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 alpha-tubulin gene X alpha T14, which contains the rare hexanucleotide CAUAAA, requires a surprisingly large amount of 3' flanking DNA to be used efficiently in Xenopus oocytes. To investigate the nature of the interaction between the X alpha T14 3' flank and upstream 3' processing sites, we have developed a modified oocyte assay based on the stimulation of processing at a single poly(A) signal. We mutated both the hexanucleotide and GU/U-rich components of a strong synthetic poly(A) signal (SPA) in order to weaken it severely. We found that efficient 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. Functional dissection of the X alpha T14 3' flank defined two separate regions that were each capable of partially restoring processing efficiency, presumably because they contain multiple, relatively weak processing enhancers. We discuss how the stimulation of 3' processing by flanking regions in oocytes could be explained by mechanisms that operate on the processing machinery directly or by indirect effects mediated by transcriptional pausing.