Alan Zahler, MCD Biology

	C. elegans expressing green fluorescent protein alternative splicing reporter constructs.

Alternative Splicing Regulation

It is now apparent from the sequencing of the human genome that about 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 35,000 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. 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, that function to change splice site choice and are applying what we learned by this genetic approach to the alteration of splicing in human cells.

MicroRNA Function

A recent project in the lab, initiated by graduate student Sam Gu, 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. We have identified an endonuclease activity associated with miRNPs that is distinct from the slicer activity found associated with siRNAs. The miRNP endonuclease activity cleaves the substrate RNAs in the regions flanking the region of miRNA basepairing, with a preference for cleavage 3' of the site of interaction. We are continuing to investigate miRNPs and their activities and are developing this as a second major focus of the lab.