Human phosphorylated CTD-interacting protein, PCIF1, negatively modulates gene expression by RNA polymerase II.

Phosphorylation of the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) regulates transcription cycle and coordinates recruitment of RNA processing factors and chromatin regulators. Recently, we reported the identification of human PCIF1 as a novel protein that directly binds to the phosphorylated CTD via its WW domain, which is highly homologous to the WW domain of human peptidylprolyl isomerase Pin1. Although PCIF1 has been shown to associate with phosphorylated Pol II, functional consequence of the interaction remains unclear. Here we further characterized the cytological, structural, and functional properties of human PCIF1. Immunofluorescence microscopy revealed that endogenous PCIF1 was colocalized with the phosphorylated Pol II and the transcription elongation factor DSIF in the cell nucleus. We also found that PCIF1 WW domain inhibits the CTD phosphatase activity of SCP1 in vitro. By examining the effect of either PCIF1 overexpression or knockdown on the transactivation of reporter gene expression by various transcriptional activation domains, we found that PCIF1 significantly repressed the transactivation depend on its CTD binding ability. These data suggest that PCIF1 modulates phosphorylation status of the CTD and negatively regulates gene expression by Pol II.

PCIF1, a novel human WW domain-containing protein, interacts with the phosphorylated RNA polymerase II.

Phosphorylation of the carboxy-terminal domain (CTD) of RNA polymerase II (RNAP II) largest subunit has an important role in transcription elongation and in coupling transcription to pre-mRNA processing. To identify proteins that can directly bind to the phosphorylated CTD, we screened a human cDNA expression library using 32P-labeled CTD as a probe. Here we report the cloning and characterization of a novel human WW domain-containing protein, PCIF1 (phosphorylated CTD interacting factor 1). PCIF1 is composed of 704 amino acids. The WW domain of PCIF1 can directly and preferentially bind to the phosphorylated CTD compared to the unphosphorylated CTD. PCIF1 binds to the hyperphosphorylated RNAP II (RNAP IIO) in vitro and in vivo. Double immunofluorescence labeling in HeLa cells demonstrated that PCIF1 and endogenous RNAP IIO are co-localized in the cell nucleus. Thus, PCIF1 may play a role in mRNA synthesis by modulating RNAP IIO activity.

Roles for the two-hybrid system in exploration of the yeast protein interactome.

Comprehensive analysis of protein-protein interactions is a challenging endeavor of functional proteomics and has been best explored in the budding yeast. The yeast protein interactome analysis was achieved first by using the yeast two-hybrid system in a proteome-wide scale and next by large-scale mass spectrometric analysis of affinity-purified protein complexes. While these interaction data have led to a number of novel findings and the emergence of a single huge network containing thousands of proteins, they suffer many false signals and fall short of grasping the entire interactome. Thus, continuous efforts are necessary in both bioinformatics and experimentation to fully exploit these data and to proceed another step forward to the goal. Computational tools to integrate existing biological knowledge buried in literature and various functional genomic data with the interactome data are required for biological interpretation of the huge protein interaction network. Novel experimental methods have to be developed to detect weak, transient interactions involving low abundance proteins as well as to obtain clues to the biological role for each interaction. Since the yeast two-hybrid system can be used for the mapping of the interaction domains and the isolation of interaction-defective mutants, it would serve as a technical basis for the latter purpose, thereby playing another important role in the next phase of protein interactome research.