M BoC | ARTICLE Mex3c regulates insulin-like growth factor 1 (IGF1) expression and promotes postnatal growth Yan Jiao, Colin E. Bishop, and Baisong Lu Institute for Regenerative Medicine, Wake Forest University Health Sciences, Winston-Salem, NC 27157 ABSTRACT Insulin-like growth factor 1 (IGF1) mediates the growth-promoting activities of Monitoring Editor growth hormone. How Igf1 expression is regulated posttranscriptionally is unclear. Caenorhab- A. Gregory Matera ditis elegans muscle excess 3 (MEX-3) is involved in cell fate specification during early embry- University of North Carolina onic development through regulating mRNAs involved in specifying cell fate. The function of Received: Nov 29, 2011 its mammalian homologue, MEX3C, is unknown. Here we show that MEX3C deficiency in Revised: Feb 14, 2012 Mex3c homozygous mutant mice causes postnatal growth retardation and background-de- Accepted: Feb 17, 2012 pendent perinatal lethality. Hypertrophy of chondrocytes in growth plates is significantly im- paired. Circulating and bone local production of IGF1 are both decreased in mutant mice. Mex3c mRNA is strongly expressed in the testis and the brain, and highly expressed in resting and proliferating chondrocytes of the growth plates. MEX3C is able to enrich multiple mRNA species from tissue lysates, including Igf1. Igf1 expression in bone is decreased at the protein level but not at the mRNA level, indicating translational/posttranslational regulation. We propose that MEX3C protein plays an important role in enhancing the translation of Igf1 mRNA, which explains the perinatal lethality and growth retardation observed in MEX3C- deficient mice. INTRODUCTION Insulin-like growth factor 1 (IGF1) is a prime mediator of the growth- growth retardation, although the manipulation does not affect circu- promoting effects of growth hormone (GH; Rodriguez et al., 2007), lating IGF1 levels (Govoni et al., 2007a, 2007b). which promotes postnatal growth through stimulating endocrinal Humans and mice have multiple forms of Igf1 transcripts because and local production of IGF1 (Ohlsson et al., 2009). The liver pro- of the usage of different polyadenylation sites; some have a short 3′ duces 75% of circulating IGF1 in the bloodstream. Eliminating he- untranslated region (3’ UTR) of 150∼400 nucleotides (nt) (e.g., mouse patic production of IGF1 in mice does not affect postnatal growth, NM_184052 and human NM_001111285), but some have a long 3′ indicating that 25% of normal circulating IGF1 is sufficient for nor- UTR of more than 6000 nt (e.g., mouse NM_010512 and human mal postnatal growth (Sjogren et al., 1999; Yakar et al., 1999; Stra- NM_001111283). Polysome association analysis found that, of the tikopoulos et al., 2008). Local production of IGF1 in developing liver Igf1 mRNAs with different lengths of 3′ UTR, only the short spe- bone is also essential for normal postnatal growth. Chondrocyte- or cies were found on polysomes, suggesting that some aspect of the osteoblast-specific knockout of Igf1 in mouse causes postnatal long 3′ UTR may prevent translation (Foyt et al., 1991). Whereas the liver expresses more Igf1 transcripts with a short 3′ UTR (Bell et al., 1986), bone-forming cells predominantly express Igf1 transcripts This article was published online ahead of print in MBoC in Press (http://www with a long 3′ UTR (Delany and Canalis, 1995). Recently it was found .molbiolcell.org/cgi/doi/10.1091/mbc.E11-11-0960) on February 22, 2012. that nocturnin regulates Igf1 mRNA stability through binding to its 3′ Address correspondence to: Baisong Lu ([email protected]). UTR (Kawai et al., 2010). It remains unclear how the translation of Igf1 Abbreviations used: AA, amino acids; β-gal, β-galactosidase; ELISA, enzyme-linked mRNA, especially in transcripts with a long 3′ UTR, is regulated. immu nosorbent assay; GH, growth hormone; IGF1, Insulin-like growth factor 1; hnRNP, heterogeneous nuclear ribonucleoprotein; KH domain, hnRNP K homology Caenorhabditis elegans muscle excess 3 (MEX-3) is a heteroge- domain; MEX-3, muscle excess 3; Mex3c, mex3 homologue C (Caenorhabditis neous nuclear ribonucleoprotein (hnRNP) K homology (KH) do- elegans); MRE, MEX-3 recognition element; nt, nucleotides; qRT-PCR, quantitative RT-PCR; 3’ UTR, 3’ untranslated region; ZNF domain, zinc finger domain. main-containing RNA binding protein. It is involved in cell fate © 2012 Jiao et al. This article is distributed by The American Society for Cell Biol- specification in the early embryonic stage ofC. elegans and in the ogy under license from the author(s). Two months after publication it is available maintenance of the totipotency of the germ line in adult worms to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). (Draper et al., 1996; Hunter and Kenyon, 1996; Ciosk et al., 2006). “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of MEX-3 is thought to prevent the translation of pal-1 and