Research

Research in my laboratory is focused on double-stranded RNA (dsRNA)--its biologic functions and the proteins that bind it to mediate these functions. Our studies are divided between two dsRNA-mediated pathways: RNA editing by adenosine deaminases that act on RNA (ADARs), and RNA interference (RNAi). For both pathways we perform in vitro studies to answer mechanistic questions, and in vivo studies in C. elegans to understand biologic function. dsRNA binding proteins (dsRBPs) bind tightly to dsRNA of any sequence, and a dsRNA substrate for one dsRBP is also a substrate for others. This suggests that different dsRNA-mediated pathways intersect and affect each other, and thus, we also study how RNA editing affects RNAi.

ADARs deaminate adenosines in double-stranded regions of cellular and viral RNAs to create the nucleoside inosine. Inosine is read as guanosine by the translational machinery, and one function of ADARs is to deaminate adenosines within codons, so that multiple protein isoforms can be synthesized from a single, encoded mRNA. In this way ADARs create protein isoforms of hepatitis delta antigen, and many proteins involved in neurotransmission, such as serotonin receptors and glutamate receptors. Consistent with the observation that ADARs are highly expressed in the nervous system, mutant animals lacking ADARs have behavioral defects. For example, our characterizations of C. elegans strains lacking ADARs show that these animals have aberrant chemotaxis and thermotaxis behaviors.

Several years ago my laboratory developed a method to identify inosine-containing RNA, and applied the method to mRNA isolated from C. elegans and human brain. We found inosines in remarkably stable structures that sometimes contain hundreds of nearly contiguous base-pairs. Surprisingly, the edited structures were in non-coding regions of mRNAs, such as 5' and 3' untranslated regions (UTRs), and introns. Our data suggest non-coding sequences are the primary targets of ADARs and that ADARs have functions in addition to altering codon meaning. Researchers in my lab are attempting to understand the function of these long double-stranded structures and the inosines within them. One possibility is that the structures induce silencing via the RNAi pathway, and that editing regulates this silencing. In support of this idea, we find that behavioral defects of C. elegans strains lacking ADARs can be rescued by a second mutation in a gene required for RNAi.

Recently, in collaboration with the Hill lab, we solved the crystal structure of the catalytic domain of human ADAR2. This study shows that ADAR catalysis involves a catalytic zinc coordinated to two cysteines and a histidine, as predicted from sequence similarities to the cytidine deaminase family. Future biochemical studies will focus on testing hypotheses about mechanism, substrate recognition, and substrate specificity that are suggested by the structure.

In addition to our studies on how RNA editing affects RNAi, we are also interested in the RNAi pathway in its own right, in particular, as it occurs in C. elegans . 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.

References

  1. Welker, N.C., Habig, J.W., Bass, B.L. (2007).  Genes misregulated in C. elegans deficient in Dicer, RDE-4, or RDE-1, are enriched for innate immunity genes. RNA, 13: 1090-1102.
  1. Parker GS, Eckert DM, Bass BL (2006) RDE-4 preferentially binds long dsRNA and its dimerization is necessary for cleavage of dsRNA to siRNA. RNA 12:807-818
  1. Bass BL, Hellwig S, Hundley HA (2005) A Nuclear RNA Is Cut Out for Translation. Cell 123:181-193
  1. Macbeth MR, Schubert HL, VanDemark AP, Lingam AT, Hill CP, Bass BL (2005) Inositol hexakisphosphate is bound in the ADAR2 core and required for RNA editing. Science 309:1534-1539
  1. Haudenschild BL, Maydanovych O, Véliz EA, Macbeth MR, Bass BL, Beal PA (2004) A Transition State Analog for an RNA-editing Reaction. J. Am. Chem. Soc. 126:11213-9
  1. Macbeth MR, Lingam AT, Bass BL (2004) Evidence for auto-inhibition by the N-terminus of hADAR2 and activation by dsRNA binding. RNA 10:1563-1571
  1. Tonkin LA, Bass BL (2003) Mutations in RNAi rescue aberrant chemotaxis of ADAR mutants. Science 302:1725
  1. Tonkin LA, Saccomanno L, Morse DP, Brodigan T, Krause M, Bass BL (2002) RNA editing by ADARs is important for normal behavior in Caenorhabditis elegans. EMBO J. 21:6025-6035
  1. Knight SW. Bass BL (2002) The Role of RNA Editing by ADARs in RNAi. Molecular Cell 10:809-817
  1. Morse DP, Aruscavage PJ, Bass BL (2002) RNA hairpins in noncoding regions of human brain and Caenorhabditis elegans mRNA are edited by adenosine deaminases that act on RNA. roc. Natl. Acad. Sci., USA 99:7906-7911
  1. Bass BL (2002) RNA editing by adenosine deaminases that act on RNA. Annu. Rev. Biochem. 71:817-846
  1. Knight SW, Bass BL (2001) A role for the RNase III enzyme DCR-1 in RNA interference and germ line development in C. elegans. Science 293:2269-71
  1. Bass BL (2001) The short answer [news and views]. Nature 411:428-429
  1. Bass BL, Editor (2001) RNA Editing: Frontiers in Molecular Biology, Oxford University Press
  1. Hough RF, Bass BL (2001) Adenosine deaminases that act on RNA. In RNA Editing: Frontiers in Molecular Biology, (ed. B.L. Bass) Oxford University Press, pp. 77-108
  1. Lehmann KA, Bass BL (2000) Double-stranded RNA adenosine deaminases ADAR1 and ADAR2 have overlapping specificities. Biochemistry 39:12875-12884
  1. Domeier ME, Morse DP, Knight SW, Portereiko M, Bass BL, Mango SE (2000) A link between RNA interference and nonsense-mediated decay in Caenorhabditis elegans. Science 289:1928-1930
  1. Öhman M, Källman AM, Bass BL (2000) In vitro analysis of the binding of ADAR2 to the pre-mRNA encoding the GluR-B R/G site. RNA 6:687-697
  1. Aruscavage PJ, Bass BL (2000) A phylogenetic analysis reveals an unusual sequence conservation within introns involved in RNA editing. RNA 6:257-269
  1. Bass BL (2000) Double-stranded RNA as a template for gene silencing [minireview]. Cell 101:235-238
  1. Hough RF, Lingam AT, Bass BL (1999) Caenorhabditis elegans mRNAs that encode a protein similar to ADARs derive from an operon containing six genes. Nucleic Acids Res. 27:3424-3432