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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM026154-23
Application #
3484590
Study Section
Molecular Biology Study Section (MBY)
Project Start
1979-05-01
Project End
1994-04-30
Budget Start
1993-05-01
Budget End
1994-04-30
Support Year
23
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Yale University
Department
Type
Schools of Medicine
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code
06520
Herbert, Kristina M; Sarkar, Susanta K; Mills, Maria et al. (2016) A heterotrimer model of the complete Microprocessor complex revealed by single-molecule subunit counting. RNA 22:175-83
Brown, Jessica A; Kinzig, Charles G; DeGregorio, Suzanne J et al. (2016) Hoogsteen-position pyrimidines promote the stability and function of the MALAT1 RNA triple helix. RNA 22:743-9
Vilborg, Anna; Passarelli, Maria C; Yario, Therese A et al. (2015) Widespread Inducible Transcription Downstream of Human Genes. Mol Cell 59:449-61
Brown, Jessica A; Bulkley, David; Wang, Jimin et al. (2014) Structural insights into the stabilization of MALAT1 noncoding RNA by a bipartite triple helix. Nat Struct Mol Biol 21:633-40
Xie, Mingyi; Steitz, Joan A (2014) Versatile microRNA biogenesis in animals and their viruses. RNA Biol 11:673-81
Riley, Kasandra J; Steitz, Joan A (2013) The ""Observer Effect"" in genome-wide surveys of protein-RNA interactions. Mol Cell 49:601-4
Herbert, Kristina M; Pimienta, Genaro; DeGregorio, Suzanne J et al. (2013) Phosphorylation of DGCR8 increases its intracellular stability and induces a progrowth miRNA profile. Cell Rep 5:1070-81
Nag, Anita; Steitz, Joan A (2012) Tri-snRNP-associated proteins interact with subunits of the TRAMP and nuclear exosome complexes, linking RNA decay and pre-mRNA splicing. RNA Biol 9:334-42
Alexandrov, Andrei; Colognori, David; Shu, Mei-Di et al. (2012) Human spliceosomal protein CWC22 plays a role in coupling splicing to exon junction complex deposition and nonsense-mediated decay. Proc Natl Acad Sci U S A 109:21313-8
Brown, Jessica A; Valenstein, Max L; Yario, Therese A et al. (2012) Formation of triple-helical structures by the 3'-end sequences of MALAT1 and MEN? noncoding RNAs. Proc Natl Acad Sci U S A 109:19202-7

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