Cloning And Function Of The Human Ctd Phosphorylation Enzyme Ubcp1 Tim50l1 And Mcp1 The Study

RNA polymerase II (RNAPII) is a multisubunits complex which transcribes protein-encoding genes. The largest subunit of RNAPII contains a unique C-terminal domain (CTD). Reversible phosphorylation of CTD plays an essential role in mRNA metabolism, including transcription initiation, elongation, capping, splicing and 3' end processing. Many CTD kinases have been identified, whereas only three kinds of CTD phosphatases, FCP1 , SCP1 and PP1, have been reported at present.We firstly cloned four genes from a human fetal cDNA library and multiple tissue cDNA panel. All these genes, UBCP1, Tim50L1, MCP1 and DUH, encode proteins containing the same CTD phosphatase domain as that in FCP1 and SCP1. The full length cDNA of UBCPl is 2215 bp, encoding a putative protein of 318 amino acids. The result of BLAST-N search against human genome database of GenBank shows that the gene is located on 5q33.3. It spans more than 22.7 Kb of genomic DNA and consists of 11 exons. Expression of UBCP1 mRNA was detected with relatively higher level in placenta, lung, testis and ovary. The expression level was relatively weak in heart, liver, kidney, spleen, thymus, colon and peripheral blood leukocyte. Strikingly, combined the results of EST, SAGE analysis and RT-PCR, the expression level of UBCPl in tumor tissues were up regulated obviously, indicating its correlation with tumorigenesis. Purified GST-UBCP1 shows distinct activity toward pNPP and its enzymatic properties are similar to those of FCP1 and SCP1. Its optimal pH is near 5.0 and activity is Mg²⁺ dependent. Site-directed mutagenesis defined six essential residues and four important residues for enzymatic activity. Most of these residues are also highly conserved in FCP1 and SCP1, suggesting UBCPl is structurally and mechanistically related with FCP1 and SCP1. Most importantly, CTD phosphatase assay showed UBCPl could effectively dephosphorylate Ser-5 site within CTD repeat whereas relatively low activity on Ser-2, indicating UBCPl is a CTD phosphatase with possible preference for Ser-5 in vitro. UBCPl has another ubiquitin-like domain (UBLD) in N-terminal, which contains a proteasome-interacting motif, indicating its potential ability of interacting with proteasome. Indeed, yeast two-hybrid system screen and in vitro binding assay revealed UBCPl could interact specifically with a subunit of proteasome named HsN3, whereas the UBLD is not necessary for this interaction. Expression of EGFP fusion protein in COS-7 confirmed the specific localization of UBCPl in nucleus and...