Ribonucleoproteins and Eukaryotic Gene Expression

RNA molecules are essential participants in many aspects of cellular function. We aim to understand the mechanisms of RNA-mediated steps during eukaryotic gene expression. Our current research is focused on two critically important pathways: pre-mRNA splicing and RNA silencing.

The machinery that removes introns from mRNA precursors is a large macromolecular machine called the spliceosome. Five small nuclear RNAs (snRNAs) are essential for spliceosome function, and establish a dynamic network of interactions that mediate spliceosome assembly, activation, and catalysis. The spliceosome protein Prp8 is very large (~280 kDa) and highly conserved, and genetic analyses suggest that it regulates the RNA structural rearrangements that underlie spliceosome catalysis. Despite its prominent role, little is known about its precise mechanism of action. We have recently established a link between Prp8 and another conserved protein called ubiquitin (a 76 amino acid protein that modulates the fates and functions of numerous target proteins via covalent attachment). We have found that an essential domain in Prp8 is homologous to other proteins that cleave ubiquitin from target proteins. In Prp8, this domain appears to serve a ubiquitin-binding function. In addition, we have obtained biochemical evidence that ubiquitin can regulate a specific step in spliceosome assembly. We are currently investigating the mechanistic basis for this regulation.

Over the past several years, a new role for RNA has been uncovered in eukaryotic gene expression: double-stranded RNA (dsRNA) molecules trigger the silencing of homologous genes in a phenomenon known as RNA interference (RNAi). RNAi is an important control pathway that regulates the expression of cellular genes and protects cells from viruses and transposons; it has also emerged as a broadly applicable experimental approach to determine the functions of novel genes. In collaboration with Professor Richard Carthew, we are investigating the mechanism of RNAi. The Carthew lab has initiated a genetic screen for RNAi-defective mutants in Drosophila, and we are using extracts from the mutants to characterize the mechanistic basis for the RNAi defects in vitro. In the course of our studies, we have delineated the assembly pathway for the RNA-induced silencing complex (RISC), and identified an ATP-dependent ~80S complex that mediates mRNA target cleavage in response to dsRNA. We are continuing our biochemical analysis of the RNAi pathway in Drosophila. In addition, we are using the biochemical tools developed in the Drosophila model system to examine the mechanism of RISC assembly and RNAi in human cell extracts.

E. J. Sontheimer, S. Sun, and J. A. Piccirilli (1997). "Metal ion catalysis during splicing of premessenger RNA." Nature 388: 801-805.

E. J. Sontheimer, P. M. Gordon, J. A. Piccirilli, and (1999). "Metal ion catalysis during group II intron self-splicing: parallels with the spliceosome." Genes and Development 13: 1729-1741.

Sontheimer, E.J. (2001). "The spliceosome shows its metal." Nature Structural Biology 8(1): 11-13.

Sontheimer, E.J. "R2D2 leads the silencing trigger to mRNA's death star." Cell 115: 132-133.

Y. S. Lee, K. Nakahara, J. W. Pham, K. Kim, Z. He, E. J. Sontheimer, and R. W. Carthew (2004). "Distinct roles for Drosophila Dicer-1 and Dicer-2 proteins in the siRNA/miRNA silencing pathways." Cell 117: 69-81.

J. W. Pham, J. L. Pellino, Y. S. Lee, R. W. Carthew, and E. J. Sontheimer (2004). "A Dicer-2-dependent 80S complex cleaves targeted mRNAs during RNAi in Drosophila." Cell 117: 83-94.

Pham, J. W., Radhakrishnan, I., and Sontheimer, E. J. (2004). "Thermodynamic and structural characterization of 2'-nitrogen-modified RNA duplexes." Nucleic Acids Research 32:3446-3455.

Sontheimer, E. J., and Carthew, R. W. (2004). "Argonaute journeys into the heart of RISC." Science 305:1409-1410.

Sontheimer, E. J. (2005). "Assembly and function of RNA silencing complexes." Nature Reviews Molecular Cell Biology 6:127-138.

Sontheimer, E. J., and Carthew, R. W. (2005). "Silence from within: Endogenous siRNAs and miRNAs." Cell 122:9-12.

Pellino, J. L., Jaskiewicz, L., Filipowicz, W., and Sontheimer, E. J. (2005). "ATP modulates siRNA interactions with an endogenous human Dicer complex." RNA 11:1719-1724.

Pham, J. W., and Sontheimer, E. J. (2005). "Molecular Requirements for RISC Assembly in the Drosophila RNAi Pathway." Journal of Biological Chemistry 280:39278-39283.

Preall, J. B., He, Z., Gorra, J., and Sontheimer, E. J. (2006). Short interfering RNA strand selection is independent of double-stranded RNA processing polarity during RNA interference in Drosophila. Current Biology 16, 530-535.

Bellare, P., Kutach, A. K., Rines, A., Guthrie, C., and Sontheimer, E. J. (2006). Ubiquitin binding by a variant Jab1/MPN domain in the essential pre-mRNA splicing factor Prp8p. RNA 12, 292-302.

View all publications by publications by Erik J. Sontheimer listed in the National Library of Medicine (PubMed).