Fig. 7. Binding of Smd1p to pre-U RNAs in extracts. (A) Extracts from wild-type (lane 2) and smd1–1 lhp1::LEU2 cells (lane 3) were subjected to Western blotting to detect Smd1p. Lane 1 contains extract from a strain in which an influenza HA epitope tag was added to Smd1p (Seto et al., 1999). (B) Mixtures of 32P-labeled pre-U4 and pre-U5 RNAs (lanes 1 and 3–7) or mature U4 and U5L RNAs (lanes 2 and 8–12) were incubated in wild-type extracts. Extracts were aliquoted and phenol extracted (lanes 3 and 8) or subjected to immunoprecipitation with the indicated sera. The pre-immune serum is from the anti-Lhp1p rabbit. Lanes 1 and 2 show the input RNAs. As mature U4 RNA terminates in UUUOH, it is bound by Lhp1p in extracts (lane 11). (C) 32P-Labeled pre-U4 and pre-U5 RNAs were incubated in wild-type (lanes 2–6), SMD1 lhp1::LEU2 (lanes 7–16) or smd1–1 lhp1::LEU2 extracts (lanes 17–26). Extracts were aliquoted and subjected to phenol extraction (lanes 2, 7, 12, 17 and 22) or immuno- precipitation as in (B). In lanes 12–16 and 22–26, 100 ng of Lhp1p (an amount equivalent to that in the wild-type extract) were included in the reactions. Lane 1 shows the input RNA. Both full-length pre-RNAs and their shorter degradation products were included in the quantitation of the data.