Molecular Virology: Virology laboratories
Yale Combined Program in the Biological and Biomedical Sciences (BBS): Biochemistry, Biophysics and Structural Biology: RNA Catalysis and Ribonucleoprotein Machines
RNA molecules are the most functionally diverse biopolymers on Earth, but we know little about their structures and behaviors. In the Pyle lab, we explore the structural complexity of RNA molecules and the proteins that bind them, focusing on three major areas: A. The tertiary structures and folding pathways of long noncoding RNAs, such as the self-splicing group II introns. B. The molecular mechanism of RNA helicase proteins and RNA-triggered mechanical devices, such as the RIG-I innate immune sensor. C. Development of new experimental and computational tools for studying RNA structure. Our investigations have carried us into the fields of virology, innate immunity, RNA processing and molecular evolution. But our findings are relevant to all of the many tasks of RNA in the cell.
Extensive Research Description
In the Pyle Lab, we focus on two related questions: (1) How do large RNAs assemble into specific, stable tertiary structures? (2) How is RNA recognized and remodeled by ATP-dependent enzymes in the cell? Our studies involve a combination of solution biochemistry, enzymology, crystallography, and cell-based functional approaches. In parallel, we develop new computational methods for solving, analyzing and predicting RNA structures.
Group II Introns and Other Large RNA Tertiary Structures
Our studies of RNA tertiary architecture have focused on group II introns, which are large self-splicing ribozymes that are essential for gene expression in many organisms. Second only to the ribosome in size, group II introns have provided key insights into our understanding of RNA structure and evolution.
Initially, my laboratory used solution biochemistry and enzymology to characterize the chemical reaction mechanisms and architecture of group II introns. While this work yielded important insights into RNA splicing, we required high-resolution information on group II intron structures to define their functions precisely. We therefore spent many years attempting to identify a stable, homogeneous group II intron suitable for structural studies and were finally successful in crystallizing and solving the structure of a group IIC intron from the bacterium Oceanobacillus iheyensis (Oi IIC, ~400 nucleotides in size). This molecule, which is among the largest free RNA structures ever solved, revealed new architectural motifs and novel strategies for catalysis by RNA molecules (Figure 1, left).
Figure 1.Crystal structure of the Oi group IIC intron (left, 4FAR). Homology model of the yeast group IIB ai5γ ( (right). The catalytic center (domain 5) is shown in red, surrounded by intron domains (gray). The ai5γ model was built using the Oi core, biochemical constraints, and the RCrane modeling program. Srinivas Somarowthu
We have since solved the Oi IIC structure as it moves through the stages of splicing, showing that both steps are catalyzed by a conserved RNA stem loop (domain 5, red in Figure 1) that contains a reactive metal ion cluster composed of magnesium and potassium ions. In addition, we captured a conformational change that occurs between the two steps of splicing, allowing the active site to exchange splice sites and carry out multistep reactions. Using these structures, we have adapted homology-modeling programs and applied them to RNA, thereby modeling the structures of much larger group II introns, such as the ai5γ group IIB intron from yeast mitochondria (Figure 1, right).
Group II introns are particularly useful model systems for understanding the eukaryotic spliceosome, which processes pre-mRNA molecules in the nucleus. It had long been hypothesized that U6 snRNA (small nuclear RNA) within eukaryotic spliceosomes behaves in a manner similar to domain 5 of group II introns. Using our crystal structures as a guide, we created a road map for identifying U6 catalytic groups and we predicted the molecular organization of the spliceosomal active site. Recent work by our colleagues in the spliceosome field has confirmed our predictions and shown that the spliceosome is a ribozyme that is organized much like a group II intron. This work provides a strong foundation for exploiting the potential of group II introns in gene therapy and for developing group II introns and spliceosomes as therapeutic targets.
Our work on group II introns has provided the methodologies and strategies needed for solving the structures of even larger RNA molecules, such as long intergenic noncoding RNAs (lincRNAs), that play a central role in epigenetic control and other processes.To that end, we have developed new methods for isolating, folding, and solving the structures of lncRNA molecules (large RNAs, usually > 2 kb).We recently published the first structural map of the regulatory lncRNA HOTAIR, and we are applying these approaches to identify the structural components of lncRNAs such as RepA and lincRNA p21.By obtaining some of the first structural information on lncRNAs, we aim to provide a mechanistic foundation for their elusive functions in the cell.
