Alternative splicing of mRNA precursors allows the synthesis of multiple mRNAs from a single primary transcript, significantly expanding the information content and regulatory possibilities of higher eukaryotic genomes. High-throughput enabling technologies, particularly large-scale sequencing and splicing-sensitive microarrays, are providing unprecedented opportunities to address key questions in this field. The picture emerging from these pioneering studies is that alternative splicing affects most human genes and a significant fraction of the genes in other multicellular organisms, with the potential to greatly influence the evolution of complex genomes. A combinatorial code of regulatory signals and factors can deploy physiologically coherent programs of alternative splicing that are distinct from those regulated at other steps of gene expression. Pre-mRNA splicing and its regulation play important roles in human pathologies, and genome-wide analyses in this area are paving the way for improved diagnostic tools and for the identification of novel and more specific pharmaceutical targets.Removal of introns from pre-mRNAs is an essential step in eukaryotic gene expression (see Fig. 1A). Alternative patterns of intron removal allow the synthesis of multiple mRNAs from a single gene encoding different proteins (see Fig. 1B). This minireview focuses on how the recent application of high-throughput technologies is providing a novel and empowered perspective to address the following four outstanding questions in the field of alternative pre-mRNA splicing (AS). 3 PrevalenceAlthough early estimates suggested that AS affects only a small fraction of human genes, large-scale genome and transcriptome sequencing projects allowing extensive alignments of mRNA with genomic sequences (see Fig. 2A) indicate that the majority of human genes are alternatively spliced (1). Splicing-sensitive microarrays (see Fig. 2B) provide independent confirmation of this high incidence. Using a variety of experimental designs and biological samples, several of these studies have produced consistent estimates of 70 -80% of alternatively spliced genes in the human genome (2-4). The production of alternatively spliced transcripts is therefore a general feature of human genes to be incorporated in biological and medical studies, from the design of gene knock-out/down experiments to molecular diagnostics or drug screens.In addition, in-depth analysis of individual genes frequently reveals novel AS isoforms, suggesting that transcript diversity is far from fully annotated in current data bases. Furthermore, recent analyses of HapMap cell lines document variations in AS among different individuals, an observation with significant basic and medical implications (5,6).Is the prevalence of AS uniform across organisms, or does it increase with the evolution of complexity? In silico comparative analyses have provided contradictory answers to this question. Although some data support an increased incidence of AS in vertebrates (7), it is also clear that ...

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Maintaining adequate proteasomal proteolytic activity is essential for eukaryotic cells. For metazoan cells, little is known about the composition of genes that are regulated in the proteasome network or the mechanisms that modulate the levels of proteasome genes. Previously, two distinct treatments have been observed to induce 26S proteasome levels in Drosophila melanogaster cell lines, RNA interference (RNAi)-mediated inhibition of the 26S proteasome subunit Rpn10/S5a and suppression of proteasome activity through treatment with active-site inhibitors. We have carried out genome array profiles from cells with decreased Rpn10/S5a levels using RNAi or from cells treated with proteasome inhibitor MG132 and have thereby identified candidate genes that are regulated as part of a metazoan proteasome network. The profiles reveal that the majority of genes that were identified to be under the control of the regulatory network consisted of 26S proteasome subunits. The 26S proteasome genes, including three new subunits, Ubp6p, Uch-L3, and Sem1p, were found to be up-regulated. A number of genes known to have proteasome-related functions, including Rad23, isopeptidase T, sequestosome, and the genes for the segregase complex TER94/VCP-Ufd1-Npl4 were also found to be up-regulated. RNAi-mediated inhibition against the segregase complex genes demonstrated pronounced stabilization of proteasome substrates throughout the Drosophila cell. Finally, transcriptional reporter assays and deletion mapping studies in Drosophila demonstrate that proteasome mRNA induction is dependent upon the 5 untranslated regions (UTRs). Transfer of the 5 UTR from the proteasome subunit Rpn1/S2 to a noninducible promoter was sufficient to confer transcriptional upregulation of the reporter mRNA after proteasome inhibition.Proteasome-dependent degradation serves an essential role in the removal of a wide variety of key nuclear and cytosolic proteins (35,42,45,52). This pathway also carries out an important housekeeping function by clearing cells from potentially harmful abnormal proteins that arise as the result of mutations, translational errors, misfolding, or postsynthetic damage and functions in the cytoplasm as a part of the protein quality control system for the endoplasmic reticulum (25).Structurally, the 26S proteasome consists of a 20S catalytic core and a 19S regulatory complex that associates with the ends of the 20S proteasome in an ATP-dependent manner (3,20,46). The eukaryotic 20S proteasome is composed of 14 different subunits arranged in four stacked, seven-membered rings that form the barrel-shaped complex (18, 43). The 19S regulatory complex is itself composed of two distinct subcomplexes, the base and the lid (15). Six distinct ATPase subunits proposed to function in substrate unfolding and gating of the 20S pore have been localized to the base along with two additional subunits (7,12,16,36). At least eight subunits form a lid subcomplex that is thought to be necessary for the processing of polyubiquitinated proteins and exhibit high...

