) project aimed at gene discovery in chickens (http://www.chick.umist.ac.uk). In total, 339,314 ESTs have been sequenced from 64 cDNA libraries generated from 21 different embryonic and adult tissues. These were clustered and assembled into 85,486 contiguous sequences (contigs). We find that a minimum of 38% of the contigs have orthologs in other organisms and define an upper limit of 13,000 new chicken genes. The remaining contigs may include novel avian specific or rapidly evolving genes. Comparison of the contigs with known chicken genes and orthologs indicates that 30% include cDNAs that contain the start codon and 20% of the contigs represent full-length cDNA sequences. Using this dataset, we estimate that chickens have approximately 35,000 genes in total, suggesting that this number may be a characteristic feature of vertebrates.

RNA interference (RNAi) provides an effective method to silence gene expression and investigate gene function. However, RNAi tools for the chicken embryo have largely been adapted from vectors designed for mammalian cells. Here we present plasmid and retroviral RNAi vectors specifically designed for optimal gene silencing in chicken cells. The vectors use a chicken U6 promoter to express RNAs modelled on microRNA30, which are embedded within chicken microRNA operon sequences to ensure optimal Drosha and Dicer processing of transcripts. The chicken U6 promoter works significantly better than promoters of mammalian origin and in combination with a microRNA operon expression cassette (MOEC), achieves up to 90% silencing of target genes. By using a MOEC, we show that it is also possible to simultaneously silence two genes with a single vector. The vectors express either RFP or GFP markers, allowing simple in vivo tracking of vector delivery. Using these plasmids, we demonstrate effective silencing of Pax3, Pax6, Nkx2.1, Nkx2.2, Notch1 and Shh in discrete regions of the chicken embryonic nervous system. The efficiency and ease of use of this RNAi system paves the way for large-scale genetic screens in the chicken embryo.