Alan M. Zahler Professor of Molecular, Cell and Developmental Biology B.S., Carnegie-Mellon University Ph.D., University of Colorado, Boulder Postdoctorate, Fred Hutchison Cancer Research Center, Seattle, WA

Alternative Splicing Regulation
It is now apparent from the sequencing of the human genome that more than half of human genes are alternatively spliced and that this alternative splicing is important for the generation of the diversity of the human proteome. However, we are just beginning to understanding how alternative splicing of pre-mRNAs is regulated. Experiments over the last fifteen years indicate that alternative splicing is regulated by cis-acting sequences in the pre-mRNA, found both in introns and exons, that are binding sites for trans-acting splicing regulatory protein factors, many of which are expressed in a tissue-specific manner. These factors promote or inhibit spliceosome assembly at the regulated splice sites. Understanding alternative splicing is important for increasing our understanding of how the vast diversity of the human proteome is generated from only about 25,000 genes.

Research in our laboratory is focused on the identification of the cis splicing-regulatory elements, the trans-acting factors that bind them, and the mechanisms by which splicing is regulated. A major area of focus in our laboratory involves using powerful genetic, molecular biology and bioinformatics tools to identify cis-acting sequences and trans-acting protein factors involved in alternative splice site selection in Caenorhabditis elegans. C. elegans has intron/exon structure and alternative splicing similar to higher organisms, however the introns are smaller and regulatory elements are easier to identify. We have taken a bioinformatics approach to studying splicing in this organism. In our genome browser, the Intronerator, we have aligned over 200,000 cDNAs and ESTs against the C. elegans genome sequence in order to identify introns and alternative splicing. With this approach, we have assembled a database of 680 alternative cassette exons. We have developed computational tools to identify conserved cis-regulatory elements around alternatively spliced regions that serve as splicing regulatory elements by aligning the full C. elegans and C. briggsae genomes with each other. We are taking a molecular and biochemical approach to understanding how these splicing regulatory elements function. This includes an important tool developed in the lab, a DNA microarray capable of simultaneously measuring the alternative splicing of 352 different genes. In another set of projects we are studying several genes that affect the choice of cryptic splice sites, both at the 5' and 3' ends of introns. These cryptic splice sites are activated when the wild type splice site is mutated, and this phenomenon occurs often in human disease mutations. We are characterizing several suppressors, including U1 snRNA mutants and proteins, that function to change splice site choice and using these discoveries to further our understanding of the fidelity of the splicing machinery.

MicroRNA Function
A recent project in the lab, initiated by graduate student Sam Gu and continued by Nicole Lambert, is to apply biochemical approaches towards understand the structure and function of microRNA containing ribonucleoprotein complexes (miRNPs). miRNAs are ~22 nucleotide long sequences that can interact with the 3' UTRs of target mRNAs and interfere with their ability to be translated. There is evidence in the scientific literature that this interference occurs at both the level of translational initiation and progression, and at the level of mRNA stability. To further investigate the mechanism of miRNA-induced translational down-regulation, we developed protocols for purification of miRNPs from C. elegans. We have identified protein and RNA components of miRNPs and have demonstrated that the RNAi pathway and miRNAs assemble distinct effector complexes. Sequence analysis of the RNA component of purified miRNPs has led to identification of new and interesting miRNAs and other classes of small RNAs. We are continuing to characterize the miRNPs and their components in the hope of better understanding miRNP biogenesis and function.

Selected Publications

Barberan-Soler, S. and Zahler, A.M. 2008. Alternative splicing regulation during C. elegans development: splicing factors as regulated targets. PLoS Genetics 4:e1000001.

Gu, S.G., Pak, J., Barberan-Soler, S., Ali, M., Fire, A., and Zahler, A.M. 2007. Distinct ribonucleoprotein reservoirs for microRNA and siRNA populations in C. elegans. RNA 13:1492-1504

Kabat, J.L., Barberan-Soler, S., McKenna, P., Clawson, H., Farrer, T. and Zahler, A.M. 2006. Intronic alternative splicing regulators identified by comparative genomics in nematodes. PLOS Computational Biology 2:e86.

Zahler, A.M. 2005. Alternative splicing in C. elegans. WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.31.1.

Zahler, A.M., Tuttle, J.D. and Chisholm, A.D. 2004. Genetic suppression of intronic +1G mutations by compensatory U1 snRNA changes in Caenorhabditis elegans. Genetics 167:1689-1696.

Zahler, A.M., Damgaard, C.K., Kjems, J. and Caputi, M. 2004. SC35 and heterogeneous nuclear ribonucleoprotein A/B proteins bind to a juxtaposed exonic splicing enhancer/exonic splicing silencer element to regulate HIV-1 tat exon 2 splicing. J. Biol. Chem. 279:10077-10084.

Farrer, T., Roller, A.B., Kent, W.J. and Zahler, A.M. 2002. Analysis of the role of C. elegans GC-AG introns in regulated splicing. Nucleic Acids Research 30:3360-3367.

Kent, W.J., Sugnet, C.W., Furey, T.S., Roskin, K.M., Pringle, T.H., Zahler, A.M. and Haussler, D. 2002. The human genome browser at UCSC. Genome Research 12:996-1006.

Caputi, M. and Zahler, A.M. 2002. SR proteins and hnRNP H regulate the splicing of the HIV-1 tev-specific exon 6D. EMBO J. 21:845-855.

Caputi, M. and Zahler, A.M. 2001. Determination of the RNA-binding specificity of the heterogeneous nuclear ribonucleoprotein (hnRNP) H/H'/F/2H9 family. J. Biol. Chem. 276:43850-43859.

Zahler, A.M. 2001. Tale of a tail kinase. Nature Structural Biology 8:104-106.

Kent, W.J. and Zahler, A.M. 2000. Conservation, regulation, synteny, and introns in a large scale C. briggsae/C. elegans genomic alignment. Genome Research 10:1115-1125.

Roller, A.B., Hoffman, D.C., and Zahler, A.M. 2000. The allele-specific suppressor sup-39 alters use of cryptic splice sites in C. elegans. Genetics 154:1169-1179.

Kent, W.J. and Zahler, A.M. 2000. The Intronerator: exploring introns and alternative splicing in C.elegans. Nucleic Acids Research 28:91-93.

Caputi, M., Mayeda, A., Krainer, A.R., and Zahler, A.M. 1999. hnRNP A/B proteins are required for inhibition of HIV-1 pre-mRNA splicing. EMBO J. 18:4060-4067.