Protein Machines for RNA Remodeling and Sensing
Eukaryotic cells express a large family of RNA-dependent ATPases (SF2 ATPases/helicases) that contribute to every aspect of RNA metabolism. Many of these proteins unwind RNA structures during the remodeling of RNA-protein complexes (acting as helicases), while others stabilize RNA structures (behaving as annealing enzymes), and yet others serve as biosensors and signals for the detection of pathogenic RNA (signaling enzymes). These proteins share certain architectural elements, including a common set of conserved domains that selectively bind RNA targets and create an active-site cleft for ATP binding and hydrolysis. The ATP-dependent motions of this cleft are coupled to mechanical functions, such as the unwinding of RNA, or domain motions that promote cell signaling. In studying the nanomechanical behavior of these proteins, we have explored new areas of molecular virology and immunology.
We are particularly interested in SF2 RNA helicase enzymes that play a role in the life cycle of viruses. For example, the NS3 helicase from hepatitis C virus (HCV) plays a key role in the replication and packaging of HCV. We have elucidated the stepwise mechanism by which NS3 unwinds RNA molecules, and we have used it as a paradigm for understanding ATP-powered translocation within the SF2 family. We have begun to dissect the network of interactions between NS3 and other components of the HCV replication complex, and we have shown that this multifunctional enzyme plays many roles in HCV pathogenicity.
During early studies of a protein involved in cancer reversion (MDA-5), we identified a subfamily of SF2 proteins that displays highly unusual behavior. The ATPase activity of these proteins, which include proteins MDA-5, RIG-I, and metazoan Dicer, is specifically stimulated by duplex RNA, rather than single-stranded RNA, and it is not accompanied by RNA unwinding. Family members such as RIG-I and MDA-5 play a central role in the human innate immune system, and Dicer proteins are key components of small interfering RNA (siRNA)- and microRNA (miRNA)-processing systems. Despite the biological significance of all these proteins, there was no high-resolution information on their structures or RNA binding interfaces and limited information on their enzymology.
We set out to change this with an intensive study of RIG-I, a surveillance protein that detects and responds to viral RNA infection within vertebrate cells. Through in vitro and in vivo experiments, we demonstrated that a 5'-triphosphorylated 10–base pair RNA duplex is sufficient for activating RIG-I and inducing a robust interferon response in vertebrates. We solved the crystal structure of RIG-I in complex with a variety of ligands, revealing an intricate machine that mechanically couples viral RNA binding with ATPase activity and signaling (Figure 2). This work paves the way for the design of new therapeutics that modulate the innate immune response and for new vaccine adjuvants. It also lays the groundwork for mechanistic understanding and pharmacological control of innate immune receptors, Dicer and related proteins.
Figure 2: Crystal structure of RIG-I in complex with RNA hairpin and Adenosine Diphosphate (top and side view, 4AY2). The color-coded key to domain organization is shown in the cartoon, below. RNA is yellow; ADP is pink. Hel1 and Hel2 are the conserved motor domains. The Pincer (P), Hel2i, and CTD are mechanical adapter domains. CARD1 and CARD2 are signaling domains. David Rawling
- Somarowthu, S., Legiewicz, M., Chillón, I., Marcia, M., Liu, F. and Pyle A.M. (2015) HOTAIR forms an intricate and modular secondary structure. Mol Cell. 58, 353-361.
- Rawling, D.C., Kohlway, A.S., Luo, D., Ding, S.C. and Pyle AM. (2014) The RIG-I ATPase core has evolved a functional requirement for allosteric stabilization by the Pincer domain. Nucleic Acids Res. 42, 11601-11.
- Kohlway, A., Pirakitikulr, N., Ding, S.C., Yang, F., Luo, D., Lindenbach, B.D. and Pyle, A.M. (2014) The linker region of NS3 plays a critical role in the replication and infectivity of hepatitis C virus. J Virol., 88, 10970-10974.
- Rawling, D.C. and Pyle, A.M. (2014) Parts, assembly and operation of the RIG-I family of motors. Curr Opin Struct Biol., 25, 25-33.
- Marcia, M. and Pyle, A.M. (2014) Principles of ion recognition in RNA: insights from the group II intron structures. RNA, 20, 516-27.
- Kohlway, A., Luo, D., Rawling, D.C., Ding, S.C. and Pyle, A.M. (2013) Defining the functional determinants for RNA surveillance by RIG-I. EMBO Rep. 14, 772-9.