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.
Full List of PubMed Publications
- Liu F, Somarowthu S, Pyle AM: Visualizing the secondary and tertiary architectural domains of lncRNA RepA. Nat Chem Biol. 2017 Mar; 2017 Jan 9. PMID: 28068310
- Fitzgerald ME, Rawling DC, Potapova O, Ren X, Kohlway A, Pyle AM: Selective RNA targeting and regulated signaling by RIG-I is controlled by coordination of RNA and ATP binding. Nucleic Acids Res. 2017 Feb 17. PMID: 28180316
- Schlick T, Pyle AM: Opportunities and Challenges in RNA Structural Modeling and Design. Biophys J. 2017 Feb 2; 2017 Feb 2. PMID: 28162235
- Fitzgerald ME, Rawling DC, Potapova O, Ren X, Kohlway A, Pyle AM: Selective RNA targeting and regulated signaling by RIG-I is controlled by coordination of RNA and ATP binding. Nucleic Acids Res. 2016 Sep 12; 2016 Sep 12. PMID: 27625396
- Pyle AM: Group II Intron Self-Splicing. Annu Rev Biophys. 2016 Jul 5. PMID: 27391926
- Zhao C, Pyle AM: Crystal structures of a group II intron maturase reveal a missing link in spliceosome evolution. Nat Struct Mol Biol. 2016 Jun; 2016 May 2. PMID: 27136328
- Pyle AM, Schlick T: Challenges in RNA Structural Modeling and Design. J Mol Biol. 2016 Feb 27; 2016 Feb 12. PMID: 26876599
- Lilley DM, Pyle AM: Editorial overview: Nucleic acids and their protein complexes. Curr Opin Struct Biol. 2016 Feb; 2016 Mar 2. PMID: 26948825
- Zhao C, Rajashankar KR, Marcia M, Pyle AM: Crystal structure of group II intron domain 1 reveals a template for RNA assembly. Nat Chem Biol. 2015 Dec; 2015 Oct 26. PMID: 26502156
- Rawling DC, Fitzgerald ME, Pyle AM: Establishing the role of ATP for the function of the RIG-I innate immune sensor. Elife. 2015 Sep 15; 2015 Sep 15. PMID: 26371557
- Somarowthu S, Legiewicz M, Chillón I, Marcia M, Liu F, Pyle AM: HOTAIR forms an intricate and modular secondary structure. Mol Cell. 2015 Apr 16; 2015 Apr 9. PMID: 25866246
- Pyle AM: Rediscovering RNA. RNA. 2015 Apr. PMID: 25780205
- Foxman EF, Storer JA, Fitzgerald ME, Wasik BR, Hou L, Zhao H, Turner PE, Pyle AM, Iwasaki A: Temperature-dependent innate defense against the common cold virus limits viral replication at warm temperature in mouse airway cells. Proc Natl Acad Sci U S A. 2015 Jan 20; 2015 Jan 5. PMID: 25561542
- Chillón I, Marcia M, Legiewicz M, Liu F, Somarowthu S, Pyle AM: Native Purification and Analysis of Long RNAs. Methods Enzymol. 2015; 2015 Feb 27. PMID: 26068736
- Pyle AM: Looking at LncRNAs with the ribozyme toolkit. Mol Cell. 2014 Oct 2. PMID: 25280101
- Rawling DC, Kohlway AS, Luo D, Ding SC, Pyle AM: The RIG-I ATPase core has evolved a functional requirement for allosteric stabilization by the Pincer domain. Nucleic Acids Res. 2014 Oct; 2014 Sep 12. PMID: 25217590
- Fitzgerald ME, Rawling DC, Vela A, Pyle AM: An evolving arsenal: viral RNA detection by RIG-I-like receptors. Curr Opin Microbiol. 2014 Aug; 2014 Jun 7. PMID: 24912143
- Rawling DC, Pyle AM: Parts, assembly and operation of the RIG-I family of motors. Curr Opin Struct Biol. 2014 Apr; 2013 Dec 20. PMID: 24878341
- Fitzgerald ME, Vela A, Pyle AM: Dicer-related helicase 3 forms an obligate dimer for recognizing 22G-RNA. Nucleic Acids Res. 2014 Apr; 2014 Jan 15. PMID: 24435798
- Somarowthu S, Legiewicz M, Keating KS, Pyle AM: Visualizing the ai5γ group IIB intron. Nucleic Acids Res. 2014 Feb; 2013 Nov 6. PMID: 24203709
- Pyle AM: Coordinating the party: assembly factors and ribogenesis. Mol Cell. 2013 Nov 21. PMID: 24267447
- Marcia M, Humphris-Narayanan E, Keating KS, Somarowthu S, Rajashankar K, Pyle AM: Solving nucleic acid structures by molecular replacement: examples from group II intron studies. Acta Crystallogr D Biol Crystallogr. 2013 Nov; 2013 Oct 12. PMID: 24189228
- Nagy V, Pirakitikulr N, Zhou KI, Chillón I, Luo J, Pyle AM: Predicted group II intron lineages E and F comprise catalytically active ribozymes. RNA. 2013 Sep; 2013 Jul 23. PMID: 23882113
