The exon͞intron architecture of genes determines whether components of the spliceosome recognize splice sites across the intron or across the exon. Using in vitro splicing assays, we demonstrate that splice-site recognition across introns ceases when intron size is between 200 and 250 nucleotides. Beyond this threshold, splice sites are recognized across the exon. Splice-site recognition across the intron is significantly more efficient than splice-site recognition across the exon, resulting in enhanced inclusion of exons with weak splice sites. Thus, intron size can profoundly influence the likelihood that an exon is constitutively or alternatively spliced. An EST-based alternative-splicing database was used to determine whether the exon͞intron architecture influences the probability of alternative splicing in the Drosophila and human genomes. Drosophila exons flanked by long introns display an up to 90-foldhigher probability of being alternatively spliced compared with exons flanked by two short introns, demonstrating that the exon͞ intron architecture in Drosophila is a major determinant in governing the frequency of alternative splicing. Exon skipping is also more likely to occur when exons are flanked by long introns in the human genome. Interestingly, experimental and computational analyses show that the length of the upstream intron is more influential in inducing alternative splicing than is the length of the downstream intron. We conclude that the size and location of the flanking introns control the mechanism of splice-site recognition and influence the frequency and the type of alternative splicing that a pre-mRNA transcript undergoes.alternative splicing ͉ bioinformatics ͉ EST database ͉ intron length P re-mRNA splicing is an essential process that accounts for many aspects of regulated gene expression. Of the Ϸ25,000 genes encoded by the human genome (1), Ͼ60% are believed to produce transcripts that are alternatively spliced. Thus, alternative splicing of pre-mRNAs can lead to the production of multiple protein isoforms from a single pre-mRNA, exponentially enriching the proteomic diversity of higher eukaryotic organisms (2, 3). Because regulation of this process can determine when and where a particular protein isoform is produced, changes in alternative-splicing patterns modulate many cellular activities.The spliceosome assembles onto the pre-mRNA in a coordinated manner by binding to sequences located at the 5Ј and 3Ј ends of introns. Spliceosome assembly is initiated by the stable associations of the U1 small nuclear ribonucleoprotein particle with the 5Ј splice site, branch-point-binding protein͞SF1 with the branch point, and U2 snRNP auxiliary factor with the pyrimidine tract (4). ATP hydrolysis then leads to the stable association of U2 snRNP at the branch-point and functional splice-site pairing (5).Intron size has been correlated with rates of evolution (6) and the regulation of genome size (7,8). The exon͞intron architecture has also been shown to influence splice-site recognition (9-11)....

Tandem affinity strategies reach exceptional protein purification grades and have considerably improved the outcome of mass spectrometry-based proteomic experiments. However, current tandem affinity tags are incompatible with two-step purification under fully denaturing conditions. Such stringent purification conditions are desirable for mass spectrometric analyses of protein modifications as they result in maximal preservation of posttranslational modifications. Here we describe the histidine-biotin (HB) tag, a new tandem affinity tag for twostep purification under denaturing conditions. The HB tag consists of a hexahistidine tag and a bacterially derived in vivo biotinylation signal peptide that induces efficient biotin attachment to the HB tag in yeast and mammalian cells. HB-tagged proteins can be sequentially purified under fully denaturing conditions, such as 8 M urea, by Ni 2؉ chelate chromatography and binding to streptavidin resins. The stringent separation conditions compatible with the HB tag prevent loss of protein modifications, and the high purification grade achieved by the tandem affinity strategy facilitates mass spectrometric analysis of posttranslational modifications. Ubiquitination is a particularly sensitive protein modification that is rapidly lost during purification under native conditions due to ubiquitin hydrolase activity. The HB tag is ideal to study ubiquitination because the denaturing conditions inhibit hydrolase activity, and the tandem affinity strategy greatly reduces nonspecific background. We tested the HB tag in proteome-wide ubiquitin profiling experiments in yeast and identified a number of known ubiquitinated proteins as well as so far unidentified candidate ubiquitination targets. In addition, the stringent purification conditions compatible with the HB tag allow effective mass spectrometric identification of in vivo cross-linked protein com- Mass spectrometric analysis of proteins has tremendously contributed to our understanding of biological systems. Mapping of covalent protein modifications by mass spectrometric approaches has made it possible to identify and rapidly evaluate the biological significance of modifications. In addition, identification of protein complexes by mass spectrometry has allowed investigators to connect cellular pathways and to describe the dynamics of protein complexes (1, 2). These approaches typically require a high degree of purification of proteins or protein complexes. Importantly to get a genuine picture of the in vivo situation it is essential to avoid any changes in protein modification or protein complex composition that might occur during the purification procedure. Two-step purification strategies have been proven to be very effective in reducing nonspecific background, which is particularly important for the analyses of complex protein samples (3). The first widely and successfully used tandem affinity tag was the TAP 1 tag, which consists of the immunoglobulin-interacting domain of Protein A and a calmodulinbinding peptide (CBP) a...

A collection of 4457 Saccharomyces cerevisiae mutants deleted for nonessential genes was screened for mutants with increased or decreased mobilization of the gypsylike retroelement Ty3. Of these, 64 exhibited increased and 66 decreased Ty3 transposition compared with the parental strain. Genes identified in this screen were grouped according to function by using GOnet software developed as part of this study. Gene clusters were related to chromatin and transcript elongation, translation and cytoplasmic RNA processing, vesicular trafficking, nuclear transport, and DNA maintenance. Sixty-six of the mutants were tested for Ty3 proteins and cDNA. Ty3 cDNA and transposition were increased in mutants affected in nuclear pore biogenesis and in a subset of mutants lacking proteins that interact physically or genetically with a replication clamp loader. Our results suggest that nuclear entry is linked mechanistically to Ty3 cDNA synthesis but that host replication factors antagonize Ty3 replication. Some of the factors we identified have been previously shown to affect Ty1 transposition and others to affect retroviral budding. Host factors, such as these, shared by distantly related Ty retroelements and retroviruses are novel candidates for antiviral targets.

Database and datasets are available on http://cdb.ics.uci.edu.

Alternative pre-mRNA splicing may be the most efficient and widespread mechanism to generate multiple protein isoforms from single genes. Here, we describe the genomic analysis of one of the most frequent types of alternative pre-mRNA splicing, alternative 59-and 39-splice-site selection. Using an EST-based alternative splicing database recording >47,000 alternative splicing events, we determined the frequency and location of alternative 59-and 39-splice sites within the human genome. The most common alternative splice sites used in the human genome are located within 6 nucleotides (nt) of the dominant splice site. We show that the EST database overrepresents alternative splicing events that maintain the reading frame, thus supporting the concept that RNA quality-control steps ensure that mRNAs that encode for potentially harmful protein products are destroyed and do not serve as templates for translation. The most frequent location for alternative 59-splice sites is 4 nt upstream or downstream from the dominant splice site. Sequence analysis suggests that this preference is a consequence of the U1 snRNP binding sequence at the 59-splice site, which frequently contains a GU dinucleotide 4 nt downstream from the dominant splice site. Surprisingly, ;50% of duplicated 39-YAG splice junctions are subject to alternative splicing. This high probability of alternative 39-splice-site activation in close proximity of the dominant 39-splice site suggests that the second step of the splicing may be prone to violate splicing fidelity.