Specific intron-dependent loading of DAZAP1 onto the cox6c transcript suppresses pre-mRNA splicing efficacy and induces cell growth retardation

Abstract

DAZAP1 is an evolutionarily conserved RNA-binding protein expressed in many tissues in mice and humans. DAZAP1-knockout mice carrying a partial loss-of-function (hypomorphic) allele exhibited severe deficiencies in spermatogenesis and cell growth, indicating that DAZAP1 plays a pivotal role in the development of germ and somatic cells. We have identified cox6c mRNA, which encodes a subunit of complex IV of the mitochondrial respiratory chain, as a target transcript regulated by DAZAP1. We found that DAZAP1 bound to cox6c mRNA derived from either the genomic DNA or a genome-type expression vector in cells, but not to cox6c mRNA derived from an intronless expression vector. Interestingly, the presence of the last intron was sufficient for DAZAP1 binding to the mRNA, suggesting specific intron dependent DAZAP1 loading onto cox6c mRNA. Overexpression of DAZAP1 resulted in the accumulation of cox6c pre-mRNA for all introns, implying that DAZAP1 reduces pre-mRNA splicing efficiency. In addition, the reduction of mature cox6c mRNA levels led to decreases in the COX6C protein levels. Both DAZAP1 knockdown and COX6C overexpression retarded cell growth. The lines of evidence presented here reveal that DAZAP1 is a negative regulator of pre-mRNA splicing and may control energy production in mitochondria by regulating COX6C expression. The DAZAP1 functions described in this study may also account for the phenotypes observed in the DAZAP1 hypomorphic mice.

Keywords: Cell growth; Mitochondria; Posttranscriptional regulation; RNA binding protein; mRNA splicing.