Double-Stranded RNA-Binding Proteins and Their Substrates
Summary: Brenda Bass is interested in double-stranded RNA-binding proteins and the biologic functions of dsRNA. Her research is particularly focused on dsRNA-binding proteins involved in RNA editing and RNA interference.
Complementary strands of RNA interact through base-pairing to form the basic unit of RNA structure, the double helix. The most well characterized RNAs, such as tRNAs, rRNAs, and ribozymes, fold back onto themselves to create a series of short double helices that form the foundation for their three-dimensional shapes. Surprisingly, some RNA molecules contain much longer double helices that can have hundreds of nearly contiguous base pairs. I am interested in the cellular functions of these RNAs and the proteins that bind them, the double-stranded RNA (dsRNA)-binding proteins (dsRBPs).
Adenosine Deaminases That Act on RNA
My interest in dsRBPs began many years ago when I discovered a member of an enzyme family known as adenosine deaminases that act on RNA (ADARs). ADARs are RNA editing enzymes that deaminate adenosines within dsRNA to create inosines. Our recent crystal structure analysis, performed in collaboration with Christopher Hill's laboratory (University of Utah), reveals that catalysis involves a zinc ion that is coordinated in the active site by a His and two Cys, in a geometry that is essentially identical to that observed at the active site of the cytidine deaminase family.
Like guanosine, inosine prefers to pair with cytidine, and most cellular enzymes treat inosine as if it were guanosine. Thus, ADARs change the sequence information in an RNA, as well as its structure, and provide another way for metazoa to diversify the information encoded by their genes. For example, ADARs sometimes deaminate adenosines within codons so that multiple protein isoforms can be synthesized from the mRNA of a single gene. In this way ADARs produce isoforms of mammalian serotonin receptors, several mammalian glutamate receptor subunits, the virally encoded hepatitis delta antigen, and most likely, scores of proteins encoded by ADAR substrates not yet discovered.
The first ADAR substrates to be discovered were identified by chance, when genomically encoded adenosines were identified as guanosines in cDNAs. Such A to G transitions are diagnostic of A to I conversions in an RNA, since by pairing with cytidine, inosine is changed to a G during cDNA synthesis. My laboratory developed a method to identify ADAR substrates systematically, rather than by chance. We used this method to identify inosine-containing mRNAs in Caenorhabditis elegans and human brain. In both cases we identified substrates that are remarkable in their structures: in some cases inosines are located in intramolecular hairpins of almost 800 contiguous base pairs. The substrates are also surprising in that none of their inosines occur in codons. Rather, the inosines are in noncoding RNAs or within noncoding regions of mRNAs, such as 5'- and 3'-untranslated regions and introns. These data suggest that inosines in noncoding regions are much more common than those within codons, and that the primary function of ADARs involves deamination of noncoding dsRNA. Recent bioinformatic studies support this idea and indicate that 5 percent of the protein-coding genes of the human genome yield transcripts that are edited in noncoding regions.
ADARs Are Important for Normal Function of the Nervous System
ADARs are highly expressed in the nervous system and are important for normal neuronal function. The contribution of ADARs to neuronal function has been analyzed in mice and the fruit fly Drosophila melanogaster, and my laboratory studies ADARs in the nematode worm C. elegans. The nervous system of C. elegans is composed of only 302 neurons, all of which have been characterized with respect to cell lineage, location, and synaptic connectivity. The simplicity of the C. elegans nervous system, combined with the wealth of existing information about how it works, offers the possibility of correlating specific editing events with changes in behavior. Although ADARs likely contribute to many aspects of normal C. elegans behavior, studies so far show that strains lacking ADARs exhibit abnormal olfactory responses and cannot chemotax normally to volatile chemicals.
Do dsRNA-Mediated Pathways Intersect?
The structure of dsRNA, particularly the very narrow major groove, makes it difficult for proteins to make sequence-specific contacts. Correspondingly, all characterized dsRBPs bind to any dsRNA, regardless of its sequence. Given this, it seems likely that a dsRNA substrate of one dsRBP in a cell will also be bound by other dsRBPs and that different dsRNA-mediated pathways will affect each other. To test this hypothesis, we investigated whether ADARs affect the RNA interference (RNAi) pathway. In theory, not only could ADARs compete with dsRBPs involved in RNAi for binding to dsRNA, but dsRNA modified by an ADAR would no longer induce RNAi since its sequence would not match the targeted mRNA.
We have several lines of evidence indicating that RNA editing and RNAi do intersect. For example, if dsRNA is expressed in the nucleus of a wild-type or adr mutant, dsRNA-mediated gene silencing is much more robust in the adr mutant. This is consistent with the idea that ADARs, which are localized to the nucleus, antagonize the RNAi machinery in a wild-type worm. This observation raised the heretical idea that at least some of the behavioral defects observed in ADAR mutants are due to aberrant gene silencing rather than a lack of editing of specific codons. To explore this idea, we crossed C. elegans strains lacking ADARs with RNAi-defective strains. The RNAi-defective strains we chose contain mutations in the rde-1 or rde-4 gene and are incapable of eliciting an RNAi response, but otherwise appear normal. Remarkably, two rde-1 alleles and an rde-4 allele rescued the chemotaxis defects of the adr mutant animals. Animals containing only a mutation in the rde-1 or rde-4 gene showed normal chemotaxis behavior. Rescue was specific for the chemotaxis defects caused by mutations in the adr genes, since crossing rde-1 alleles into other chemotaxis-deficient strains did not lead to rescue. Future work will focus on understanding the molecular basis for this observation.
In addition to our studies on how RNA editing affects RNAi, we are also interested in the RNAi pathway in its own right, particularly as it occurs in C. elegans. Our biochemical studies are focused on the dsRBPs required for RNAi in C. elegans, and we analyze their RNA-binding properties and their catalytic activities in vitro. In regard to biologic function we want to understand the natural roles of dsRNA-mediated gene silencing. We are using microarray analyses to identify genes that are misregulated in C. elegans strains containing mutations in the Dicer gene, as well as those with mutations in other genes required for RNAi.
Graduate students in the Bass laboratory are supported by the National Institutes of Health.
Last updated April 22, 2009