All of these metabolic actions are performed by large multisubunit machineries, appear to be mechanistically linked with each other, and they are subject to regulation (12, 21, 27)

All of these metabolic actions are performed by large multisubunit machineries, appear to be mechanistically linked with each other, and they are subject to regulation (12, 21, 27). During mRNA processing, the removal of introns is usually facilitated by the spliceosome, which is composed of various small nuclear ribonucleoprotein particles (snRNPs) and several non-snRNP protein components (15, 29, 36), including users of the serine- and arginine-rich (SR) protein family (10, 23). RNA processing and apoptosis. Gene expression in eukaryotic cells is usually a multistep process that begins in the nucleus with the generation of an mRNA precursor by gene transcription. In subsequent actions the mRNA transcript is usually subject to processing and export into the cytoplasm, where it undergoes quality control, translation, and finally decay. All of these metabolic actions are performed by large multisubunit machineries, appear to be mechanistically linked with each NSI-189 other, and are subject to regulation (12, 21, 27). During mRNA processing, the removal of introns is usually facilitated by the spliceosome, which is composed of various small nuclear ribonucleoprotein particles (snRNPs) and several non-snRNP protein components (15, 29, 36), including users of the serine- and arginine-rich (SR) protein family (10, 23). In mammalian cells, SR proteins and other splicing factors exist in nuclear regions called splicing factor compartments, nuclear speckles, or interchromatin granules and have proposed functions in NSI-189 several actions Rabbit Polyclonal to RAB33A of NSI-189 the gene expression process (26, 31). For example, a spliceosome-associated RNA-binding protein with a serine-rich domain name, RNPS1, is thought to be involved in the regulation of splicing, mRNA export, and nonsense-mediated mRNA decay (16, 17, 19, 20, 25). Regulation of RNA processing allows alternate splicing of transcripts, a process that enables the cell to generate multiple unique transcript species from a common precursor. Alternate splicing adds an additional layer of control to the gene expression process, since it leads to the production of discrete protein isoforms that can have distinct functions in cellular events (18, 22). One important cellular event is the process of programmed cell death or apoptosis (32). Interestingly, numerous apoptotic genes, including death receptors and intracellular components of the death machinery, are subject to option splicing (3, 13). Apoptosis is usually distinguished from other forms of cell death, such as necrosis, on the basis of morphological criteria, which include membrane blebbing, fragmentation of nuclear DNA, and chromatin condensation (11). Most of the apoptotic morphological changes are caused by a set of cysteinyl aspartate-specific proteinases (caspases), which are specifically activated in apoptotic cells and cleave selected target proteins (6, 14). Among these target proteins is usually a nuclear protein named Acinus (for apoptotic chromatin condensation inducer in the nucleus) (30). Acinus is usually expressed in different isoforms, termed Acinus-L, Acinus-S, and Acinus-S, with molecular masses of ca. 220, 98, and 94 kDa, respectively (Fig. ?(Fig.1a).1a). Acinus is usually cleaved during apoptosis by NSI-189 caspase 3 and by an unidentified protease to produce a 23-kDa isoform NSI-189 (p23) that has been implicated in mediating apoptotic chromatin condensation prior to DNA fragmentation (30). Open in a separate windows FIG. 1. Purification of a complex consisting of SAP18, RNPS1, and Acinus-L. (a) Schematic representation of Acinus isoforms. Described protein domains as well as protease cleavage sites and their corresponding amino acids (1, 30) are indicated as follows: red box, P-loop for nucleotide binding; green box, SAP domain; dark blue box, region much like RNA recognition motif of Sxl; black box, region specific for Acinus-S; black arrowhead, cleavage site for unknown protease; reddish arrowhead, cleavage site for caspase 3. Regions recognized by polyclonal antibodies against Acinus ([N], [S(N10)], [A19], [M], and [C]) (observe also Materials and Methods) are shown above the representation of Acinus-L. (b) Purification plan used to isolate the ASAP complexes from HeLa cell nuclear pellets. (c) Silver stain of an SDS-4 to 15% (wt/vol) polyacrylamide gradient gel made up of aliquots of fractions (input [I] and the indicated column fractions) derived from the MonoP column. High-molecular-mass requirements [M] are indicated at the left, and components of the ASAP complex are.