Processing of the Primary Transcript for the Rat Growth Hormone Gene In Vivo

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Abstract
Procesing of the rat growth hormone (rGH) gene primary transcript and the effects of thyroid and glucocoreticoid hormoens on rGH pre-mRNA levels have beens tudied using subcloned radiolabeled DNA fragments from each of the four introns of this gene as probes. Blot-hybridization analysis of poly(A)+RNA from GC cells, GH3 cells, and normal pituitary gland indicates that processing of intron sequences from the precursor transcript takes place in a qualitatively similar fashion in each of these cell types. The data indicate that, in general, those introns closest to the termini of the primary transcript are removed first followed by removal of the internal introns. The suggested order of removal is IA, ID, IC, and IB. This process is unaffected qualitatively by thyroid or glucocorticoid hormones, both of which increase the rate of transcription of the gene. In addition to the primary transcript and the partially processed intermediate transcripts, GC and GH3 cells were found to contain a heterogenous group of intron-containing polyadenylated rGH gene transcripts which cannot be accounted for by any combination of intron deletions. These transcripts could arise either from internal start sites in the gene, premature termination of transcription, or inefficient processing of rGH mRNA precursors in the transformed cells. Thyroid hormone rapidly increases the levels of intron C-containing transcripts with kinetics that parallel the binding of thyroid hromone receptor to nuclei, but does not alter the ratio of primary to partially processed transcripts. These data sugest that most of the stimulatory activity of this hormone is due to effects on rGH gene transcription and not on pre-mRNA processing. In response to thyroid hormone, the levels of intron-containing rGH transcription products peak at .simeq. 8 hr and dro to a steady-state level less than 25% that of the peak levels by 24 hr. This decrease is paralleled by a smaller (approximately 50%) decrease in thyroid hormone receptor levels. This indicates that the attentuation of transcription is not directly proportional to the decrease in thyroid hormone receptor occupancy and could reflect either heterogeneity in the receptor population or the existence of an additional post-receptor effect.