Multiple splicing factors are released from endogenous complexes during in vitro pre-mRNA splicing.

Pre-mRNA splicing occurs in a macromolecular complex called the spliceosome. Efforts to isolate spliceosomes from in vitro splicing reactions have been hampered by the presence of endogenous complexes that copurify with de novo spliceosomes formed on added pre-mRNA. We have found that removal of these large complexes from nuclear extracts prevents the splicing of exogenously added pre-mRNA. We therefore examined these complexes for the presence of splicing factors and proteins known or thought to be involved in RNA splicing. These fast-sedimenting structures were found to contain multiple small nuclear ribonucleoproteins (snRNPs) and a fragmented heterogeneous nuclear ribonucleoprotein complex. At least two splicing factors other than the snRNPs were also associated with these large structures. Upon incubation with ATP, these splicing factors as well as U1 and U2 snRNPs were released from these complexes. The presence of multiple splicing factors suggests that these complexes may be endogenous spliceosomes released from nuclei during preparation of splicing extracts. The removal of these structures from extracts that had been preincubated with ATP yielded a splicing extract devoid of large structures. This extract should prove useful in the fractionation of splicing factors and the isolation of native spliceosomes formed on exogenously added pre-mRNA.     

Proteolysis of cardiac gap junctions during their isolation from rat hearts

Summary Gap junctions (GJ) isolated from rat hearts in presence of the protease inhibitor phenylmethylsulfonylfuoride (PMSF) contain a M_r 44,000 to 47.000 major polypeptide and have a urea-resistant layer of fuzz on their cytoplasmic surfaces, whereas junctions isolated without PMSF are proteolyzed to a M_r 29.500 polypeptide by a serine protease and have smooth cytoplasmic surfaces (C.K. Manjunath, G.E. Goings & E. PageAm. J. Physiol. 246:H865–H875, 1984). Rat liver GJ isolated with or without PMSF contain a M_r 28,000 polypeptide and have smooth cytoplasmic surfaces. Here we examine the origin, type and inhibitor sensitivity of the heart protease; why similar proteolysis is absent during isolation of rat liver gap junctions; and whether the M_r 44.000 to 47,000 cardiac GJ polypeptide is the precursor of the M_r 29,500 subunit. We show that the M_r 44,000 to 47,000 polypeptide corresponds to the unproteolyzed connexon subunit; that proteolysis of this polypeptide occurs predominantly during exposure to high ionic strength solution (0.6m KI) which releases serine protease from mast cell granules; that this protease is inhibitable with PMSF and (less completely) soybean trypsin inhibitor and chymostatin; and thatin vivo degranulation of mast cells by injecting rats with compound 48/80 fails to prevent breakdown of cardiac GJ during isolation. The results support the concept that GJ from rat heart and liver differ in protein composition.     

Proteolysis and the cell cycle

Without doubt, one of the more dramatic breakthroughs in recent cell cycle history has been the discovery that growth regulators are controlled by proteolysis. This concept blossomed within the last six or seven years, but the story really began when cyclins were discovered, soon followed by the suggestion that proteolysis events might control cell cycle transitions. Proteolytic targets that are now known include most of the cyclins, cyclin dependent kinase inhibitors, DNA replication factors, the securin class of proteins that inhibit loss of sister chromatid cohesion following DNA replication and, of course, the cohesion factor itself. Protein degradation is controlled in various ways including ubiquitin-dependent targeting to proteasomes, activation of ubiquitin ligases by ubiquitin-like molecule conjugation, phosphorylation of proteolytic targets, and activation of the separin class of proteases.     

Proteolysis of cortactin by calpain regulates membrane protrusion during cell migration

Calpain 2 regulates membrane protrusion during cell migration. However, relevant substrates that mediate the effects of calpain on protrusion have not been identified. One potential candidate substrate is the actin binding protein cortactin. Cortactin is a Src substrate that drives actin polymerization by activating the Arp2/3 complex and also stabilizes the cortical actin network. We now provide evidence that proteolysis of cortactin by calpain 2 regulates membrane protrusion dynamics during cell migration. We show that cortactin is a calpain 2 substrate in fibroblasts and that the preferred cleavage site occurs in a region between the actin binding repeats and the alpha-helical domain. We have generated a mutant cortactin that is resistant to calpain proteolysis but retains other biochemical properties of cortactin. Expression of the calpain-resistant cortactin, but not wild-type cortactin, impairs cell migration and increases transient membrane protrusion, suggesting that calpain proteolysis of cortactin limits membrane protrusions and regulates migration in fibroblasts. Furthermore, the enhanced protrusion observed with the calpain-resistant cortactin requires both the Arp2/3 binding site and the Src homology 3 domain of cortactin. Together, these findings suggest a novel role for calpain-mediated proteolysis of cortactin in regulating membrane protrusion dynamics during cell migration.     