- Marcia M, Somarowthu S, Pyle AM: Now on display: a gallery of group II intron structures at different stages of catalysis. Mob DNA. 2013 May 1; 2013 May 1. PMID: 23634971
- Luo D, Kohlway A, Pyle AM: Duplex RNA activated ATPases (DRAs): platforms for RNA sensing, signaling and processing. RNA Biol. 2013 Jan; 2012 Dec 10. PMID: 23228901
- Vela A, Fedorova O, Ding SC, Pyle AM: The thermodynamic basis for viral RNA detection by the RIG-I innate immune sensor. J Biol Chem. 2012 Dec 14; 2012 Oct 10. PMID: 23055530
- Luo D, Kohlway A, Vela A, Pyle AM: Visualizing the determinants of viral RNA recognition by innate immune sensor RIG-I. Structure. 2012 Nov 7; 2012 Sep 27. PMID: 23022350
- Marcia M, Pyle AM: Visualizing group II intron catalysis through the stages of splicing. Cell. 2012 Oct 26. PMID: 23101623
- Fedorova O, Pyle AM: The brace for a growing scaffold: Mss116 protein promotes RNA folding by stabilizing an early assembly intermediate. J Mol Biol. 2012 Sep 21; 2012 Jun 13. PMID: 22705286
- Humphris-Narayanan E, Pyle AM: Discrete RNA libraries from pseudo-torsional space. J Mol Biol. 2012 Aug 3; 2012 Mar 13. PMID: 22425640
- Keating KS, Pyle AM: RCrane: semi-automated RNA model building. Acta Crystallogr D Biol Crystallogr. 2012 Aug; 2012 Jul 17. PMID: 22868764
- Keating KS, Humphris EL, Pyle AM: A new way to see RNA. Q Rev Biophys. 2011 Nov; 2011 May 18. PMID: 21729350
- Luo D, Ding SC, Vela A, Kohlway A, Lindenbach BD, Pyle AM: Structural insights into RNA recognition by RIG-I. Cell. 2011 Oct 14. PMID: 22000018
- Pyle AM: RNA helicases and remodeling proteins. Curr Opin Chem Biol. 2011 Oct; 2011 Aug 20. PMID: 21862383
- Cao W, Coman MM, Ding S, Henn A, Middleton ER, Bradley MJ, Rhoades E, Hackney DD, Pyle AM, De La Cruz EM: Mechanism of Mss116 ATPase reveals functional diversity of DEAD-Box proteins. J Mol Biol. 2011 Jun 10; 2011 Apr 9. PMID: 21501623
- Phan T, Kohlway A, Dimberu P, Pyle AM, Lindenbach BD: The acidic domain of hepatitis C virus NS4A contributes to RNA replication and virus particle assembly. J Virol. 2011 Feb; 2010 Nov 3. PMID: 21047963
- Zingler N, Solem A, Pyle AM: Dual roles for the Mss116 cofactor during splicing of the ai5γ group II intron. Nucleic Acids Res. 2010 Oct; 2010 Jun 16. PMID: 20554854
- Matranga C, Pyle AM: Double-stranded RNA-dependent ATPase DRH-3: insight into its role in RNAsilencing in Caenorhabditis elegans. J Biol Chem. 2010 Aug 13; 2010 Jun 7. PMID: 20529861
- Pyle AM: The tertiary structure of group II introns: implications for biological function and evolution. Crit Rev Biochem Mol Biol. 2010 Jun. PMID: 20446804
- Keating KS, Pyle AM: Semiautomated model building for RNA crystallography using a directed rotameric approach. Proc Natl Acad Sci U S A. 2010 May 4; 2010 Apr 19. PMID: 20404211
- Taylor SD, Solem A, Kawaoka J, Pyle AM: The NPH-II helicase displays efficient DNA x RNA helicase activity and a pronounced purine sequence bias. J Biol Chem. 2010 Apr 9; 2010 Jan 28. PMID: 20110368
- Fedorova O, Solem A, Pyle AM: Protein-facilitated folding of group II intron ribozymes. J Mol Biol. 2010 Apr 2; 2010 Feb 6. PMID: 20138894
- Roitzsch M, Fedorova O, Pyle AM: The 2'-OH group at the group II intron terminus acts as a proton shuttle. Nat Chem Biol. 2010 Mar; 2010 Jan 31. PMID: 20118939
- Toor N, Keating KS, Fedorova O, Rajashankar K, Wang J, Pyle AM: Tertiary architecture of the Oceanobacillus iheyensis group II intron. RNA. 2010 Jan; 2009 Dec 1. PMID: 19952115
- Keating KS, Toor N, Perlman PS, Pyle AM: A structural analysis of the group II intron active site and implications for the spliceosome. RNA. 2010 Jan; 2009 Nov 30. PMID: 19948765
- Pyle AM: How to drive your helicase in a straight line. Cell. 2009 Oct 30. PMID: 19879832
- Toor N, Keating KS, Pyle AM: Structural insights into RNA splicing. Curr Opin Struct Biol. 2009 Jun; 2009 May 13. PMID: 19443210
- Beran RK, Lindenbach BD, Pyle AM: The NS4A protein of hepatitis C virus promotes RNA-coupled ATP hydrolysis by the NS3 helicase. J Virol. 2009 Apr; 2009 Jan 19. PMID: 19153239
