In vivo analysis of ribonucleoprotein complexes using nucleotide analog interference mapping.

Multicomponent RNA-protein complexes are essential for eukaryotic gene expression. Some, like the spliceosome, have been studied successfully in vitro using biochemical and structural approaches, but many have not been reconstituted in cell-free systems. Nucleotide analog interference mapping (NAIM) can report detailed atomic information about requirements for ribonucleoprotein particle assembly and function in living cells, providing a method to study complexes in a cellular context at a level of detail comparable to many biochemical assays.

Molecular basis for RNA kink-turn recognition by the h15.5K small RNP protein.

The interaction between box C/D small nucleolar (sno)RNAs and the 15.5K protein nucleates snoRNP assembly. Many eukaryotic snoRNAs contain two potential binding sites for this protein, only one of which appears to be utilized in vivo.

Exclusive interaction of the 15.5 kD protein with the terminal box C/D motif of a methylation guide snoRNP.

Box C/D small nucleolar RNAs (snoRNAs) direct site-specific methylation of ribose 2'-hydroxyls in ribosomal and spliceosomal RNAs. To identify snoRNA functional groups contributing to assembly of an active box C/D snoRNP in Xenopus oocytes, we developed an in vivo nucleotide analog interference mapping procedure. Deleterious substitutions consistent with requirements for binding the 15.