Anna Marie Pyle

The hepatitis C viral NS3 protein is a processive DNA helicase with cofactor enhanced RNA unwinding

The RNA helicase/protease NS3 plays a central role in the RNA replication of hepatitis C virus (HCV), a cytoplasmic RNA virus that represents a major worldwide health problem. NS3 is, therefore, an important drug target in the effort to combat HCV. Most work has focused on the protease, rather than the helicase, activities of the enzyme. In order to further characterize NS3 helicase activity, we evaluated individual stages of duplex unwinding by NS3 alone and in complex with cofactor NS4A. Despite a putative replicative role in RNA unwinding, we found that NS3 alone is a surprisingly poor helicase on RNA, but that RNA activity is promoted by cofactor NS4A. In contrast, NS3 alone is a highly processive helicase on DNA. Phylogenetic analysis suggests that this robust DNA helicase activity is not vestigial and may have specifically evolved in HCV. Given that HCV has no replicative DNA intermediate, these findings suggest that NS3 may have the capacity to affect host DNA.

The architectural organization and mechanistic function of group II intron structural elements.

Group II introns are large, self-splicing RNAs and mobile genetic elements that provide good model systems for studies of RNA folding. The structures and mechanistic functions of individual domains are being elucidated, and long-range tertiary interactions between the domains are being identified, thus helping to define the three-dimensional architecture of the intron.

The DEAH-box protein PRP22 is an ATPase that mediates ATP-dependent mRNA release from the spliceosome and unwinds RNA duplexes.

Of the proteins required for pre‐mRNA splicing, at least four, the DEAH‐box proteins, are closely related due to the presence of a central ‘RNA helicase‐like’ region, and extended homology through a large portion of the protein. A major unresolved question is the function of these proteins. Indirect evidence suggests that several of these proteins are catalysts for important structural rearrangements in the spliceosome. However, the mechanism for the proposed alterations is presently unknown. We present evidence that PRP22, a DEAH‐box protein required for mRNA release from the spliceosome, unwinds RNA duplexes in a concentration‐ and ATP‐dependent manner. This demonstrates that PRP22 can modify RNA structure directly. We also show that the PRP22‐dependent release of mRNA from the spliceosome is an ATP‐dependent process and that recombinant PRP22 is an ATPase. Non‐hydrolyzable ATP analogs did not substitute for ATP in the RNA‐unwinding reaction, suggesting that ATP hydrolysis is required for this reaction. Specific mutation of a putative ATP phosphate‐binding motif in the recombinant protein eliminated the ATPase and RNA‐unwinding capacity. Significantly, these data suggest that the DEAH‐box proteins act directly on RNA substrates within the spliceosome.

Catalytic Role of 2′-Hydroxyl Groups Within a Group II Intron Active Site

Domain 5 is an essential active-site component of group II intron ribozymes. The role of backbone substituents in D5 function was explored through synthesis of a series of derivatives containing deoxynucleotides at each position along the D5 strand. Kinetic screens revealed that eight 2′-hydroxyl groups were likely to be critical for activity of D5. Through two separate methods, including competitive inhibition and direct kinetic analysis, effects on binding and chemistry were distinguished. Depending on their function, important 2′-hydroxyl groups lie on opposite faces of the molecule, defining distinct loci for molecular recognition and catalysis by D5.

Group II intron splicing in vivo by first-step hydrolysis

Group I, group II and spliceosomal introns splice by two sequential transesterification reactions. For both spliceosomal and group II introns, the first-step reaction occurs by nucleophilic attack on the 5′ splice junction by the 2′ hydroxyl of an internal adenosine, forming a 2′–5′ phosphodiester branch in the intron. The second reaction joins the two exons with a 3′–5′ phosphodiester bond and releases intron lariat. In vitro, group II introns can self-splice by an efficient alternative pathway in which the first-step reaction occurs by hydrolysis. The resulting linear splicing intermediate participates in normal second-step reactions, forming spliced exon and linear intron RNAs,. Here we show that the group II intron first-step hydrolysis reaction occurs in vivo in place of transesterification in the mitochondria of yeast strains containing branch-site mutations. As expected, the mutations block branching, but surprisingly still allow accurate splicing. This hydrolysis pathway may have been a step in the evolution of splicing mechanisms.

