FIG. 7.

The CBC inhibits termination at an internal poly(A) addition site in the RNA14 ORF. (A) Schematic of the RNA14 gene and its three transcripts (A, B, and C) generated by the major poly(A) sites labeled with asterisks (43). The position of the DNA fragment used to probe all three transcripts by Northern blot analysis is indicated by the black bar. (B) Strains BY4741 (WT), 2074 (cbp20Δ), 6566 (cbp80Δ), and 4268 (npl3Δ), transformed with YCplac33, were grown in synthetic complete medium lacking uracil with 2% glucose; BY4741 (WT) was treated separately with 6AU (at 50 μg/ml) for 4 h; and poly(A)⁺ mRNAs were isolated from 150 μg total RNA and subjected to Northern blot analysis, with probing for RNA14 and PYK1 (as loading control). The hybridization signals for the RNA14 transcripts were quantified by phosphorimaging analysis, and the ratios of the B and A transcripts relative to the C transcript were calculated and normalized to the corresponding ratios measured in WT cells. The middle panel shows a longer exposure of the upper panel. (C) A hypothetical model depicting cooperation between the CBC and Npl3p in preventing transcription termination at weak termination sites. (Top) CBC is recruited cotranscriptionally to the capped 5′ end of the nascent transcript and interacts with Pol II during elongation, forming a pseudocircular mRNA. CBC also interacts with Npl3p and stabilizes Npl3p binding to weak termination sites in the nascent mRNA (gray). Npl3p competes with CF I for recruitment to weak terminators, preventing transcript cleavage. The circularization of the mRNA by CBC might also impede cleavage by CF I. (Bottom) At the strong termination site, CF I displaces Npl3p and cleaves the mRNA to prepare the 3′ end for polyadenylation. CBC dissociates from Pol II by an unknown mechanism.