Figure 2.

U2 internal modifications occur exclusively in the nucleus. α[³²P]UTP uniformly labeled G- or A-capped U2 was injected into the cytoplasm of oocytes (A, lanes 1 and 2; B, lanes 1–6) or directly into nuclei (A, lanes 4 and 5; B, lanes 10–15). After cytoplasmic injection, only G-capped U2 entered the nucleus; after nuclear injection, both G- and A-capped U2 RNAs were retained in the nucleus. RNAs recovered from nuclei (A, lanes 1, 4, and 5; B, lanes 1–3, 10–15) or from cytoplasm (A, lane 2; B, lanes 4–6) were assayed for pseudouridylation (A) and 2′-O-methylation (B). In the 2′-O-methylation assay, two different chimeras were used to test two positions (G11 and A30). The control is uninjected U2 RNA. (C) Oocyte nuclei were separated from the cytoplasm under oil. α[³²P]UTP uniformly labeled U2 was then injected into the isolated nuclei (lane 1) or enucleated oocytes (lane 2). After a 5-h incubation, U2 RNAs were recovered and assayed for pseudouridylation. The positions of uridylate, pseudouridylate, uncleaved U2, and 3′ fragments generated by RNase H site-specific cleavage are indicated on the side of each gel. In these experiments, >80% of the expected level of pseudouridylation and 2′-O-methylation was observed.