FIG. 4.

Polyadenylation stimulatory activity maps to NRS5′, but hnRNP H-binding sites are not required. (A) RNA substrates were uniformly labeled with ³²P and incubated in HeLa nuclear extract for the times (in minutes) indicated above each lane. The NRS5′ and NRS3′ regions are the same as those bracketed in Fig. 3A, except that this NRS3′ version contained nt 801 to 932. RNA was subjected to electrophoresis on a 6% polyacrylamide gel that contains 8 M urea, and the image was obtained with a PhosphorImager; the results are representative of at least three independent experiments. Polyadenylation appears as a slower-migrating smear. The results of quantitation of polyadenylation at 30 min are the following: NRS-RSV, 13%; NRS5′-RSV, 12%; and NRS3′-RSV, 2%. (B) NRS-RSV and mutH-RSV, which contain the mutated hnRNP H-binding sites (Fig. 3A), were treated as described for panel A. The results of quantitation of polyadenylation at 30 min are the following: NRS-RSV, 13%; and NRSmutH-RSV, 13%. M, ³²P-end-labeled pBR322/MspI markers, the sizes of which are indicated at left.