The goal of our studies is to define the molecular mechanisms underlying pre-mRNA splicing, an essential step in expression of most eukaryotic genes. Typically, efficient splicing requires that the substrate sites that directly participate in catalysis: the 5'splice site (5'SS), branch site (BS) and the 3'splice site (3'SS), adhere to the conserved consensus;sequence deviations from this consensus often result in inhibition of splicing or aberrant splicing events. Typically, splice site signals of alternatively spliced introns deviate from the consensus, and are often accompanied by splicing enhancer motifs. By contrast, splice site signals in Saccharomyces cerevisiae fit well to the consensus and alternative splicing is thought not to occur in yeast. Nonetheless, we have recently identified several pre-mRNA sequence motifs that strongly affect splicing in yeast;these motifs are strikingly similar to their mammalian counterparts. The overall goal of the proposed research is to study basic mechanisms involved in pre-mRNA splicing, with the emphasis on understanding how enhancer/silencer sequences flanking the splice site signals influence splicing catalysis and positioning of the splice sites at the active site of the spliceosome.
The specific aims are: 1. To identify additional yeast enhancers;study the importance of position, copy number, context;2. To identify and characterize enhancer binding sites within the spliceosome;3. To study interactions at the catalytic center of the spliceosome. These objectives will be achieved through a combination of genetic approaches in vivo and biochemical experiments in vitro - using S. cerevisiae as a system. A series of selection screens will identify new enhancer motifs, and another series of genetic screens will identify splicing factors that recognize them. In vitro analysis will provide detailed information concerning direct interactions at the spliceosomal catalytic center. Because function of the catalytic core of the spliceosome appears to be conserved in all eukaryotes, information gained from these studies will apply to mammalian spliceosome as well. Defects in splicing lead to many human genetic diseases, for example, mutations in Prp8, a core spliceosomal factor, are associated with retinitis pigmentosa. Aberrant alternative splicing events are also commonly associated with human disease;insights into the basic mechanisms of premRNA splicing and splice site recognition are therefore fundamental to understanding regulated gene expression and human disease. These studies, aimed at basic mechanisms of splicing, are necessary not only because of their inherent importance, but also because they lead to understanding of both the evolutionary origins of this reaction and the regulation of alternative splicing in human cells.

Public Health Relevance

Pre-mRNA splicing is an obligatory step in expression of most eukaryotic genes. Splicing defects are at the basis of multiple genetic disorders and cancer;understanding this process at the basic, mechanistic level is critical for our ability to diagnose and treat these diseases. The proposed studies, aimed at understanding the function of the yeast spliceosome, are expected to explain some fundamental properties of alternative splicing in human cells.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular Genetics A Study Section (MGA)
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Bender, Michael T
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Rockefeller University
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New York
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Kurtovic-Kozaric, A; Przychodzen, B; Singh, J et al. (2015) PRPF8 defects cause missplicing in myeloid malignancies. Leukemia 29:126-36
Query, Charles C; Konarska, Maria M (2012) CEF1/CDC5 alleles modulate transitions between catalytic conformations of the spliceosome. RNA 18:1001-13
Smith, Duncan J; Konarska, Maria M; Query, Charles C (2009) Insights into branch nucleophile positioning and activation from an orthogonal pre-mRNA splicing system in yeast. Mol Cell 34:333-43
Smith, Duncan J; Konarska, Maria M (2009) Identification and characterization of a short 2'-3' bond-forming ribozyme. RNA 15:8-13
Smith, Duncan J; Query, Charles C; Konarska, Maria M (2008) ""Nought may endure but mutability"": spliceosome dynamics and the regulation of splicing. Mol Cell 30:657-66
Simoes-Barbosa, Augusto; Meloni, Dionigia; Wohlschlegel, James A et al. (2008) Spliceosomal snRNAs in the unicellular eukaryote Trichomonas vaginalis are structurally conserved but lack a 5'-cap structure. RNA 14:1617-31
Smith, Duncan J; Query, Charles C; Konarska, Maria M (2007) trans-splicing to spliceosomal U2 snRNA suggests disruption of branch site-U2 pairing during pre-mRNA splicing. Mol Cell 26:883-90
Liu, Li; Query, Charles C; Konarska, Maria M (2007) Opposing classes of prp8 alleles modulate the transition between the catalytic steps of pre-mRNA splicing. Nat Struct Mol Biol 14:519-26
Konarska, Maria M; Vilardell, Josep; Query, Charles C (2006) Repositioning of the reaction intermediate within the catalytic center of the spliceosome. Mol Cell 21:543-53
Konforti, B B; Konarska, M M (1994) U4/U5/U6 snRNP recognizes the 5' splice site in the absence of U2 snRNP. Genes Dev 8:1962-73

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