Biochemical Society Symposia最新文献

Diverse classes of non-coding RNAs, including snRNAs (small nuclear RNAs), play fundamental regulatory roles in gene expression. For example, 7SK RNA and the components of the splicing apparatus U1-U6 snRNAs are implicated in the regulation of transcriptional elongation. The first evidence for the involvement of RNA in the regulation of transcriptional initiation is now emerging. TFIIH (transcription factor IIH), a general transcription initiation factor, appears to associate specifically with U1 snRNA, a core splicing component. Reconstituted transcription in vitro demonstrates an increase in the rate of formation of the first phosphodiester bond by RNA polymerase II in presence of U1 snRNA. Reconstituted re-initiation is also stimulated by U1 snRNA. These results suggest that U1 snRNA functions in the regulation of transcription by RNA polymerase II in addition to its role in RNA processing. The implications of these data extend to the development of new technologies that will allow the identification and analysis of diverse RNA species present as regulatory components in transcription-related ribonucleoprotein complexes.

Wilms' tumour is a paediatric malignancy of the kidneys that affects one in every 10,000 live births, making it the most common solid tumour in the young. This cancer arises due to a failure of the metanephric mesenchyme to differentiate and form the kidney filtration units and tubules, which instead undergo uncontrolled proliferation. WT1 (Wilms' tumour 1) was identified as a factor that is frequently mutated in Wilms' tumours. WT1 plays a central role in the development of the genito-urinary organs and also other regions of the embryo. A major function of WT1 is to act as a regulator of transcription, controlling the expression of genes that are involved in proliferation and differentiation. WT1 can either activate or repress transcription of its target genes. Thus the transcription function of WT1 is highly context-specific, and can be modulated by a number of cofactors. Here, the known interaction partners of WT1 and the mechanisms by which they modulate WT1 transcription function will be discussed.

SCA7 (spinocerebellar ataxia type 7) is a neurodegenerative disorder caused by a CAG repeat expansion in the SCA7 gene that leads to elongation of a polyglutamine tract in ataxin-7, a protein of unknown function. Sgf73, a putative yeast orthologue of ataxin-7, has been identified as a new component of the yeast SAGA (Spt/Ada/Gcn5 acetyltransferase) multisubunit complex, a co-activator required for the transcription of a subset of RNA polymerase II-dependent genes. We show here that ataxin-7 is an integral component of mammalian SAGA-like complexes, i.e. the TFTC [TBP (TATA-binding protein)-free TAF (TBP-associated factor) complex] and the STAGA (SPT3/TAF9/GCN5 acetyltransferase) complex. In agreement with this, immunoprecipitation of ataxin-7 retained a histone acetyltransferase activity characteristic of TFTC-like complexes. Moreover, polyglutamine expansion in ataxin-7 did not affect its incorporation into TFTCs/STAGA complexes purified from cells from a SCA7 patient. We demonstrate here that ataxin-7 is the human orthologue of a the yeast SAGA Sgf73 subunit, and is a bona fide subunit of human TFTC-like transcriptional complexes.

The archaeal basal transcriptional machinery consists of TBP (TATA-binding protein), TFB (transcription factor B; a homologue of eukaryotic TFIIB) and an RNA polymerase that is structurally very similar to eukaryotic RNA polymerase II. This constellation of factors is sufficient to assemble specifically on a TATA box-containing promoter and to initiate transcription at a specific start site. We have used this system to study the functional interaction between basal transcription factors and RNA polymerase, with special emphasis on the post-recruitment function of TFB. A bioinformatics analysis of the B-finger of archaeal TFB and eukaryotic TFIIB reveals that this structure undergoes rapid and apparently systematic evolution in archaeal and eukaryotic evolutionary domains. We provide a detailed analysis of these changes and discuss their possible functional implications.

