The maturation of pre-messenger RNAs in the nucleoplasm of vertebrate and other higher eucaryotic cells is orchestrated by ten small nuclear ribonucleoproteins (snRNPs) that are central players in the action of two spliceosomes and the histone mRNA 3'-end processing machinery. In the nucleolus, are many more (approximately 200) small nucleolar RNPs (snoRNPs) that direct cleavages and specific nucleotide modifications, converting pre-ribosomal RNA into mature 18S, 5.8S and 28S rRNAs. Additional nucleoplasmic snRNPs guide the modification of spliceosomal snRNAs. The functions, mechanisms of action and biogenesis of vertebrate snRNPs and snoRNPs will be investigated with emphasis on the underlying molecular interactions using mammalian cells and cell extracts and the Xenopus oocyte. Our growing appreciation of coupling between steps in gene expression is a central focus - particularly with respect to the relationship between splicing and snoRNP biogenesis, as well as mRNA export. For the class of snoRNPs characterized by conserved sequences called boxes C and D, we will define the molecular basis of their asymmetric protein distribution, identify the molecular interactions that lead to synergy between splicing and the release of an intron-encoded snoRNA, and investigate the signals in dimeric snoRNAs responsible for their unusual nucleoplasmic localization that enables their action on spliceosomal snRNAs. We will investigate how both the major (U2-type) and minor (U12-type) spliceosome direct the deposit of a multiprotein complex (called the exon junction complex or EJC) on the newly spliced mRNA upstream of exon-exon junctions and attempt to capture spliceosomes arrested at particular stages in vivo. Studies of the regulation of the U 12-type spliceosome will exploit the presence of a twintron that is alternatively excised by the U12- or U2-type spliceosome in the Drosophila prospero pre-mRNA. The role of SR proteins, essential splicing factors, as adapter molecules for the nuclear export of both intronless and spliced mRNAs by the major mRNA export receptor TAP will be analyzed. Nucleotide analog interference mapping (NAIM) will assign RNA functional groups in the U7 snRNP and its substrate essential for 3'-end maturation ofhistone mRNAs. These studies will illuminate a variety of human disease processes that can be traced to perturbations in snRNP biogenesis, functions and associations.
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