HRS/SRp40-Mediated Inclusion of the Fibronectin EIIIB Exon, a Possible Cause of Increased EIIIB Expression in Proliferating Liver

Abstract

Serine-arginine (SR)-rich proteins are believed to be important in mediating alternative pre-mRNA splicing. HRS/SRp40 expression is elevated in liver cell proliferation during development, regeneration, and oncogenesis. We tested whether HRS expression correlates with the appearance of alternatively spliced fibronectin transcripts during liver growth. HRS was highly expressed during the proliferative phase of liver development, correlating with expression of the fibronectin EIIIB alternative exon. In regenerating liver, HRS protein was induced in a time course consistent with the observed increase in fibronectin transcripts containing the EIIIB exon, particularly in nonparenchymal liver cells. Furthermore, in an in vivo assay, HRS, and not other SR proteins, directly mediated EIIIB exon inclusion in the fibronectin transcript. This alternative splicing was dependent on a purine-rich region within the EIIIB exon to which HRS specifically bound. We have established that HRS has the potential to contribute to the regulation of fibronectin pre-mRNA splicing during liver growth. Changes in fibronectin forms may be important in modifying liver architecture during the proliferative response, thus providing a potential mechanism by which SR proteins may participate in cellular growth control.The splicing factor HRS (hepatic arginine-serine protein) is expressed in proliferating liver cells. HRS is induced as an immediate-early gene in insulin-treated, mitogen-activated rat hepatoma H35 cells and in our study represented 12% of all insulin-induced cDNAs (7). HRS is a delayed-early gene in regenerating liver and is expressed at a very high level during liver development when the liver is rapidly proliferating (7, 46; reviewed in reference 9). HRS belongs to the serine-arginine (SR) protein family, which is comprised of at least nine structurally related proteins, including SRp20, SRp30a (SF2/ASF), SRp30b (SC35), SRp40, SRp55, and SRp75 (11,12,27,31,39,[52][53][54]. Comparing the partial sequence of human SRp40 and HRS sequence revealed that HRS is rat SRp40 (7, 52). Recently, the human homolog of HRS was identified and shown to have properties of other splicing factors in both in vitro and in vivo splicing assays (39).Pre-mRNA alternative splicing provides an important mechanism of gene and protein expression regulation (19,29,31,40). Through alternative splicing, one primary transcript can generate multiple proteins which may have different functions. Alternative splicing occurs via several pathways, including skipped exons, included introns, alternative 5Ј splice sites, alternative 3Ј splice sites, and mutually exclusive exons. SR proteins have been shown to play essential roles in both constitutive pre-mRNA splicing and regulation of pre-mRNA alternative splicing. In vitro and in vivo splicing assays indicate that SR proteins regulate alternative splicing by promoting the use of proximal 5Ј splice sites (10,23,26,55), and different SR proteins have distinct abilities to regulate alternative splicing of various model pr...

Influence of Polymerase II Processivity on Alternative Splicing Depends on Splice Site Strength

“…Nevertheless, it is important to point out that not all alternatively spliced regions are subjected to transcriptional control. For example, inclusion levels of the alternatively spliced EDII exon of human fibronectin (also named EDB and EIIIB) are not affected by promoter swapping, transcription factor activation, or the slow polymerase but are stimulated by overexpression of the SR protein Srp40 (27). This indicates that control by SR proteins might or might not coexist with transcriptional control.…”

Mitogenic Up-regulation of the PRL-1 Protein-tyrosine Phosphatase Gene by Egr-1

“…Primary antibody anti-Egr 1 antiserum (Santa Cruz Biotechnology) was diluted 1:1000 and incubated with the membrane for 1 h at room temperature. Horseradish peroxidase-linked secondary antibody was added at a dilution of 1:10000 for 30 min and then detected by chemiluminescence (Amersham Pharmacia Biotech) according to the instructions of the manufacturer (27).…”

Identification of Two RNA cis-Elements That Function to Regulate the 5′ Splice Site Selection of Bcl-x Pre-mRNA in Response to Ceramide

“…For example, Taub and co-workers (40) first cloned the SR protein, SRp40/HRS, using a subtractive cDNA library in a model of regenerating rat liver responsive to insulin. Subsequently, they showed that this increase in the expression of SRp40 in response to insulin was required for the inclusion of exon EIIIB of the fibronectin gene (22). Krainer and co-workers (34) demonstrated that increased levels of SRp30a during T-cell activation correlated with changes in the alternative splicing patterns of CD44 and CD45 pre-mRNA.…”

“…Several studies in the literature (22,34,40) demonstrate that changes in the level of expression of SR proteins in response to growth factors and cellular proliferation can also regulate the alternative splicing of specific pre-mRNAs. For example, Taub and co-workers (40) first cloned the SR protein, SRp40/HRS, using a subtractive cDNA library in a model of regenerating rat liver responsive to insulin.…”

“…A family of ceramide-regulated enzymes has been identified, ceramide-activated protein phosphatases, which include the serine/threonine-specific protein phosphatases PP1 1 and PP2A (11)(12)(13)(14). Interestingly, SR proteins, a family of arginine/serinerich domain containing proteins, have been shown to be specific PP1 substrates and are well established regulators of alternative pre-mRNA processing (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). Furthermore, endogenous ceramide has been found recently to modulate the phosphorylation status of SR proteins in a PP1-dependent manner (26).…”