dRTA (distal renal tubular acidosis) results from the failure of the a-intercalated cells in the distal tubule of the nephron to acidify the urine. A truncated form of AE1 (anion-exchanger 1; Band 3), kAE1 (kidney isoform of AE1), is located in the basolateral membrane of the intercalated cell. Mutations in the AE1 gene cause autosomal dominant and recessive forms of dRTA. All the dominant dRTA mutations investigated cause aberrant trafficking of kAE1, resulting in its intracellular retention or mistargeting to the apical plasma membrane. Therefore the intracellular retention of hetero-oligomers containing wild-type and dRTA mutants, or the mistargeted protein in the apical membrane neutralizing acid secretion, explains dominant dRTA. The kAE1 (Arg(901)-->stop) mutant has been studied in more detail, since the mistargeting kAE1 (Arg(901)-->stop) from the basolateral to the apical membrane is consistent with the removal of a basolateral localization signal. The C-terminal amino acids deleted by the Arg(901)-->stop mutation, contain a tyrosine motif and a type II PDZ interaction domain. The tyrosine residue (Tyr(904)), but not the PDZ domain, is critical for basolateral localization. In the absence of the N-terminus of kAE1, the C-terminus was not sufficient to localize kAE1 to the basolateral membrane. This suggests that a determinant within the kAE1 N-terminus co-operates with the C-terminus for kAE1 basolateral localization. Interestingly, Tyr(359), in the N-terminal domain, and Tyr(904) in the C-terminus of AE1 are phosphorylated in red blood cells. A potential scheme is suggested where successive phosphorylation of these residues is necessary for correct localization and recycling of kAE1 to the basolateral membrane.

Cargo proteins moving along the secretory pathway are sorted at the TGN (trans-Golgi network) into distinct carriers for delivery to the plasma membrane or endosomes. Recent studies in yeast and mammals have shown that formation of these carriers is regulated by PtdIns(4)P. This phosphoinositide is abundant at the TGN and acts to recruit components required for carrier formation to the membrane. Other phosphoinositides are also present on the TGN, but the extent to which they regulate trafficking is less clear. Further characterization of phosphoinositide kinases and phosphatases together with identification of new TGN-associated phosphoinositide-binding proteins will reveal the extent to which different phosphoinositides regulate TGN trafficking, and help define the molecular mechanisms involved.

GPCRs (G-protein-coupled receptors) play key roles in many cellular processes, and malfunction may lead to a range of pathologies, including psychiatric and neurological disorders. It is therefore not surprising that this group of receptors supplies a majority of the targets for pharmaceutical drug development. Despite their importance, the mechanisms that regulate their function and signalling still remain only partially understood. Recently, it has become evident that a subset of GPCRs is not homogeneously distributed in the plasma membrane, but localizes instead to specific membrane microdomains known as lipid rafts. Lipid rafts are characterized by their enrichment in cholesterol and sphingolipids, and have been suggested to serve as platforms for a range of cellular signalling complexes. In the present review, we will be discussing the effects of the lipid raft environment on trafficking, signalling and internalization of raft-associated GPCRs.

Clathrin-coated vesicles carry traffic from the plasma membrane to endosomes. We report here the first real-time visualization of cargo sorting and endocytosis by clathrin-coated pits in living cells. We have visualized the formation of coats by monitoring the incorporation of fluorescently tagged clathrin or its adaptor AP-2 (adaptor protein 2), and have followed clathrin-mediated uptake of transferrin, single LDL (low-density lipoprotein) and single reovirus particles. The intensity of a cargo-loaded clathrin cluster grows steadily during its lifetime, and the time required to complete assembly is proportional to the size of the cargo particle. These results are consistent with a nucleation-growth mechanism and an approximately constant growth rate. There are no preferred nucleation sites. A proportion of the nucleation events appear to be abortive. Cargo incorporation occurs primarily or exclusively in a newly formed coated pit, and loading appears to commit that pit to finish assembly. Our data led to a model in which coated pits initiate randomly, but collapse with high likelihood unless stabilized, presumably by cargo capture.

Reversible phosphorylation has long been an attractive mechanism to control cycles of coat assembly and disassembly during clathrin-mediated endocytosis. Many of the coat proteins are phosphorylated in vivo and in vitro. Our work has focused on the role of phosphorylation of the mu2 subunit of AP-2 (adaptor protein 2), which appears to be necessary for efficient cargo recruitment. Studies to probe the regulation of mu2 phosphorylation demonstrated that clathrin is a specific activator of the mu2 kinase, and, in permeabilized cells, cargo sequestration, driven by exogenously added clathrin, results in elevated levels of m2 phosphorylation. Furthermore, phosphorylated mu2 is mainly associated with assembled clathrin in vivo and its steady-state level is strongly reduced in cells depleted of clathrin heavy chain. Our results imply a central role for clathrin in the regulation of cargo selection via modulation of phospho-mu2 levels. This is therefore a novel regulatory role for clathrin that is independent of its structural role and that provides elegant spatial control of AP-2 and cargo interactions, ensuring that AP-2 is only activated at the correct cellular location and in the correct functional context. Ongoing studies are exploring further the roles of reversible phosphorylation in the coated vesicle cycle.