- Roitzsch M, Pyle AM: The linear form of a group II intron catalyzes efficient autocatalytic reverse splicing, establishing a potential for mobility. RNA. 2009 Mar; 2009 Jan 23. PMID: 19168748
- Serebrov V, Beran RK, Pyle AM: Establishing a mechanistic basis for the large kinetic steps of the NS3 helicase. J Biol Chem. 2009 Jan 23; 2008 Nov 14. PMID: 19010782
- Keating KS, Toor N, Pyle AM: The GANC tetraloop: a novel motif in the group IIC intron structure. J Mol Biol. 2008 Nov 14; 2008 Aug 26. PMID: 18773908
- Toor N, Rajashankar K, Keating KS, Pyle AM: Structural basis for exon recognition by a group II intron. Nat Struct Mol Biol. 2008 Nov; 2008 Oct 26. PMID: 18953333
- Beran RK, Pyle AM: Hepatitis C viral NS3-4A protease activity is enhanced by the NS3 helicase. J Biol Chem. 2008 Oct 31; 2008 Aug 22. PMID: 18723512
- Fedorova O, Pyle AM: A conserved element that stabilizes the group II intron active site. RNA. 2008 Jun; 2008 Apr 25. PMID: 18441048
- Toor N, Keating KS, Taylor SD, Pyle AM: Crystal structure of a self-spliced group II intron. Science. 2008 Apr 4. PMID: 18388288
- Waldsich C, Pyle AM: A kinetic intermediate that regulates proper folding of a group II intron RNA. J Mol Biol. 2008 Jan 11; 2007 Oct 24. PMID: 18022197
- Pyle AM: Translocation and unwinding mechanisms of RNA and DNA helicases. Annu Rev Biophys. 2008. PMID: 18573084
- Zingler N, Solem A, Pyle AM: Protein-facilitated ribozyme folding and catalysis. Nucleic Acids Symp Ser (Oxf). 2008. PMID: 18776256
- de Lencastre A, Pyle AM: Three essential and conserved regions of the group II intron are proximal to the 5'-splice site. RNA. 2008 Jan; 2007 Nov 26. PMID: 18039742
- Beran RK, Serebrov V, Pyle AM: The serine protease domain of hepatitis C viral NS3 activates RNA helicase activity by promoting the binding of RNA substrate. J Biol Chem. 2007 Nov 30; 2007 Oct 5. PMID: 17921146
- Fedorova O, Waldsich C, Pyle AM: Group II intron folding under near-physiological conditions: collapsing to the near-native state. J Mol Biol. 2007 Mar 2; 2006 Dec 6. PMID: 17196976
- Pyle AM, Fedorova O, Waldsich C: Folding of group II introns: a model system for large, multidomain RNAs? Trends Biochem Sci. 2007 Mar; 2007 Feb 7. PMID: 17289393
- Waldsich C, Pyle AM: A folding control element for tertiary collapse of a group II intron ribozyme. Nat Struct Mol Biol. 2007 Jan; 2006 Dec 3. PMID: 17143279
- Wagner GP, Pyle AM: Tinkering with transcription factor proteins: the role of transcription factor adaptation in developmental evolution. Novartis Found Symp. 2007. PMID: 17710850
- Solem A, Zingler N, Pyle AM: A DEAD protein that activates intron self-splicing without unwinding RNA. Mol Cell. 2006 Nov 17. PMID: 17188036
- Beran RK, Bruno MM, Bowers HA, Jankowsky E, Pyle AM: Robust translocation along a molecular monorail: the NS3 helicase from hepatitis C virus traverses unusually large disruptions in its track. J Mol Biol. 2006 May 12; 2006 Mar 20. PMID: 16569413
- Su LJ, Waldsich C, Pyle AM: An obligate intermediate along the slow folding pathway of a group II intron ribozyme. Nucleic Acids Res. 2005; 2005 Nov 27. PMID: 16314300
- Fedorova O, Pyle AM: Linking the group II intron catalytic domains: tertiary contacts and structural features of domain 3. EMBO J. 2005 Nov 16; 2005 Oct 27. PMID: 16252007
- Pyle AM: Capping by branching: a new ribozyme makes tiny lariats. Science. 2005 Sep 2. PMID: 16141065
- Serebrov V, Pyle AM: Periodic cycles of RNA unwinding and pausing by hepatitis C virus NS3 helicase. Nature. 2004 Jul 22. PMID: 15269774
- Fedorova O, Mitros T, Pyle AM: Domains 2 and 3 interact to form critical elements of the group II intron active site. J Mol Biol. 2003 Jul 4. PMID: 12823961
- Adamidi C, Fedorova O, Pyle AM: A group II intron inserted into a bacterial heat-shock operon shows autocatalytic activity and unusual thermostability. Biochemistry. 2003 Apr 1. PMID: 12653544