The S13 subunit (also called Pad1, Rpn11, and MPR1) is a component of the 19S complex, a regulatory complex essential for the ubiquitin-dependent proteolytic activity of the 26S proteasome. To address the functional role of S13, we combined double-stranded RNA interference (RNAi) against the Drosophila proteasome subunit DmS13 with expression of wild-type and mutant forms of the homologous human gene, HS13. These studies show that DmS13 is essential for 26S function. Loss of the S13 subunit in metazoan cells leads to increased levels of ubiquitin conjugates, cell cycle defects, DNA overreplication, and apoptosis. In vivo assays using short-lived proteasome substrates confirmed that the 26S ubiquitin-dependent degradation pathway is compromised in S13-depleted cells. In complementation experiments using Drosophila cell lines expressing HS13, wild-type HS13 was found to fully rescue the knockdown phenotype after DmS13 RNAi treatment, while an HS13 containing mutations (H113A-H115A) in the proposed isopeptidase active site was unable to rescue. A mutation within the conserved MPN/JAMM domain (C120A) abolished the ability of HS13 to rescue the Drosophila cells from apoptosis or DNA overreplication. However, the C120A mutant was found to partially restore normal levels of ubiquitin conjugates. The S13 subunit may possess multiple functions, including a deubiquitinylating activity and distinct activities essential for cell cycle progression that require the conserved C120 residue.The ubiquitin-proteasome system is the major nonlysosomal pathway responsible for the degradation of intracellular proteins in eukaryotic cells. This system participates in the regulation of a vast number of cellular pathways through timely and specific conjugation of target proteins with multiple ubiquitin molecules followed by proteolysis by the 26S proteasome (52). The 26S proteasome is a large protease (2,500 kDa) composed of a 20S catalytic core and a 19S regulatory complex that associates with the ends of the 20S proteasome in an ATPdependent manner. The 20S core (700 kDa) is a compartmentalized multicatalytic complex composed of a total of 28 ␣ and ␤ subunits arranged in four stacked heptameric rings. The subunits generate a hollow cylindrical structure that separates the cytosolic environment from the catalytic sites located at the lumenal face of the ␤-rings (16,30,46,60). In the absence of regulatory factors, the 20S proteasome exists in an autoinhibited (latent) state in which the free N-terminal tails of its ␣ subunits extend into the 20S pores and thus sterically block substrate access to the lumen (17). Activation of the 20S proteasome requires interactions of the ␣ subunits with specific regulators, such as the PA28␣␤ or -␥ heptamer (35), PA200 (47), or the 19S regulatory complex (8).The 19S complex has been directly implicated in nonproteolytical regulatory functions, such as nucleotide excision-repair (10, 38) and transcription elongation (7,14). Its main function however, is to carry out several distinct steps critical for u...

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