We have sequenced and annotated the genome of ®ssion yeast (Schizosaccharomyces pombe), which contains the smallest number of protein-coding genes yet recorded for a eukaryote: 4,824. The centromeres are between 35 and 110 kilobases (kb) and contain related repeats including a highly conserved 1.8-kb element. Regions upstream of genes are longer than in budding yeast (Saccharomyces cerevisiae), possibly re¯ecting more-extended control regions. Some 43% of the genes contain introns, of which there are 4,730. Fifty genes have signi®cant similarity with human disease genes; half of these are cancer related. We identify highly conserved genes important for eukaryotic cell organization including those required for the cytoskeleton, compartmentation, cell-cycle control, proteolysis, protein phosphorylation and RNA splicing. These genes may have originated with the appearance of eukaryotic life. Few similarly conserved genes that are important for multicellular organization were identi®ed, suggesting that the transition from prokaryotes to eukaryotes required more new genes than did the transition from unicellular to multicellular organization.We report here the completion of the fully annotated genome sequence of the simple eukaryote Schizosaccharomyces pombe, a ®ssion yeast. It becomes the sixth eukaryotic genome to be sequenced, following Saccharomyces cerevisiae 1 , Caenorhabditis elegans 2 , Drosophila melanogaster 3 , Arabidopsis thaliana 4 and Homo sapiens 5,6 . The entire sequence of the unique regions of the three chromosomes is complete, with gaps in the centromeric regions of about 40 kb, and about 260 kb in the telomeric regions. The completion of this sequence, the availability of sophisticated research methodologies, and the expanding community working on S. pombe, will accelerate the use of S. pombe for functional and comparative studies of eukaryotic cell processes.

Base pairing between U2 snRNA and the branchpoint sequence (BPS) is essential for pre-mRNA splicing. Because the metazoan BPS is short and highly degenerate, this interaction alone is insufficient for specific binding of U2 snRNP. The splicing factor U2AF binds to the pyrimidine tract at the 3 splice site in the earliest spliceosomal complex, E, and is essential for U2 snRNP binding in the spliceosomal complex A. We show that the U2 snRNP protein SAP 155 UV cross-links to pre-mRNA on both sides of the BPS in the A complex. SAP 155's downstream cross-linking site is immediately adjacent to the U2AF binding site, and the two proteins interact directly in protein-protein interaction assays. Using UV cross-linking, together with functional analyses of pre-mRNAs containing duplicated BPSs, we show a direct correlation between BPS selection and UV cross-linking of SAP 155 on both sides of the BPS. Together, our data are consistent with a model in which U2AF binds to the pyrimidine tract in the E complex and then interacts with SAP 155 to recruit U2 snRNP to the BPS.The pre-mRNA splicing reaction is carried out with extreme precision in order to generate mRNAs that encode functional proteins. The accuracy of splicing depends on multiple sequence elements located at the 5Ј and 3Ј splice sites, at the branch site, and within exons. Networks of RNA-RNA, RNAprotein, and protein-protein interactions involving each of these sequence elements contribute to the specificity of splicing. Additional specificity is derived from the recognition of each sequence element multiple times prior to the two catalytic steps of splicing. These successive recognition events occur as the spliceosomal complexes E, A, B, and C assemble on premRNA in a stepwise pathway (for reviews, see references 17, 25, and 32).One of the critical sequence elements in the intron is the branchpoint sequence (BPS). This element contains an adenosine that functions as the nucleophile for catalytic step I of splicing. Despite its key role in splicing, the BPS is weakly conserved in metazoans, and additional elements are required for BPS recognition. The most important of these is the pyrimidine tract located immediately downstream from the BPS. The splicing factor U2AF, which is composed of two subunits (U2AF 65 and U2AF 35 ), binds to the pyrimidine tract in the E complex, with U2AF 65 directly contacting the pre-mRNA (4, 33 [for reviews, see references 17, 25, and 32]). The essential splicing factor SF1 (also known as mBBP) interacts with U2AF 65 and also has sequence specificity for the BPS (2, 5, 16). Thus, this network of interactions is thought to function in the initial recognition of the pyrimidine tract and BPS.The BPS is recognized a second time during spliceosome assembly by formation of a duplex between the BPS and U2 snRNA (for review, see reference 19). This duplex is first established in the A complex and plays an essential role in splicing by specifying the branch-site adenosine as the nucleophile for catalytic step I (23). Two multisubunit splicin...

