STIMULATION OF RNA 3' PROCESSING BY FLANKING DNA IN XENOPUS OOCYTES

We have previously shown that the second poly(A) signal of the Xenopus laevis a-tubulin gene X alpha T14, which contains the rare hexanucleo tide CAUAAA, requires a surprisingly large amount of 3' flanking DNA t o be used efficiently in Xenopus oocytes. To investigate the nature of the interaction between the X alpha T14 3' flank and upstream 3' proc essing sites, we have developed a modified oocyte assay based on the s timulation of processing at a single poly(A) signal. We mutated both t he hexanucleotide and GU/U-rich components of a strong synthetic poly( A) signal (SPA) in order to weaken it severely. We found that efficien t use of the mutant signal could be fully restored by the addition of 1.2 kb of X alpha T14 3' flank, but only in its natural orientation. F unctional dissection of the X alpha T14 3' flank defined two separate regions that were each capable of partially restoring processing effic iency, presumably because they contain multiple, relatively weak proce ssing enhancers. We discuss how the stimulation of 3' processing by fl anking regions in oocytes could be explained by mechanisms that operat e on the processing machinery directly or by indirect effects mediated by transcriptional pausing. (C) 1998 Elsevier Science B.V.