Protein PSF Research Articles

Splicing and transcription-associated proteins PSF and p54nrb/nonO bind to the RNA polymerase II CTD.

The carboxyl-terminal domain (CTD) of the largest subunit of eukaryotic RNA polymerase II (pol II) plays an important role in promoting steps of pre-mRNA processing. To identify proteins in human cells that bind to the CTD and that could mediate its functions in pre-mRNA processing, we used the mouse CTD expressed in bacterial cells in affinity chromatography experiments. Two proteins present in HeLa cell extract, the splicing and transcription-associated factors, PSF and p54nrb/NonO, bound specifically and could be purified to virtual homogeneity by chromatography on immobilized CTD matrices. Both hypo- and hyperphosphorylated CTD matrices bound these proteins with similar selectivity. PSF and p54nrb/NonO also copurified with a holoenzyme form of pol II containing hypophosphorylated CTD and could be coimmunoprecipitated with antibodies specific for this and the hyperphosphorylated form of pol II. That PSF and p54nrb/NonO promoted the binding of RNA to immobilized CTD matrices suggested these proteins can interact with the CTD and RNA simultaneously. PSF and p54nrb/NonO may therefore provide a direct physical link between the pol II CTD and pre-mRNA processing components, at both the initiation and elongation phases of transcription.

Identification of tyrosine-phosphorylated proteins associated with the nuclear envelope.

The nuclear envelope separates the nucleoplasm from the rest of the cell. Throughout the cell cycle, its structural integrity is controlled by reversible protein phosphorylation. Whereas its phosphorylation-dependent disassembly during mitosis is well characterized, little is known about phosphorylation events at this structure during interphase. The few characterized examples cover protein phosphorylation at serine and threonine residues, but not tyrosine phosphorylation at the nuclear envelope. Here, we demonstrate that tyrosine phosphorylation and dephosphorylation occur at the nuclear envelope of intact Neuro2a mouse neuroblastoma cells. Tyrosine kinase and phosphatase activities remain associated with purified nuclear envelopes. A similar pattern of tyrosine-phosphorylated nuclear envelope proteins suggests that the same tyrosine kinases act at the nuclear envelope of intact cells and at the purified nuclear envelope. We have also identified eight tyrosine-phosphorylated nuclear envelope proteins by 2D BAC/SDS/PAGE, immunoblotting with phosphotyrosine-specific antibodies, tryptic in-gel digestion, and MS analysis of tryptic peptides. These proteins are the lamina proteins lamin A, lamin B1, and lamin B2, the inner nuclear membrane protein LAP2beta, the heat shock protein hsc70, and the DNA/RNA-binding proteins PSF, hypothetical 16-kDa protein, and NonO, which copurify with the nuclear envelope.

Aberrant folate response and premature development in a mutant of Dictyostelium discoideum

Growth and development are mutually exclusive in Dictyostelium discoideum. The transition between the two stages of the life cycle is regulated by the relative abundance of nutrients and proteins secreted by the cells which reflect population density. At the transition from growth to development, the discoidin genes – developmental markers – are induced by the “quorum” protein PSF. The effect of PSF is counteracted by food bacteria and by folate [8]. We show that folate treatment during growth delays morphologic development. Furthermore, we demonstrate that in a mutant of Dictyostelium discoideum (V188, renamed HBW3), which expresses discoidinI during growth and which develops rapidly [46], discoidinI expression is less sensitive to folate than in wild type cells. Finally, we present evidence that fragments of the discoidinIγ promoter which are unresponsive to PSF and CM are sufficient for misregulation in the mutant. The only known regulator of these promoter elements is folate. Changes in the expression of other early developmental genes are also shown. Taken together, these data suggest that the reduced sensitivity to folate might be the cause for the “rapid development” phenotype of the mutant and that folate regulates developmental timing.