Differential Translatability in Vitro of Multiple Messenger RNAs Encoding the β-Subunit of Mouse Thyrotropin

The expression of TSH subunit genes (TSH␣ and -␤) in pituitary thyrotropes is primarily regulated via circulating thyroid hormone levels (T 3 ) and the hypothalamic TRH. Hypophyseal pars tuberalis (PT)-specific cells also express both hormonal subunits of TSH, but do not resemble thyrotropes of the pars distalis (PD) with respect to their distinct morphology, secretion, and direct modulation of TSH expression by photoperiodic inputs and melatonin. To investigate whether this distinct regulation of TSH is related to a different molecular structure or different signaling cascades, we analyzed PT-specific TSH and its transcriptional regulation in ovine PT-specific cells. After construction of PT-and PD-specific complementary DNA (cDNA) libraries, the cloning and sequencing of several TSH␣ and -␤ subunit clones revealed identical sizes and sequences for the translated and untranslated regions in both hypophyseal compartments. Transcription start site analysis also displayed three identical start sites for the transcription of TSH␤ in PT and PD. After cloning of the ovine TRH receptor cDNA and a partial T 3 receptor cDNA, in situ hybridization, Northern blot analysis, and PCR experiments showed that TRH and T 3 receptors are not expressed in specific cells of the PT. The transcription factor Pit-1 that is involved in TSH expression of thyrotropes could only be detected in the PD.In additional experiments rats were treated with T 4 or TRH, and subsequent in situ hybridization studies showed that TSH␤ messenger RNA (mRNA) formation was not altered in the PT. In the PD, however, TSH␤ mRNA was significantly reduced in the T 4 -treated group, but was enhanced in the TRH-treated group. We conclude that PT-specific cells of the pituitary are characterized by the transcription of TSH subunits that are identical to TSH expressed in thyrotropes of the PD. The absence of TRH, T 3 receptor mRNA, and Pit-1, respectively, as well as the different reactions compared to PD thyrotropes in in vivo experiments lead to the conclusion that the expression of TSH in PT-specific cells of the pituitary is not regulated via the classical thyrotrope receptors and their intracellular pathways, but through a novel, photoperiod-dependent mechanism. (Endocrinology 138: 1019 -1028, 1997) T HE HYPOPHYSEAL glycoprotein hormone, TSH, consists of two noncovalently bound subunits, ␣ and ␤, which are encoded by separate genes on different chromosomes (1). The ␣-subunit is common to the other members of the glycoprotein hormone family, pituitary LH and FSH and placental CG, whereas the unique ␤-subunit determines the specificity of the hormonal dimer (2). The expression and secretion of TSH␣ and -␤ subunits in pituitary thyrotropes are closely regulated by hypothalamic factors released from the median eminence into the hypophyseal blood flow, in particular TRH, and a feedback control by thyroid hormones (e.g. T 3 ) (3, 4). T 3 acts as a suppressor of TSH transcription after attachment to a nuclear T 3 receptor (TR␤) that binds as a T 3 -receptor c...

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“…differential translatability in vitro (22,44). In the sheep, these splicing events at the 3Ј-end could not be detected.…”

Section: Discussionmentioning

confidence: 86%

Thyrotropin Expression in Hypophyseal Pars Tuberalis-Specific Cells is 3,5,3′-Triiodothyronine, Thyrotropin-Releasing Hormone, and Pit-1 Independent*

et al. 1997

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“…The expression of porphobilinogen deaminase, erythroid membrane protein 4.1, and Gsa protein requires alternative splicing as well as promoter switching; they are included because the net effect is activation of an internal AUG initiator codon by making it the first AUG in the mRNA . Not listed are some interesting genes that produce small amounts of 5' truncated transcripts in extraneous tissues (63,160,234,405,410) . Even when such mRNAs can be shown to direct synthesis of a polypeptide fragment (63), which is almost inevitable if the transcript enters the cytoplasm, the phenomenon might reflect inadvertent expression; what needs to be established is that the NH 2 terminally truncated polypeptide serves a unique function in the ectopic tissue .…”

Section: Bifunctional Genes and Bifunctional Mrnasmentioning

confidence: 99%

An analysis of vertebrate mRNA sequences: intimations of translational control.