the Cell®” are registered trademarks of The American Society of Cell Biology. nanos mRNA in the anterior blastomeres or germ cells 1404 | Y. Jiao et al. Molecular Biology of the Cell (Draper et al., 1996; Hunter and Kenyon, 1996; Jadhav et al., RESULTS 2008). Human and mouse genomes encode four MEX-3 homo- Background-dependent postnatal lethality of Mex3c logues, named MEX3A, MEX3B, MEX3C, and MEX3D (Buchet- mutant mice Poyau et al., 2007). They all have two KH RNA-binding domains in Mex3c mutant mice were generated from a gene trap ES cell line, the N termini which are believed to bind RNA molecules, and a where the Mex3c gene was mutated by gene trapping. Based on zinc finger (ZNF) domain at the C termini which is believed to me- the National Center for Biotechnology Information (NCBI) EST se- diate protein–protein interactions. MEX3D, once described as quence CJ072395.1 and the mRNA sequence BC125427, the mouse TINO, is a BCL2 mRNA AU-rich element-binding protein and neg- Mex3c gene contains three exons, with CJ072395.1 spanning exons atively regulates the stability of BCL2 mRNA (Donnini et al., 1 and 2, and BC125427 spanning exons 2 and 3. This Mex3c mRNA 2004). transcript is predicted to encode a protein of 464 AA, containing MEX3C (once called RKHD2) is highly conserved among mam- two hnRNP KH RNA-binding domains (Grishin, 2001) and one ZNF malian species. Mouse MEX3C (464 amino acids [AA] according to domain (Figure 1A). The NCBI mouse Mex3c reference sequence DDBJ Accession Number BR000945) is 99% identical with human, NM_001039214.4 regards intron 1 in Figure 1A as exon sequences, chimpanzee, and bovine MEX3Cs. A combination of sibling-pair resulting in a protein with an extra 189 AA at the N terminus. In our linkage analysis and case–control association studies suggests a gene trap mouse, the trapping vector pGT1xrT2 was integrated into contribution of MEX3C to the genetic susceptibility of hypertension, the second intron of Mex3c (Figure 1B). Reverse transcription PCR although a possible mechanism is unknown (Guzman et al., 2006). (RT-PCR) and DNA sequencing analyses revealed that most tran- So far, no mRNA targets have been reported for MEX3C, and little scripts from the trapped allele (written as “tr” hereafter) contained is known about its role in RNA metabolism. the LacZ cDNA from the trapping vector, but lacked the third exon In our effort to generate rodent mutants for reproductive biol- of Mex3c, producing a peptide containing the N-terminal 56 AA of ogy using a Sleeping Beauty transposon-mediated mutagenesis the 464 AA MEX3C and the full-length β-galactosidase (β-gal) pep- strategy (Lu et al., 2007), we encountered a mutant line with post- tide. Because this fusion peptide lacks the hnRNP KH RNA-binding natal growth retardation. DNA analysis found three transposon and the ZNF domains, it is expected to be nonfunctional. The β-gal insertions: two in the regions without genes and one in the intron moiety in the fusion protein permits tracing of the expression of of Mex3c. To eliminate the possible influence of transposons out- Mex3c mRNA through detecting β-gal activity. side the Mex3c gene, we generated a new Mex3c mutant line Transcripts from the trapped allele, however, also included a low from a gene trap embryonic stem cell line (Skarnes et al., 2004). percentage of intact full-length Mex3c mRNA due to alternative splic- Here we show that Mex3c plays an important role in promoting ing. Quantitative RT-PCR (qRT-PCR) revealed that authentic Mex3c postnatal growth, possibly through translational regulation of Igf1 mRNA in +/tr and tr/tr mice comprised ∼55% and 2.2% of that of +/+ mRNA. mice, respectively (Figure 1C). Thus tr/tr mice were not Mex3c null but expressed <5% of normal levels of Mex3c mRNA. Because neither customized nor commercially available anti-MEX3C antibod- ies were specific enough to detect endoge- nous MEX3C protein, the expression level of MEX3C protein in mutant mice is unclear. Trapping both alleles of the Mex3c gene did not affect embryonic development, be- cause the tr/tr pups from +/tr × +/tr matings were born normal with Mendelian ratios. Postnatal survival, however, was affected to variant degrees depending on background. Under a C57BL/6 background, more than 85% of mutants died within several hours after birth. Only seven homozygous mutant mice from more than 160 pups of heterozy- gous parents survived to weaning time. Mu- FIGURE 1: Mutation of Mex3c in mouse by gene trapping. (A) Structure of normal Mex3c gene tant pups most likely died from poor breath- and its gene products. The black line indicates the introns of mouse Mex3c gene, and the solid ing, and their bodies were cyanotic. On boxes indicate the three Mex3c exons. Exons 2 and 3 contain coding regions. The N-terminal histological analysis, alveolar spaces in lungs KH RNA-binding domains (KH) and the C-terminal ZNF domain (zinc F) are indicated.