ORGANIZATION OF SMALL NUCLEOLAR RIBONUCLEOPROTEINS (SNORNPS) BY FLUORESCENCE IN-SITU HYBRIDIZATION AND IMMUNOCYTOCHEMISTRY

The organization of the U3, U8, and U13 small nucleolar ribonucleoprot eins (snoRNPs) has been investigated in HeLa cells using antisense DNA and 2'-OMe RNA oligonucleotides. Oligomers corresponding to deoxynucl eotides that target RNase H degradation of intact RNP particles were s ynthesized and used for fluorescence in situ hybridization. U3 and U13 are distributed throughout the nucleolus and colocalize with anti-fib rillarin antibodies. U8, however, is organized in discrete ring-like s tructures near the center of the nucleolus and surround bright punctat e regions visualized with anti-RNA polymerase I and anti-UBF/NOR-90 an tibodies. In decondensed nucleoli, a necklace of smaller ring-like str uctures of U8 RNA appear. A model for the recruitment of U8 (and presu mably other processing factors) to the sites of rRNA transcription is discussed. Hybridization to mitotic cells showed that unlike pol I and NOR-90, U8 is dispersed into the cytoplasm during mitosis. The subnuc leolar organization of U8 is consistent with its demonstrated particip ation in early intermediate steps in pre-rRNA processing. In contrast, the more dispersed intranucleolar distribution of U3 agrees with its putative involvement in both early and late steps of rRNA maturation. These studies illustrate the feasibility of mapping functional domains within the nucleolus by correlating the in vitro activities of small nuclear RNPs with their in situ locations.