AUTOANTIBODY PROBES FOR MAMMALIAN GENE EXPRESSION

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Our long-term objectives are to elucidate the structures and functions of a variety of small RNA-protein (RNP) complexes present in the nucleus (snRNPs), nucleolus (snoRNPs), or cytoplasm (scRNPs) of mammalian cells. Patient autoantibodies (directed against the protein components of these particles) and in vitro systems active in RNA processing or other steps in gene expression are among the primary tools to be employed. Now that the most abundant Sm snRNPs containing U1, U2, U4/U6 and U5 RNAs have been identified as spliceosome components, their roles in pre-mRNA splicing will be more precisely defined by: examining the effect of heat shock on the U4/U6-U5 snRNP complex; ordering early steps in spliceosome assembly using U2 snRNP affinity columns; screening "antibiotics" for their ability to generate splicing intermediates and defining how aurintricarboxylic acid acts to inhibit in vitro splicing; attempting to obtain correct alternative splice site pairing in vitro in order to uncover the basis of regulated splicing; ascertaining the role of the snRNP-associated 70kd 3' splice site binding protein in splicing; and assessing the ability of constructs containing snRNA and substrate sequences to carry out partial splicing reactions in the absence of proteins. The recent realization that trans- spliced leader RNAs are molecular chimeras containing a 5' exon and snRNA- like moiety that assemble with Sm proteins (a novel Sm snRNP subclass) will be exploited in experiments designed to ask whether mammalian cells likewise engage in trans-splicing. The interactions of SL snRNPs with other splicing components will be probed in HeLa nuclear extracts to define the overlaps between the cis- and trans-splicing machinery. Structures of >10 newly identified low abundance Sm snRNPs will be elucidated and their presumed functions in mRNA biogenesis established using oligonucleotide probes to examine their tissue distribution and to test their activities and interactions with other snRNPs in vitro. Putative polyadenylation snRNPs will be identified by seeking cellular analogues of Herpesvirus saimiri U RNAs, which exhibit complementarity to AAUAAA. Base-pairing interactions between the U7 snRNP and the downstream conserved signal of histone pre-mRNAs will be manipulated to ask whether specific base pairs are required for 3' end maturation; auxiliary protein factors regulating histone mRNA processing will also be characterized. Ribosomal protein L5, which binds 5S rRNA prior to ribosome assembly will be tested for its ability to deliver viroid RNAs to their nucleolar site of replication (with consequent implications for pathogenicity). The protein composition of the 7SK snRNP will be defined using autoantibodies, and hints that it may function coordinately with Sm snRNPs will be pursued. Understanding the small RNP targets of autoimmunity will provide insights not only into rheumatic disease but also into the evolution and regulation of gene expression in normal and malignant cells.