The increased level of b1,4-galactosyltransferase required for lactose biosynthesis is achieved in part by translational control (5*-untranslated regionyin vitro translationyin vivo translationymammary glandyRNA secondary structure)

b1,4-Galactosyltransferase (b4GalT-I) participates in both glycoconjugate biosynthesis (ubiquitous activity) and lactose biosynthesis (mammary gland-specific activity). In somatic tissues, transcription of the mammalian b4GalT-I gene results in a 4.1-kb mRNA and a 3.9-kb mRNA as a consequence of initiation at two start sites separated by '200 bp. In the mammary gland, coincident with the increased b4GalT-I enzyme level ('50-fold) required for lactose biosynthesis, there is a switch from the 4.1-kb start site to the preferential use of the 3.9-kb start site, which is governed by a stronger tissue-restricted promoter. The use of the 3.9-kb start site results in a b4GalT-I transcript in which the 5*untranslated region (UTR) has been truncated from '175 nt to '28 nt. The 5*-UTR of the 4.1-kb transcript [UTR(4.1)] is predicted to contain extensive secondary structure, a feature previously shown to reduce translational efficiency of an mRNA. In contrast, the 5*-UTR of the 3.9-kb mRNA [UTR(3.9)] lacks extensive secondary structure; thus, this transcript is predicted to be more efficiently translated relative to the 4.1-kb mRNA. To test this prediction, constructs were assembled in which the respective 5*-UTRs were fused to the luciferase-coding sequence and enzyme levels were determined after translation in vitro and in vivo. The luciferase mRNA containing the truncated UTR(3.9) was translated more efficiently both in vitro ('14-fold) and in vivo (3to 5-fold) relative to the luciferase mRNA containing the UTR(4.1). Consequently, in addition to control at the transcriptional level, b4GalT-I enzyme levels are further augmented in the lactating mammary gland as a result of translational control. b1,4-Galactosyltransferase (b4GalT-I) is a constitutively expressed, trans-Golgi resident, type II membrane-bound glycoprotein that is widely distributed in vertebrates. b4GalT-I catalyzes the transfer of galactose to N-acetylglucosamine residues, forming the b4-N-acetyllactosamine (Galb4GlcNAc) or polyb4-N-acetyllactosamine structures found in glycoconjugates (1). In mammals, b4GalT-I has been recruited for a second biosynthetic function, the tissue-specific production of lactose (Galb4Glc), which takes place exclusively in the lactating mammary gland. The synthesis of lactose is carried out by a protein heterodimer assembled from b4GalT-I and a-lactalbumin, a noncatalytic mammalian protein expressed de novo exclusively in the mammary gland during lactation (2–4). The net result of the association of a-lactalbumin with b4GalT-I is to lower the Km for glucose about three orders of magnitude, consequently making it an effective acceptor substrate at physiological concentrations. Beginning in late pregnancy, the b4GalT-I enzyme level in the mammary gland has been estimated to increase '50-fold in preparation for lactose biosynthesis (5, 6). With the recruitment of b4GalT-I for lactose biosynthesis, the regulatory problem arose as to how to specifically increase the enzyme level only in the lactating mammary gland while maintaining the comparatively low level of constitutively expressed enzyme required for glycoconjugate biosynthesis in all other somatic tissues. To address this question, we have carried out a detailed analysis of the structure and transcriptional regulation of the murine b4GalT-I gene. The main observations can be summarized as follows. (i) The murine b4GalT-I gene specifies two transcripts of '4.1 and '3.9 kb in somatic cells as a consequence of initiation at two different start sites separated by '200 bp (ref. 7; Fig. 1A). The identical structural features are also found in the bovine (8) and human (9, 10) b4GalT-I genes, which suggests that they may be a distinguishing characteristic of all mammalian b4GalT-I orthologues. (ii) Structurally, the 4.1and 3.9-kb transcripts are essentially identical, with the exception of their respective 59-untranslated region (UTR). The length of the 59-UTR of the 4.1-kb transcript [UTR(4.1)] is '175 nt; in contrast, the 59-UTR of the 3.9-kb transcript [UTR(3.9)] is only '28 nt long (Fig. 1B). (iii) In murine somatic tissues, the 4.1-kb start site is used predominantly. Expression from this start site is controlled by a relatively weak promoter containing multiple Sp1 sites, consistent with the role of the 4.1-kb mRNA as the ubiquitous transcript involved in glycoconjugate biosynthesis (11, 12). (iv) In the midto late-pregnant and lactating mammary gland, the 3.9-kb start site is used preferentially. Expression from this start site is controlled by a stronger promoter that is in part regulated by lactating mammary gland-restricted transcription factors. The net result of this switch to the 3.9-kb start site in the lactating mammary gland is a '10-fold increase in the steady-state levels of b4GalT-I mRNA (11, 12). In the context of the required '50-fold increase in b4GalT-I enzymatic activity in the lactating mammary gland, two questions arose. First, could the '10-fold increase in mRNA levels by itself account for the '50-fold increase in enzyme levels observed? Second, why has nature gone to the trouble to generate a second b4GalT-I transcript that is distinguished by the absence of '180 nt, primarily from the 59-UTR? A comparison of the UTR(4.1) and UTR(3.9) provided the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1998 by The National Academy of Sciences 0027-8424y98y9514805-6$2.00y0 PNAS is available online at www.pnas.org. Abbreviations: b4GalT-I, b1,4-galactosyltransferase (EC 2.4.1.38); murine b4GalT-I refers to the a-lactalbumin-responsive UDP galactose, N-acetyl-b-D-glucosaminylglycopeptide b1,4-galactosyltransferase that has been mapped to the centromeric region of mouse chromosome 4; GlcNAc, N-acetylglucosamine; UTR, untranslated region; UTR(4.1), 59-UTR of the 4.1-kb b4GalT-I mRNA; UTR(3.9), 59-UTR of the 3.9-kb b4GalT-I mRNA; LUC, luciferase; eIF, eukaryotic initiation factor; RT-PCR, reverse transcription–PCR. ‡To whom reprint requests should be addressed at: The Johns Hopkins University School of Medicine, Oncology Center, Room 1-127, 600 North Wolfe Street, Baltimore, MD 21287-8937. e-mail: nshaper@ jhmi.edu.