Regulated specific protein binding to a conserved region of the 3'-untranslated region of thyrotropin beta-subunit mRNA.

Abstract

Thyroid hormone (T3) regulates the expression of rat TSH beta-subunit (TSH beta) mRNA, in part, at the posttranscriptional level, by reducing the half-life of TSH beta mRNA. The mechanism(s) mediating this alteration in mRNA stability are unknown, but previous work indicates that labile protein(s) are involved. The majority of cis-acting elements identified to date that have been implicated in the regulated destabilization of mRNAs have been located in the 3'-untranslated region (3'-UTR) of the mRNA. The 3'-UTR of rat, murine, and human TSH beta mRNA is highly conserved, and within this region is a 12-nucleotide consensus sequence, which is shared by the 3'-UTR of several other genes with unstable mRNAs. We reasoned that this homologous region could represent a binding motif for specific trans-acting RNA-binding protein(s), and that identification and characterization of such trans-acting factor(s) may provide critical insight into the mechanisms underlying T3-induced changes in TSH beta mRNA stability. Utilizing the RNA electrophoretic mobility shift assay and analysis of UV cross-linked RNA-protein complexes, a cytoplasmic trans-acting factor of approximately 80-85 kilodaltons was identified from rat pituitaries and several cell lines that binds in a sequence-specific manner to the 3'-UTR of rat TSH beta mRNA. Using competitive antisense oligonucleotides, the predominant binding site was mapped to the first 41 nucleotides of the 3'-UTR, which includes the consensus region. However, sequence upstream of the consensus was also shown to be important for binding. Using RNA electrophoretic mobility shift assay, two mRNAs containing sequence homology with the consensus region, c-erbA alpha-2 and a rat ferritin pseudogene, were shown to specifically compete with rat TSH beta mRNA for binding of this factor. Remarkably, the binding activity of this factor was regulated positively by T3 within 4 h, but only with rat pituitary extracts. These data suggest that in addition to binding rat TSH beta mRNA in a sequence-specific and T3-regulated manner, this novel trans-acting RNA-binding protein may also bind to other cytoplasmic mRNAs involved in diverse intracellular processes.

HADHB, HuR, and CP1 Bind to the Distal 3′-Untranslated Region of Human Renin mRNA and Differentially Modulate Renin Expression

“…Unlabeled transcripts were synthesized as described above, but with 2.5 mM rNTPs, and quantified by polyacrylamide gel electrophoresis. Binding reactions were performed as described (41)(42)(43) with 10 g of Calu-6 cytoplasmic extract, 200 ng of GST-HADHB, 200 ng of GST-HuR, or 200 ng of cleaved CP1 and 10 5 cpm RNA (ϳ10 -20 pg). Briefly, following incubation at 22°C for 30 min, 0.3 units of RNase T1 (Roche Diagnostics) was added for 10 min, followed by 50 g of heparin (Sigma) for 10 min.…”

Identification of a Novel AU-Rich Element in the 3′ Untranslated Region of Epidermal Growth Factor Receptor mRNA That Is the Target for Regulated RNA-Binding Proteins

“…RNA-protein binding reactions were carried out as described above, using 20 to 30 g of cytoplasmic extract and 1.5 ϫ 10 5 cpm of RNA (15 to 30 pg) of 32 P-riboprobe (35,55,58). Following the addition of heparin, samples were placed on ice in a microtiter tray and UV irradiated for 10 to 15 min, 1 cm below the Stratalinker UV light source (240-nm UV bulb; Stratagene, La Jolla, Calif.).…”

Thyroid Hormone Modulates the Interaction between Iron Regulatory Proteins and the Ferritin mRNA Iron-responsive Element

“…Preparation of RNA Transcripts-Linearized templates (SmaI-digested) were used with either T7 or SP6 RNA polymerase (Promega) in transcription reactions containing [␣-32 P]UTP (800 Ci/mmol; DuPont NEN), as described (30,33), to produce transcripts with a specific activity of approximately 2 ϫ 10 8 cpm/g of RNA. Full-length transcripts were isolated on 5% urea/acrylamide gels, eluted for 3 h at 25°C in 0.5 M ammonium acetate, 1 mM EDTA, and ethanol-precipitated to recover the RNA as described (30,33).…”