Cloning and characterization of PIMT, a protein with a methyltransferase domain, which interacts with and enhances nuclear receptor coactivator PRIP function

Abstract

The nuclear receptor coactivators participate in the transcriptional activation of specific genes by nuclear receptors. In this study, we report the isolation of a nuclear receptor coactivator-interacting protein from a human liver cDNA library by using the coactivator peroxisome proliferator-activated receptor-interacting protein (PRIP) (ASC2͞AIB3͞RAP250͞NRC͞TRBP) as bait in a yeast twohybrid screen. Human PRIP-interacting protein cDNA has an ORF of 2,556 nucleotides, encodes a protein with 852 amino acids, and contains a 9-aa VVDAFCGVG methyltransferase motif I and an invariant GXXGXXI segment found in K-homology motifs of many RNA-binding proteins. The gene encoding this protein, designated PRIP-interacting protein with methyltransferase domain (PIMT), is localized on chromosome 8q11 and spans more than 40 kb. PIMT mRNA is ubiquitously expressed, with a high level of expression in heart, skeletal muscle, kidney, liver, and placenta. Using the immunofluorescence localization method, we found that PIMT and PRIP proteins appear colocalized in the nucleus. PIMT strongly interacts with PRIP under in vitro and in vivo conditions, and the PIMT-binding site on PRIP is in the region encompassing amino acids 773-927. PIMT binds S-adenosyl-L-methionine, the methyl donor for methyltransfer reaction, and it also binds RNA, suggesting that it is a putative RNA methyltransferase. PIMT enhances the transcriptional activity of peroxisome proliferator-activated receptor ␥ and retinoid-X-receptor ␣, which is further stimulated by coexpression of PRIP, implying that PIMT is a component of nuclear receptor signal transduction apparatus acting through PRIP. Definitive identification of the specific substrate of PIMT and the role of this RNA-binding protein in transcriptional regulation remain to be determined.

An enzyme-coupled high-throughput assay for screening RNA methyltransferase activity inE. Colicell lysate

“…20 Today, measuring methyltransferase activity is mainly based on using [methyl-3 H] SAM as cosubstrate. 15,19,21 Although this approach can be used for most enzymes, it requires access to an accordingly equipped isotope laboratory, which is not always available. Downstream processing requires gel electrophoresis and analysis by autoradiography which makes the overall process long and tedious and severely limits throughput.…”

Phosphorylation of Transcriptional Coactivator Peroxisome Proliferator-activated Receptor (PPAR)-binding Protein (PBP)

“…Plasmid Constructs-pCDNA3.1-PPAR ␥1, pCMX-PBP, pCMX-RXR, Gal4-PBP, pGEX-PPAR␥1, 5xUAS-Luc, 3XPPRE-Luc, Raf-BXB, Raf-BXB301, pCMV-ERK2-HA, pCMV-ERK2-DN, pCMV-ERK5-HA, pCMV-ERK5-DN, MEK1-DN, JNK, MKK4, MKK7, and MEKK1 have been described earlier (52)(53)(54)(55). Sequences encoding peptide fragments of PBP fused to glutathione S-transferase (hereafter GST-PBP) were generated by subcloning respective PCR fragments amplified from pCMX-PBP into the EcoR1/NotI sites of pGEX4T1 (Amersham Biosciences).…”

The Nuclear Hormone Receptor Coactivator NRC Is a Pleiotropic Modulator Affecting Growth, Development, Apoptosis, Reproduction, and Wound Repair

“…In addition to CBP, NRC has been reported to associate with a number of other factors, including CoAA, PIMT, CAPER, and NIF-1 (27,28,40,71). Unlike NIF-1, CAPER, PIMT, and CoAA each contain RNA binding motifs.…”