3' RNA PROCESSING EFFICIENCY PLAYS A PRIMARY ROLE IN GENERATING TERMINATION-COMPETENT RNA POLYMERASE-II ELONGATION COMPLEXES

In several mammalian transcription units, a transcription termination mechanism in which efficient termination is dependent on the presence of an intact 3' RNA processing site has been identified. The mouse bet a(maj)-globin transcription unit is one such example, in which an inta ct poly(A) site is required for efficient transcription termination. I t is now evident that 3' mRNA processing sites are not always processe d with the same efficiency. In this study, we characterized several pr e-mRNAs as substrates for the 3' mRNA processing reaction of cleavage and polyadenylation. We then determined whether poly(A) sites which va ry in processing efficiency support a poly(A) site-dependent terminati on event. The level of processing efficiency was determined in vitro b y assays measuring the efficiency of the pre-mRNA cleavage event and i n vivo by the level of poly(A) site-dependent mRNA and gene product ex pression generated in transient transfection assays. The beta(maj) glo bin pre-mRNA is very efficiently processed. This efficient processing correlates with its function in termination assays using recombinant a denovirus termination vectors in nuclear run-on assays. When the beta( maj) globin poly(A) site was replaced by the L1 poly(A) site of the ad enovirus major late transcription unit (Ad-ml), which is a poor proces sing substrate, termination efficiency decreased dramatically. When th e beta(maj) globin poly(A) site was replaced by the Ad-ml L3 poly(A) s ite, which is 10- to 20-fold more efficiently processed than the Ad-ml L1 poly(A) site, termination efficiency remained high. Termination is therefore dependent on the yield of the processing event. We then tes ted chimeric poly(A) sites containing the L3 core AAUAAA but varied do wnstream GU-rich elements. The change in downstream GU-rich elements a ffected processing efficiency in a manner which correlated with termin ation efficiency. These experiments provide evidence that the efficien cy of 3' processing complex formation is directly correlated to the ef ficiency of RNA polymerase II termination at the 3' end of a mammalian transcription unit.