Alternative mRNA Splicing Produces a Novel Biologically Active Short Isoform of PGC-1α

Yubin Zhang,Peter Huypens,Aaron W Adamson,Ji Suk Chang,Tara M Henagan,Anik Boudreau,Natalie R Lenard,David Burk,Johannes Klein,Nina Perwitz,Jeho Shin,Mathias Fasshauer,Anastasia Kralli,Thomas W Gettys

doi:10.1074/jbc.m109.037556

Abstract

The transcriptional co-activator PGC-1alpha regulates functional plasticity in adipose tissue by linking sympathetic input to the transcriptional program of adaptive thermogenesis. We report here a novel truncated form of PGC-1alpha (NT-PGC-1alpha) produced by alternative 3' splicing that introduces an in-frame stop codon into PGC-1alpha mRNA. The expressed protein includes the first 267 amino acids of PGC-1alpha and 3 additional amino acids from the splicing insert. NT-PGC-1alpha contains the transactivation and nuclear receptor interaction domains but is missing key domains involved in nuclear localization, interaction with other transcription factors, and protein degradation. Expression and subcellular localization of NT-PGC-1alpha are dynamically regulated in the context of physiological signals that regulate full-length PGC-1alpha, but the truncated domain structure conveys unique properties with respect to protein-protein interactions, protein stability, and recruitment to target gene promoters. Therefore, NT-PGC-1alpha is a co-expressed, previously unrecognized form of PGC-1alpha with functions that are both unique from and complementary to PGC-1alpha.

Highlights

PGC-1␣ was identified as a brown adipocyte-enriched, coldinducible, and transcriptional co-activator of the nuclear receptor PPAR␥3 [1]
Examination of the intronic sequence revealed two consensus 3Ј splice acceptor sites in intron 6 as follows: an upstream site that leads to the 31-bp inclusion we identified, and a downstream site that leads to the known, reported Pgc-1␣ mRNA isoform
C, co-immunoprecipitation analysis of Alternative splicing expands the translational repertoire of numerous genes by producing multiple transcripts that encode protein-protein interaction of HA-tagged NT-PGC-1␣ with FLAG-tagged PPAR␣ in COS cells. 24 h after treatment with vehicle (DMSO) or 10 ␮M WY14693, whole cell extracts were immunoprecipitated with anti-FLAG, anti-HA, mouse preimmune IgG, or rabbit IgG

Summary

Introduction

PGC-1␣ was identified as a brown adipocyte-enriched, coldinducible, and transcriptional co-activator of the nuclear receptor PPAR␥3 [1]. Induction of the thermogenic gene program by NTPGC-1␣ was examined by treating differentiated brown adipocyte cell lines expressing NT-PGC-1␣-HA, PGC-1␣-V5, or empty vector with a ligand mixture containing 100 ␮M 8-CPTcAMP, 10 ␮M WY14693, or BRL49653, and 5 ␮M 9-cis-RA.

Results

Conclusion