Stuart A. Wilson

A Comprehensive Collection of Chicken cDNAs

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.

The chicken as a model for large-scale analysis of vertebrate gene function.

The chicken has been an important experimental system for developmental biology, immunology and microbiology, having led to many fundamental discoveries. The increase in genomic resources, easy access to the embryo and the application of RNA interference mean that it will be easy and quick to use chick embryos to screen the function of many genes during embryonic development. So, it seems likely that the chicken will increasingly be the system of choice for many vertebrate biologists who are interested in gene function.

Herpes simplex virus ICP27 protein provides viral mRNAs with access to the cellular mRNA export pathway.

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.

Sequestration of multiple RNA recognition motif-containing proteins by C9orf72 repeat expansions

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.

Molecular basis of RNA recognition and TAP binding by the SR proteins SRp20 and 9G8

The sequence‐specific RNA‐binding proteins SRp20 and 9G8 are the smallest members of the serine‐ and arginine‐rich (SR) protein family, well known for their role in splicing. They also play a role in mRNA export, in particular of histone mRNAs. We present the solution structures of the free 9G8 and SRp20 RNA recognition motifs (RRMs) and of SRp20 RRM in complex with the RNA sequence 5′CAUC3′. The SRp20‐RNA structure reveals that although all 4 nt are contacted by the RRM, only the 5′ cytosine is primarily recognized in a specific way. This might explain the numerous consensus sequences found by SELEX (systematic evolution of ligands by exponential enrichment) for the RRM of 9G8 and SRp20. Furthermore, we identify a short arginine‐rich peptide adjacent to the SRp20 and 9G8 RRMs, which does not contact RNA but is necessary and sufficient for interaction with the export factor Tip‐associated protein (TAP). Together, these results provide a molecular description for mRNA and TAP recognition by SRp20 and 9G8.

Mutually exclusive interactions drive handover of mRNA from export adaptors to TAP

Adaptor proteins stimulate the nuclear export of mRNA, but their mechanism of action remains unclear. Here, we show that REF/ALY binds mRNA; but upon formation of a ternary complex with TAP the RNA is transferred from REF to TAP, and overexpression of TAP displaces REF from mRNA in vivo. RNA is also handed over from two other adaptors, 9G8 and SRp20 to TAP upon formation of a ternary complex. Interestingly, the RNA-binding affinity of TAP is enhanced 4-fold in vitro once it is complexed with REF. 9G8 and SRp20 also enhance the TAP RNA-binding activity in vitro. Consistent with a model in which TAP directly binds mRNA handed over from adaptors during export, we show that TAP binds mRNA in vivo by an arginine-rich motif in its N-terminal domain. The importance of direct TAP–mRNA interactions is confirmed by the observation that a mutant form of TAP that fails to bind mRNA but retains the ability to bind REF does not function in mRNA export.

ATP is required for interactions between UAP56 and two conserved mRNA export proteins, Aly and CIP29, to assemble the TREX complex

The conserved TREX mRNA export complex is known to contain UAP56, Aly, Tex1, and the THO complex. Here, we carried out proteomic analysis of immunopurified human TREX complex and identified the protein CIP29 as the only new component with a clear yeast relative (known as Tho1). Tho1 is known to function in mRNA export, and we provide evidence that CIP29 likewise functions in this process. Like the known TREX components, a portion of CIP29 localizes in nuclear speckle domains, and its efficient recruitment to mRNA is both splicing- and cap-dependent. We show that UAP56 mediates an ATP-dependent interaction between the THO complex and both CIP29 and Aly, indicating that TREX assembly is ATP-dependent. Using recombinant proteins expressed in Escherichia coli, we show that UAP56, Aly, and CIP29 form an ATP-dependent trimeric complex, and UAP56 bridges the interaction between CIP29 and Aly. We conclude that the interaction of two conserved export proteins, CIP29 and Aly, with UAP56 is strictly regulated by ATP during assembly of the TREX complex.

The integrity of a lamin-B1-dependent nucleoskeleton is a fundamental determinant of RNA synthesis in human cells.

Spatial organisation of nuclear compartments is an important regulator of chromatin function, yet the molecular principles that maintain nuclear architecture remain ill-defined. We have used RNA interference to deplete key structural nuclear proteins, the nuclear lamins. In HeLa cells, we show that reduced expression of lamin B1, but not lamin A/C, severely inhibits RNA synthesis – first by RNA polymerase II and later by RNA polymerase I. Declining levels of transcription correlate with different morphological changes in major nuclear compartments, nucleoli and nuclear speckles. Ultimately, nuclear changes linked to the loss of synthetic activity result in expansion of the inter-chromatin domain and corresponding changes in the structure and spatial organisation of chromosome territories, which relocate towards the nuclear periphery. These results show that a lamin B1-containing nucleoskeleton is required to maintain RNA synthesis and that ongoing synthesis is a fundamental determinant of global nuclear architecture in mammalian cells.

UIF, a New mRNA Export Adaptor that Works Together with REF/ALY, Requires FACT for Recruitment to mRNA

Summary Messenger 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.

Drosha regulates neurogenesis by controlling Neurogenin 2 expression independent of microRNAs

Temporal regulation of embryonic neurogenesis is controlled by hypostable transcription factors. The mechanism of the process is unclear. Here we show that the RNase III Drosha and DGCR8 (also known as Pasha), key components of the microRNA (miRNA) microprocessor, have important functions in mouse neurogenesis. Loss of microprocessor in forebrain neural progenitors resulted in a loss of stem cell character and precocious differentiation whereas Dicer deficiency did not. Drosha negatively regulated expression of the transcription factors Neurogenin 2 (Ngn2) and NeuroD1 whereas forced Ngn2 expression phenocopied the loss of Drosha. Neurog2 mRNA contains evolutionarily conserved hairpins with similarities to pri-miRNAs, and associates with the microprocessor in neural progenitors. We uncovered a Drosha-dependent destabilization of Neurog2 mRNAs consistent with microprocessor cleavage at hairpins. Our findings implicate direct and miRNA-independent destabilization of proneural mRNAs by the microprocessor, which facilitates neural stem cell (NSC) maintenance by blocking accumulation of differentiation and determination factors.