RNA 3' END SIGNALS OF THE S-POMBE URA4 GENE COMPRISE A SITE DETERMINING AND EFFICIENCY ELEMENT

We have defined sequences in the 3' non-coding region of the Schizosaccharomyces pombe ura4 gene that are required for efficient mRNA 3' end formation. Three separate sequence elements have been identified. Two of these are site determining elements which specify alternative sites of polyadenylation [the major poly(A) site and a minor downstream poly(A) site]. The third sequence, located downstream of both poly(A) sites, functions as an efficiency element that enhances utilization of either polyadenylation site. By employing sensitive RT-PCR analysis, we demonstrate that although low levels of transcripts are detected up to the efficiency element, none is detected beyond this point. The downstream site determining element and efficiency element have both been delineated to specific 16 nt sequences which we show are together sufficient for ura4 mRNA 3' end formation. We have further characterized the interaction between these two elements and show that the efficiency element behaves in a position-independent, orientation-dependent manner, but cannot form 3' ends independently of the site determining element. Surprisingly, we find that the efficiency element can be functionally replaced by a second copy of either site determining element. We present a model for the mechanism of RNA 3' end formation of the ural gene and note that this bipartite structure for a poly(A) signal in S.pombe may be related to the AAUAAA and downstream GU-rich sequences of poly(A) signals in mammalian genes.