Antibodies to hnRNP core proteins inhibitin vitrosplicing of human β-globin pre-mRNA

Abstract

In vitro splicing of human beta-globin pre-mRNA can be fully inhibited by treatment of the splicing extract with polyclonal antibodies against hnRNP core proteins prior to the addition of pre-mRNA. Inhibition of the first step in the splicing pathway, cleavage at the 5' splice site and lariat formation, requires more antibodies than inhibition of the second step, cleavage at the 3' splice site and exon ligation. The anti-hnRNP antibodies can also inhibit the splicing reaction after the formation of the active nucleoprotein splicing complex which is known to occur during the initial lag period. Thus, hnRNP core proteins appear to be present in the complex that performs pre-mRNA splicing.

Immunopurification of heterogeneous nuclear ribonucleoprotein particles reveals an assortment of RNA-binding proteins.

“…The specificity of the immunopurification procedure can thus be taken advantage of for the identification of the components of intact hnRNP particles and for the study of their structure and function. The antibodies have also been useful reagents to investigate the role of hnRNP proteins in mRNA splicing (Choi et al 1986;Sierakowska et al 1986). …”

Purification and characterization of pre-mRNA splicing factor SF2 from HeLa cells.

“…Krainer, unpubl.), yet we know that SF2 and C proteins are distinct. Thus, either C protein is not necessary for splicing or the small amount in the $100 extract is sufficient for activity (Choi et al 1986;Sierakowska et al 1986). We also note that there may be subtle differences in the composition of $100 extracts prepared in different laboratories.…”

“…of action are unknown. In addition, several HeLa cell polypeptides have been described and, in some cases, purified, that interact with RNA specifically and/or are present in spliceosomes and are thus potential splicing factors (Choi et al 1986;Gerke and Steitz 1986;Sierakowska et al 1986; Tazi et al 1986;Kumar et al 1987; Swanson and Dreyfuss 1988;Garcia-Blanco et al 1989; Zamore and Green 1989;Fu and Maniatis 1990}. The snRNA components of U1 and U2 snRNPs basepair to complementary sequences within conserved splicing signals on pre-mRNA, and these interactions have been shown to play a fundamental role in splice site recognition (Zhuang andWeiner 1986, 1989;Parker et al 1987;Seraphin et al 1988;Siliciano and Guthrie 1988;Wu and Manley 1989). In addition, these and other snRNAs could potentially act as ribozymes, although it is not known at present whether RNA catalysis is involved in nuclear pre-mRNA splicing.…”

Serine/threonine phosphorylation regulates binding of C hnRNP proteins to pre-mRNA.

“…The first described spliceosome proteins were called hnRNP proteins, based on their association with large heterogeneous nuclear RNA transcripts now known to include unspliced pre-mRNAs (6-12). The C-group hnRNP proteins are abundant nuclear proteins of Mr 42,000-44,000 that have been implicated in splicing (13,14) and are known to be phosphorylated in vivo (8,(15)(16)(17) by a casein kinase II-type activity (18,19 ATP, and 20 mM creatine phosphate. Streptavidin-bound pre-mRNA was then added to the nuclear extract and incubation was continued for an additional 30 min (30°C).…”

“…The first described spliceosome proteins were called hnRNP proteins, based on their association with large heterogeneous nuclear RNA transcripts now known to include unspliced pre-mRNAs (6)(7)(8)(9)(10)(11)(12). The C-group hnRNP proteins are abundant nuclear proteins of Mr 42,000-44,000 that have been implicated in splicing (13,14) and are known to be phosphorylated in vivo (8,(15)(16)(17) by a casein kinase II-type activity (18,19). Because phosphorylation plays an important role in the regulation of protein binding to nucleic acids (20)(21)(22)(23)(24)(25), we have investigated C hnRNP protein phosphorylation/dephosphorylation in a HeLa cell nuclear extract system used for in vitro splicing of pre-mRNA and have also examined C hnRNP protein binding to pre-mRNA as a function of phosphorylation.…”