Kozak¹

1991

The Journal of Cell Biology

1,579

1,042

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Abstract. Five structural features in mRNAs have been found to contribute to the fidelity and efficiency of initiation by eukaryotic ribosomes. Scrutiny of vertebrate cDNA sequences in light of these criteria reveals a set of transcripts-encoding oncoproteins, growth factors, transcription factors, and other regulatory proteins-that seem designed to be translated poorly. Thus, throttling at the level of translation may be a critical component of gene regulation in vertebrates. An alternative interpretation is that some (perhaps many) cDNAs with encumbered 5' noncoding sequences represent mRNA precursors, which would imply extensive regulation at a posttranscriptional step that precedes translation .NITIATIO N oftranslation in multicellular eukaryotes is influenced by five aspects ofmRNA structure : (a) the m7G cap (355); (b) theprimary sequence or context surround ing the AUG codon (187,190,194); (c) the position of the AUG codon, i.e., whether it is the first AUG in the message (186); (d) leader length (198,199) ; and (e) secondary structure both upstream (188, 195) and downstream (196) from the AUG codon . Elsewhere (200) I have reviewed the evidence for these five features and explained how they work together to determine the fidelity and efficiency ofinitiation. A scanning mechanism for initiation can explain many of the effects of cap, context, position, etc . The scanning model (193) in its simplest form postulates that a 40S ribosomal subunit, carrying Met-tRNA;^" and an imperfectly defined set of initiation factors (302), enters at the 5' end of the mRNA and migrates linearly until it reaches the first AUG codon, whereupon a 60S subunit joins and the first peptide bond is formed . Evidence in support of the model has been adduced previously (62,193,197). More recent evidence for scanning includes the apparent queuing of 40S ribosomal subunits on long leader sequences (199) and the stalling of 40S subunits on the 5' side of a stable hairpin structure introduced between the cap and the AUG codon (195). The possibility of initiation by a mechanism other than scanning has been proposed (158) and is evaluated elsewhere (197) .The trick to identifying elements in 5' noncoding sequences that can modulate translation was to isolate each feature (200), an approach made possible by the techniques ofgenetic engineering . For example, by devising a transcript in which the first AUG codon was in an unfavorable context and hence "leaky," we were able to show that downstream

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“…(Januszeski andGurr, 1991, Horiuchi et al 1990;Garrett et al, 1989). Moreover, both the leader and trailer [3' untranslated region (3' UTR)] can contain sequences that keep the mRNA temporarily out of translation altogether, for example during development.…”

mentioning

confidence: 99%

Differential polysomal localization of human insulin‐like‐growth‐factor‐2 mRNAs in cell lines and foetal liver

Moor

Jansen

Sussenbach

et al. 1994

European Journal of Biochemistry

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Examination of the association of insulin-like-growth-factor-2 mRNAs with polyribosomes in five cell lines revealed that greater than SO% of the total mRNA population was present in the untranslated free mRNP fraction for each cell line. Of the different subtypes of insulin-like-growthfactor-2 messengers, the least abundant mRNAs, starting with exon 4 (leader 2, 5.0 kb) and exon 6 (leader 4, 4.8 kb), were found in the polysomes only, while the most abundant transcript, starting with exon 5 (leader 3, 6.0 kb and 2.1 kb) was found predominantly in the untranslated fractions. 20-30% of leader 3 mRNAs, however, were in the larger polysomes (four or more ribosomes), indicating that a subpopulation of this mRNA can be translated efficiently. The peak fraction for the leader 4 insulin-like-growth-factor-2 mRNA (4.8 kb) in the polysomes was migrating faster in the sucrose gradients than the peak fractions of leader 2 and 3 mRNAs (5.0 kb and 6.0 kb), implying that more ribosomes were associated with this type of mRNA. In foetal liver, the situation was similar, though in this case the leader 2 mRNA was most heavily loaded with polysomes. Treatment of cells with low concentrations of cycloheximide caused the polysomal RNAs to shift to even larger polysomes while the untranslated fraction of the leader 3 mRNAs stayed in the untranslated fractions. These results indicate that, both in established cell lines and in foetal liver, insulin-likegrowth-factor-2 translation is influenced both by mRNP sequestration and differential translation initiation efficiency of the insulin-like-growth-factor-2 mRNAs.The human gene for insulin-like-growth-factor-2 can direct the synthesis of several different mRNAs that encode the same preproprotein (Sussenbach et al., 1992;Rotwein, 1991). Four promoters (Pl-P4) give rise to mRNAs with different 5' untranslated regions (leaders), whereas an alternative polyadenylation site produces a minor species that is 4 kb smaller than the major polyadenylated product (Fig.

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Differential Translatability in Vitro of Multiple Messenger RNAs Encoding the β-Subunit of Mouse Thyrotropin

Cited by 8 publications

References 39 publications

Thyrotropin Expression in Hypophyseal Pars Tuberalis-Specific Cells is 3,5,3′-Triiodothyronine, Thyrotropin-Releasing Hormone, and Pit-1 Independent*

Thyrotropin Expression in Hypophyseal Pars Tuberalis-Specific Cells is 3,5,3′-Triiodothyronine, Thyrotropin-Releasing Hormone, and Pit-1 Independent*

An analysis of vertebrate mRNA sequences: intimations of translational control.

Differential polysomal localization of human insulin‐like‐growth‐factor‐2 mRNAs in cell lines and foetal liver

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