FIG. 2.

The binding of the 15.5K protein is an essential first step in the assembly of the box C/D snoRNP. (A) Sequence and structure of the two U4 5′ stem-loop oligonucleotides used to block 15.5K binding. The conserved internal loop nucleotides are shown in white on a black background. U4-SL1 is the wild-type sequence, while U4-SL17 contains a point mutation in the internal loop that inhibits 15.5K binding (33). (B) An excess of a U4 5′ stem-loop oligonucleotide can specifically block the assembly of the box C/D snoRNP. Radiolabeled U14 snoRNA was incubated in HeLa nuclear extract that had been preincubated with increasing amounts of either U4-SL1 or U4-SL17 RNA oligonucleotides. The binding of individual snoRNP proteins was then assayed by immunoprecipitation (see Materials and Methods). Bound RNAs were recovered and then separated on an 8% polyacrylamide-7 M urea gel. Antibodies used for immunoprecipitation are indicated on the left of the figure. Input, 10% of the RNA after incubation in nuclear extract. The amount as well as the identity of the RNA oligonucleotide used is indicated above each lane. (C) Rescue of 15.5K depletion by the addition of recombinant 15.5K. HeLa nuclear extract was preincubated with either 800 pmol of U4-SL1 RNA oligonucleotide (+) or buffer (−). Radiolabeled U14 snoRNA was subsequently added in the presence (10 pmol in lane 3, 20 pmol in lane 4, and 40 pmol in lane 5) or absence (lanes 1 and 2) of recombinant 15.5K. The binding of individual snoRNP proteins was assayed as for panel B.