Abstract
Fibrinogen gamma-chains differ in amino acid sequence at the carboxyterminus due to alternative 3′ RNA processing. Previous studies reported differences between humans and rats in the mechanism of gamma- chain RNA processing and that it was a nonregulated event. To test the hypothesis that rat gamma-chain RNA processing involves both alternative splicing and poly(A) site selection and that it is regulated in a tissue-specific manner, we determined the tissue distribution of gamma-chain mRNA expression and the pattern of gamma- chain pre-mRNA processing. The results of in situ hybridization demonstrated that gamma A and gamma B transcripts were localized to and codistributed in liver hepatocytes, indicating that no subset of cells process gamma B mRNA. The ubiquitous expression of the fibrinogen gamma- chain promoter was demonstrated in marrow, lung, brain, and liver by RNase protection using a 5′-specific gamma-chain probe. RNase protection studies to map 3′ RNA processing sites suggested that, in addition to the distal poly(A) signal previously identified, two alternative poly(A) signals within the last intron (ATTAAA and AATAAA) were used only in liver to produce gamma B transcripts. Approximately equal usage of the three poly(A) signals (27%, 37%, and 36%, respectively) to form the 3′ end of mature gamma B transcripts suggested that poly(A) site selection is random. These results indicate that splicing of the last intron to produce gamma A mRNA is the ubiquitous and constitutive pattern of gamma-chain RNA processing, while retention of the last intron to produce gamma B mRNAs is the tissue-specific and regulated pattern of gamma-chain RNA processing. The pattern of rat gamma-chain RNA processing is similar to human, implying that the mechanism is conserved. These data support a mechanism of tissue-specific splice site selection predominating over poly(A) site selection in gamma-chain pre-mRNA processing. The expression of both fibrinogen gamma-chain transcripts in liver, rather than mutually exclusive expression in liver and other tissues, provides a new model for studying tissue-specific alternative 3′ end formation regulatory mechanisms.
