Over the past five years, our laboratory has developed numerous approaches to study the regulation of pre-mRNA splicing using C. elegans as a model system. Alternative splicing is a key mechanism for controlling gene expression and creating diversity in the proteome. Using C. elegans as a model system to study this process has several distinct advantages in that this organism maintains the same types of alternative splicing events and regulators as higher eukaryotes, while the available genomics and genetics tools make this a very tractable system for studying the regulation of this process. We have developed bioinformatics techniques for identifying alternatively spliced genes and doing interspecies alignments of genomes. We have applied these approaches towards identifying alternative splicing regulatory elements in introns. The relatively small intron size of nematodes, and the large degree of sequence divergence between C. elegans and C. briggsae introns, gives us advantages over vertebrate model systems for identifying these cis splicing regulators. We will apply this knowledge to understanding the function of these sequences in regulating alternative splicing. We are characterizing the role of splicing regulatory factor MEC-8 in splice site choice and have shown that it is part of a large stable ribonucleoprotein complex. Understanding this complex and its function in splice site choice will illuminate the mechanism of MEC-8 splicing regulation. We have cloned and characterized two suppressors of mutations to the first nucleotide of an intron. We have also mapped regulators of cryptic splice site choice at the 3' end of the intron. We propose to continue and expand these studies to further understand the mechanisms of splice site selection.
The specific aims of this grant proposal are: 1- To identify intronic pre-mRNA splicing regulatory elements through combined bioinformatics and in vivo studies. These will identify a comprehensive set of intronic alternative splicing control signals and the factors that interact with them. 2- To determine the mechanism of action of the pre-mRNA splicing factor MEC-8 in regulation of pre-mRNA splicing. 3- To characterize suppressors of 5' and 3' splice site defects in order to illuminate the mechanisms of splice site selection.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM061646-05A1
Application #
6966920
Study Section
Molecular Genetics C Study Section (MGC)
Program Officer
Rhoades, Marcus M
Project Start
2000-07-01
Project End
2009-08-31
Budget Start
2005-09-01
Budget End
2006-08-31
Support Year
5
Fiscal Year
2005
Total Cost
$273,420
Indirect Cost
Name
University of California Santa Cruz
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
125084723
City
Santa Cruz
State
CA
Country
United States
Zip Code
95064
Barberan-Soler, Sergio; Medina, Pedro; Estella, Jeffrey et al. (2011) Co-regulation of alternative splicing by diverse splicing factors in Caenorhabditis elegans. Nucleic Acids Res 39:666-74
Barberan-Soler, Sergio; Lambert, Nicole J; Zahler, Alan M (2009) Global analysis of alternative splicing uncovers developmental regulation of nonsense-mediated decay in C. elegans. RNA 15:1652-60
Kabat, Jennifer L; Barberan-Soler, Sergio; Zahler, Alan M (2009) HRP-2, the Caenorhabditis elegans homolog of mammalian heterogeneous nuclear ribonucleoproteins Q and R, is an alternative splicing factor that binds to UCUAUC splicing regulatory elements. J Biol Chem 284:28490-7
Dassah, Maryann; Patzek, Sophie; Hunt, Valerie M et al. (2009) A genetic screen for suppressors of a mutated 5' splice site identifies factors associated with later steps of spliceosome assembly. Genetics 182:725-34
Barberan-Soler, Sergio; Zahler, Alan M (2008) Alternative splicing and the steady-state ratios of mRNA isoforms generated by it are under strong stabilizing selection in Caenorhabditis elegans. Mol Biol Evol 25:2431-7
Barberan-Soler, Sergio; Zahler, Alan M (2008) Alternative splicing regulation during C. elegans development: splicing factors as regulated targets. PLoS Genet 4:e1000001
Gu, Sam G; Pak, Julia; Barberan-Soler, Sergio et al. (2007) Distinct ribonucleoprotein reservoirs for microRNA and siRNA populations in C. elegans. RNA 13:1492-504
Kabat, Jennifer L; Barberan-Soler, Sergio; McKenna, Paul et al. (2006) Intronic alternative splicing regulators identified by comparative genomics in nematodes. PLoS Comput Biol 2:e86
Zahler, Alan M (2005) Alternative splicing in C. elegans. WormBook :1-13
Zahler, Alan M; Tuttle, John D; Chisholm, Andrew D (2004) Genetic suppression of intronic +1G mutations by compensatory U1 snRNA changes in Caenorhabditis elegans. Genetics 167:1689-96

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