Dephosphorylation-dependent sorting of SR splicing factors during mRNP maturation

SR proteins are a family of sequence-specific RNA binding proteins originally discovered as essential factors for pre-mRNA splicing and recently implicated in mRNA transport, stability, and translation. Here, we used a genetic complementation system derived from conditional knockout mice to address the function and regulation of SR proteins in vivo. We demonstrate that ASF/SF2 and SC35 are each required for cell viability, but, surprisingly, the effector RS domain of ASF/SF2 is dispensable for cell survival in MEFs. Although shuttling SR proteins have been implicated in mRNA export, prevention of ASF/SF2 from shuttling had little impact on mRNA export. We found that shuttling and nonshuttling SR proteins are segregated in an orderly fashion during mRNP maturation, indicating distinct recycling pathways for different SR proteins. We further showed that this process is regulated by differential dephosphorylation of the RS domain, thus revealing a sorting mechanism for mRNP transition from splicing to export.     

Alternative splicing during chondrogenesis: cis and trans factors involved in splicing of fibronectin exon EIIIA

Primary chicken mesenchymal cells from limb buds and vertebral chondrocytes have been used to study the changes that occur in alternative mRNA splicing of fibronectin exon EIIIA during chondrogenesis. The mesenchymal cell phenotype (exon EIIIA included) and chondrocyte phenotype (exon EIIIA excluded) were preserved in culture. Both primary cell types were transfected with an EIIIA minigene and alternative splicing was monitored by S1 protection assay. Differential cell‐specific splicing of the reporter was observed. The roles of two regulatory elements, an exon splicing enhancer (ESE) and an exon splicing silencer (ESS) were examined. Both elements were required for EIIIA inclusion into mRNA in mesenchymal cells. Gel mobility shift assays revealed that both chondrocyte‐ and mesenchymal cell‐derived nuclear extracts contained exon EIIIA binding factors, but the RNA binding factors present in the two cell types appeared to be distinct. The ESE and ESS appeared to cooperate in the formation of both cell type‐specific complexes. These results suggest a model in which inhibitory factors enriched in chondrocytes compete with positive factors enriched in mesenchymal cells for binding to exon EIIIA, determining whether the exon is included. J. Cell. Biochem. 76:341–351, 1999. © 1999 Wiley‐Liss, Inc.     

Alternative splicing during chondrogenesis: cis and trans factors involved in splicing of fibronectin exon EIIIA

Primary chicken mesenchymal cells from limb buds and vertebral chondrocytes have been used to study the changes that occur in alternative mRNA splicing of fibronectin exon EIIIA during chondrogenesis. The mesenchymal cell phenotype (exon EIIIA included) and chondrocyte phenotype (exon EIIIA excluded) were preserved in culture. Both primary cell types were transfected with an EIIIA minigene and alternative splicing was monitored by S1 protection assay. Differential cell-specific splicing of the reporter was observed. The roles of two regulatory elements, an exon splicing enhancer (ESE) and an exon splicing silencer (ESS) were examined. Both elements were required for EIIIA inclusion into mRNA in mesenchymal cells. Gel mobility shift assays revealed that both chondrocyte- and mesenchymal cell-derived nuclear extracts contained exon EIIIA binding factors, but the RNA binding factors present in the two cell types appeared to be distinct. The ESE and ESS appeared to cooperate in the formation of both cell type-specific complexes. These results suggest a model in which inhibitory factors enriched in chondrocytes compete with positive factors enriched in mesenchymal cells for binding to exon EIIIA, determining whether the exon is included.     

Nuclear organization of pre-mRNA splicing factors.

The splicing of mRNA precursors (pre-mRNA) in the nucleus is catalyzed by a complex machinery termed the spliceosome. In order to understand how it functions in vivo, it is essential to complement biochemical analyses with a detailed study of how spliceosome components are organized within the nucleus.