Transcriptional termination in the Balbiani ring 1 gene is closely coupled to 3′-end formation and excision of the 3′-terminal intron

Abstract

We have analyzed transcription termination, 3′-end formation, and excision of the 3′-terminal intron in vivo in the Balbiani ring 1 (BR1) gene and its pre-mRNA. We show that full-length RNA transcripts are evenly spaced on the gene from a position 300 bp upstream to a region 500–700 bp downstream of the polyadenylation sequence. Very few full-length transcripts and no short, cleaved, nascent transcripts could be observed downstream of this region. Pre-mRNA with 10–20 adenylate residues accumulates at the active gene and then rapidly leaves from the gene locus. Only polyadenylated pre-mRNAs could be detected in the nucleoplasm. Our results are consistent with the hypothesis that transcription termination occurs in a narrow region for the majority of transcripts, simultaneous with 3′-end formation. Excision of the 3′-terminal intron occurs before 3′-end formation in about 5% of the nascent transcripts. When transcription terminates, 3′ cleavage takes place and 10–20 adenylate residues are added, the 3′-terminal intron is excised from additionally about 75% of the pre-mRNA at the gene locus. Our data support a close temporal and spatial coupling of transcription termination and the cleavage and initial polyadenylation of 3′-end formation. Excision of the 3′-terminal intron is highly stimulated as the cleavage/polyadenylation complex assembles and 3′-end formation is initiated.