Human SHBG mRNA Translation Is Modulated by Alternative 5′-Non-Coding Exons 1A and 1B

Tomàs Pinós,Francina Munell,Albert Santamaria-Martínez,Antoni Hurtado,Jaume Reventós,Inés De Torres,Anna Barbosa-Desongles,Alfons Navarro

doi:10.1371/journal.pone.0013844

Abstract

BackgroundThe human sex hormone-binding globulin (SHBG) gene comprises at least 6 different transcription units (TU-1, -1A, -1B, -1C, -1D and -1E), and is regulated by no less than 6 different promoters. The best characterized are TU-1 and TU-1A: TU-1 is responsible for producing plasma SHBG, while TU-1A is transcribed and translated in the testis. Transcription of the recently described TU-1B, -1C, and -1D has been demonstrated in human prostate tissue and prostate cancer cell lines, as well as in other human cell lines such as HeLa, HepG2, HeK 293, CW 9019 and imr 32. However, there are no reported data demonstrating their translation. In the present study, we aimed to determine whether TU-1A and TU-1B are indeed translated in the human prostate and whether 5′ UTR exons 1A and 1B differently regulate SHBG translation.ResultsCis-regulatory elements that could potentially regulate translation were identified within the 5′UTRs of SHBG TU-1A and TU–1B. Although full-length SHBG TU-1A and TU-1B mRNAs were present in prostate cancer cell lines, the endogenous SHBG protein was not detected by western blot in any of them. LNCaP prostate cancer cells transfected with several SHBG constructs containing exons 2 to 8 but lacking the 5′UTR sequence did show SHBG translation, whereas inclusion of the 5′UTR sequences of either exon 1A or 1B caused a dramatic decrease in SHBG protein levels. The molecular weight of SHBG did not vary between cells transfected with constructs with or without the 5′UTR sequence, thus confirming that the first in-frame ATG of exon 2 is the translation start site of TU-1A and TU-1B.ConclusionsThe use of alternative SHBG first exons 1A and 1B differentially inhibits translation from the ATG situated in exon 2, which codes for methionine 30 of transcripts that begin with the exon 1 sequence.

Highlights

Sex hormone-binding globulin (SHBG) is a dimeric glycoprotein that transports sex steroids in the blood and regulates their access to target tissues [1]
The use of alternative sex hormone-binding globulin (SHBG) first exons 1A and 1B differentially inhibits translation from the ATG situated in exon 2, which codes for methionine 30 of transcripts that begin with the exon 1 sequence
The uAUG of exon 1A produces an uORF of only 2 codons, whereas the uAUG found in exon 2 of both TU-1A and TU-1B generates an uORF of 11 codons, and the corresponding stop codon overlaps with the predicted translation start site of the SHBG ORF (Figure 1B)

Summary

Introduction

Sex hormone-binding globulin (SHBG) is a dimeric glycoprotein that transports sex steroids in the blood and regulates their access to target tissues [1]. The human SHBG gene is located in the short arm of chromosome 17 (17p13.1), contains at least 6 different transcription units, which are constituted of a common region that spans exons 2 to 8, and 6 alternative first exons [2]. These exons are named 1, 1A, 1B, 1C, 1D and 1E, following their 59 to 39 orientation on the positive strand of chromosome 17, and are all spliced to exon 2 using the same 39 splice site [2]. We aimed to determine whether TU-1A and TU-1B are translated in the human prostate and whether 59 UTR exons 1A and 1B differently regulate SHBG translation

Objectives

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Conclusion