Ribozyme Catalysis from the Major Groove of Group II Intron Domain 5

The most highly conserved nucleotides in D5, an essential active site component of group II introns, consist of an AGC triad, of which the G is invariant. To understand how this G participates in catalysis, the mechanistic contribution of its functional groups was examined. We observed that the exocyclic amine of G participates in ground state interactions that stabilize D5 binding from the minor groove. In contrast, each major groove heteroatom of the critical G (specifically N7 or O6) is essential for chemistry. Thus, major groove atoms in an RNA helix can participate in catalysis, despite their presumed inaccessibility. N7 or O6 of the critical G could engage in critical tertiary interactions with the rest of the intron or they could, together with phosphate oxygens, serve as a binding site for catalytic metal ions.

Visualizing the solvent-inaccessible core of a group II intron ribozyme.

Group II introns are well recognized for their remarkable catalytic capabilities, but little is known about their three‐dimensional structures. In order to obtain a global view of an active enzyme, hydroxyl radical cleavage was used to define the solvent accessibility along the backbone of a ribozyme derived from group II intron ai5γ. These studies show that a highly homogeneous ribozyme population folds into a catalytically compact structure with an extensively internalized catalytic core. In parallel, a model of the intron core was built based on known tertiary contacts. Although constructed independently of the footprinting data, the model implicates the same elements for involvement in the catalytic core of the intron.

More than one way to splice an RNA: Branching without a bulge and splicing without branching in group II introns

Domain 6 (D6) of group II introns contains a bulged adenosine that serves as the branch-site during self-splicing. In addition to this adenosine, other structural features in D6 are likely to contribute to the efficiency of branching. To understand their role in promoting self-splicing, the branch-site and surrounding nucleotides were mutagenized. Detailed kinetic analysis on the self-splicing efficiency of the mutants revealed several interesting features. First, elimination of the branch-site does not preclude efficient splicing, which takes place instead through a hydrolytic first step. Second, pairing of the branch-site does not eliminate branching, particularly if the adenosine is involved in a mispair. Third, the G-U pairs that often surround group II intron branch-points contribute to the efficiency of branching. These results suggest that there is a strong driving force for promoting self-splicing by group II introns, which employ a versatile set of different mechanisms for ensuring that splicing is successful. In addition, the behavior of these mutants indicates that a bulged adenosine per se is not the important determinant for branch-site recognition in group II introns. Rather, the data suggest that the branch-site adenosine is recognized as a flipped base, a conformation that can be promoted by a variety of different substructures in RNA and DNA.

The Pathway for DNA Recognition and RNA Integration by a Group II Intron Retrotransposon

Group II intron RNPs are mobile genetic elements that attack and invade duplex DNA. In this work, we monitor the invasion reaction in vitro and establish a quantitative kinetic framework for the steps of this complex cascade. We find that target site specificity is achieved after DNA binding, which occurs nonspecifically. RNP searches the bound DNA before undergoing a conformational change that is associated with identification of its specific binding site. The study reveals a facile equilibrium between intron invasion and splicing, indicating that RNP invasion of top strand DNA is a relatively unfavorable event. Group II mobility must therefore depend on the trapping of invasion products, potentially through interaction of the intron-encoded protein with the DNA target and/or initiation of reverse transcription.

Group II introns: highly specific endonucleases with modular structures and diverse catalytic functions

Group II introns are large catalytic RNAs with a remarkable repertoire of reactions. Here we present construct designs and protocols that were used to develop a set of kinetic frameworks for studying the structure and reaction mechanisms of group II introns and ribozymes derived from them. In addition, we discuss application of these systems to structure/function analysis of the ai5gamma group II intron.