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Formidable challenges for Parkinson's disease (PD) research are to understand the processes underlying nigrostriatal degeneration and how to protect the dopamine neurons. Fundamental research relies on good animal models that demonstrate the pathological hallmarks and motor deficits of PD. Using a chronic regimen of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and probenecid (MPTP/p) in mice, dopamine cell loss exceeds 60%, extracellular glutamate is elevated, cytoplasmic inclusions are formed and inflammation is chronic. Nevertheless, isradipine, an L-type calciumchannel blocker, attenuates the degeneration. These data support the validity of the MPTP/p model for unravelling the degenerative processes in PD and testing therapies that slow their progress.

The P2X 7 receptor regulates cell growth through mediation of apoptosis. P2X 7 levels are lower in cancer epithelial cells than in normal cells, and previous studies showed that expression of P2X 7 was regulated post-transcriptionally. The objective of the study was to understand regulation of P2X 7 mRNA stability. Overexpression of a reporter containing the full-length human P2X 7 3-untranslated region (3-UTR) or reporters containing parts of the 3-UTR-P2X 7 were associated with increased abundance of the construct in normal cells and decreased abundance in cancer epithelial cells. Sequences within the 3-UTR-P2X 7 , which are putative target sites for the microRNAs, miR-186 (middle segment) and miR-150 (distal segment), decreased the abundance of the P2X 7 transcript. Overexpression in cancer cells of mutated miR-186 and miR-150 target sites was associated with lower levels of the reporter genes. In normal cells overexpression of the mutated miR-186 target site was associated with marked increased concentration, but overexpression of the miR-150 target site reporters, wild-type and mutant, did not change over time. Levels of miR-186 and miR-150 were higher in cancer than in normal cells, and treatment with miR-186 and miR-150 inhibitors increased P2X 7 mRNA. In human embryonic kidney-293 cells heterologously expressing the full-length 3-UTR-P2X 7 luciferase reporter, miR-186 and miR-150 inhibitors increased luciferase activity, whereas miR-186 and miR-150 mimics decreased luciferase activity after actinomycin D treatment. These data suggest that increased expression of miR-186 and miR-150 in cancer epithelial cells decreases P2X 7 mRNA by activation of miR-186 and miR-150 instability target sites located at the 3-UTR-P2X 7 .The receptor P2X 7 is a membrane-bound, ligand-operated channel (1-6). ATP is the naturally occurring ligand for the P2X 7 , and extracellular levels of ATP may reach low micromolar levels (7-12), which are sufficient to activate the receptor (13). Activation of the receptor may induce formation of pores in the plasma membrane (14), which in epithelial cells mediate apoptosis via the caspase-9 mitochondrial pathway (15, 16). The P2X 7 apoptosis effects can be regulated by receptor glycosylation (16), trafficking, plasma membrane expression (17-20), oligomerization (7, 21), and by receptor post-activation internalization, recycling, and degradation (14,21).In epithelial tissues the P2X 7 receptor is expressed predominantly by proliferative (germinative) epithelial cells (21-23), and it controls the growth of the epithelial cells. Previous studies in human uterine epithelial cells showed that base-line and P2X 7 -mediated apoptosis are lower in cancer cells than in normal cells (12,22,23). The differences were not the result of ligand availability because steady-state levels of ATP in conditioned media of cancer epithelial cells were similar to those of normal epithelial cells (12). Similarly, there were no significant differences in P2X 7 receptor activation, oligomerization, or cycling betwe...

Parkinson's disease (PD) is the second most prevalent neurodegenerative disease in ageing individuals. It is now clear that genetic susceptibility and environmental factors play a role in disease etiology and progression. Because environmental factors are involved with the majority of the cases of PD, it is important to understand the role nutrition plays in both neuroprotection and neurodegeneration. Recent epidemiological studies have revealed the promise of some nutrients in reducing the risk of PD. In contrast, other nutrients may be involved with the etiology of neurodegeneration or exacerbate disease progression. This review summarizes the studies that have addressed these issues and describes in detail the nutrients and their putative mechanisms of action in PD.

Several fission yeast temperature-sensitive mutants defective in pre-mRNA processing (prp- mutants) at the nonpermissive temperature have been identified. Here, the prp2+ gene has been cloned by its ability to complement the temperature-sensitive growth defect of a prp2- mutant. The gene also corrects the pre-mRNA splicing defect of prp2- mutants and encodes a 59-kilodalton polypeptide (PRP2). A molecular characterization indicates that PRP2 is a previously uncharacterized yeast splicing factor with extensive similarity to the mammalian splicing factor U2AF65. Thus, this study provides evidence that a U2AF homolog participates in RNA processing in vivo.