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.

Localization of RNAPII and 3′ end formation factor CstF subunits onC. elegansgenes and operons

“…25 In mammals, mRNA 3 0 end formation consists of an endonucleolytic cleavage, followed by polyadenylation of the free 3 0 end. Transcription terminates often several kb downstream of the poly-A site [26][27][28][29][30] and is mechanistically linked to 3 0 end formation. 31,32 The "core" of the 3 0 end formation complex contains two highly conserved multisubunit protein complexes, the cleavage and polyadenylation specificity factor (CPSF) and the cleavage stimulatory factor (CstF).…”

Synergie entre les complexes de transcription et de maturation des ARN messagers

“…Dans un premier temps, le clivage d'un intron situé immédiatement en amont du signal de polyadénylation augmentera l'efficacité d'excision et d'ajout d'une queue de poly(A) en raison de l'apparition d'interactions entre le spliceosome et le complexe de polyadénylation. Celles-ci seront reflétées principalement par l'association entre les facteurs U1 snRNP et CPSF [16]. Certaines observations laissent égale-ment croire que cette même ribonucléoprotéine U1 aurait une affinité pour des séquences du site de polyadénylation.…”

“…Certaines observations laissent égale-ment croire que cette même ribonucléoprotéine U1 aurait une affinité pour des séquences du site de polyadénylation. De plus, notons que PAP peut contribuer à stimuler l'épissage de ce même intron en stabilisant le facteur accessoire (U2AF-65) associé au segment riche en pyrimidine du site d'épissage (Figure 4) [16].…”

RNA Polymerase II Phosphorylation and Gene Expression Regulation

“…Therefore, regulation of CTD phosphorylation as the polymerase transcribes facilitates coordination of the assembly of the 3′-end processing machinery with transcription [16]. Additionally, the polyadenylation signals are required for proper transcription termination in mammals and yeast [236][237]. In fact, Rtt103, which is a 3'-end mRNA processing factor, interacts with the CTD phosphorylated on Ser2 and recruits a 5'-3' RNA exonuclease, thereby promoting the release of RNAPII from the DNA [238][239].…”