An Introduction to RNA Helicases: Superfamilies, Families, and Major Themes

ECKHARD JANKOWSKY AND MARGARET E. FAIRMAN-WILLIAMS

1.1 RNA Helicases in the Helicase Universe

1.1.1 The Sequence-Based Helicase Classification

In the late 1970s, the term helicase was proposed to describe enzymes that unwound DNA duplexes in an ATP-dependent fashion. In 1988, T.C. Hodgeman and a group around Alexander Gorbalenya and Eugene Koonin noted that proteins with DNA helicase activity contained several highly conserved-sequence motifs that were also found in a number of viral proteins. Among these motifs were the NTPase/ATPase signatures of P-loop proteins, a set of functionally diverse, NTP hydrolysing enzymes that also include G-proteins, the kinesin and myosin motor proteins, ABC transporters, and F-type ATPases. The reports speculated that the helicase-like proteins encoded by RNA viruses could be units of "RNA helicases" that unwound RNA during viral replication, in analogy to the helicases that unwound DNA.

At this time, no viral protein had been shown to have RNA helicase activity. However, a eukaryotic protein, the eukaryotic initiation factor 4A (eIF4A) had been demonstrated to unwind RNA duplexes in an ATP-dependent fashion, and thus qualified as RNA helicase (see also the foreword of this volume). Indeed, the presence of the typical "helicase" sequence motifs in eIF4A and the then "putative" RNA helicase p68 was noted shortly after the reports by Hodgeman and Gorbalenya and colleagues, and extensive similarities between proteins with DNA and RNA helicase activities were highlighted. Early 1989, Patrick Linder and coworkers showed that even more proteins shared extensive sequence similarity to eIF4A and p68 over several conserved-sequence motifs. One of these motifs read, in single letter code, "D-E-A-D", thus marking the birth of the D-E-A-D box.

The following years saw a tremendous increase in the number of proteins containing the characteristic helicase motifs, and in 1993 Gorbalenya and Koonin proposed a systematic, sequence-based classification of helicases. Based on their sequences, proteins containing helicase motifs were divided into three helicase superfamilies (two big and one small) and two smaller helicase families. The helicase superfamilies were further divided into protein families, one of which was comprised of the DEAD-box proteins. Gorbalenya and Koonin also noted that some of the amino acids in the helicase motifs were conserved across all superfamilies, whereas others were specific for only one protein family or superfamily.

This sequence-based helicase classification, along with the definition of the characteristic helicase motifs has proven remarkably robust, and is still valid despite the exponential increase in the number of available protein sequences, and the advent of helicase structures. In fact, the helicase structures confirmed the suitability of the classification by Gorbalenya and Koonin. Exceptional structural conservation is seen within the superfamilies and families, especially in the two large helicase superfamilies 1 and 2. More pronounced differences between the structures in the remaining helicases prompted Singleton et al. in 2007 to amend the helicase classification for these proteins and reassign them to four different superfamilies (3–6).

This most recent helicase systematics thus consists of the superfamilies 1–6 (Figure 1.1). All helicases are P-loop NTPases, and therefore contain the typical Walker A and B sites for NTP binding and hydrolysis (Figure 1.1). Proteins of the superfamily 1 and 2 are characterised by a helicase core formed by two structurally almost identical helicase domains. The conserved helicase sequence motifs are located in both of these helicase core domains. The helicase superfamily 2, by far the largest helicase superfamily in eukaryotes, consists of several well-defined protein families with distinct sequence, structure, and functional signatures (detailed in the Chapters 2–7). Helicases of the superfamilies 3–6 form hexameric toroids. These proteins contain only one helicase domain with overall similarity to, but also notable differences from, the helicase domains of SF1 and 2 helicases.

The classification of helicases in superfamilies and families generally correlates with functional characteristics of the enzymes. For example, proteins of the DEAD-box family employ unwinding mechanisms distinct from other SF2 helicases (see also Chapter 3). DEAH/RHA proteins and viral DExH proteins can hydrolyse different NTPs, while proteins in other SF2 families are generally specific for adenosine triphosphate (Chapters 3–7). These correlations illustrate that the recent helicase classification is a useful foundation for a systematic view of these enzymes. Given that the classification is now based on a considerable number of structures and a large number of sequences, many from completely sequenced genomes, this system is likely to endure.

1.1.2 Helicases that do not Unwind Duplexes? Discrepancies Between Sequence-Based and Functional Helicase Definitions

Based on the presence of the characteristic sequence motifs, a large number of proteins qualify as helicases. Helicases, as defined by sequence, are among the largest protein classes. In eukaryotic RNA metabolism, helicases are the largest group of enzymes. However, following the establishment of the sequence-based helicase classification by Gorbalenya and Koonin, it became clear that many of the proteins classified as "helicases", while generally able to hydrolyse ATP in a nucleic-acid-dependent fashion, did not necessarily unwind DNA or RNA duplexes. Proteins of certain SF2 families, including the SWI/SNF proteins and ATP-dependent restriction endonucleases, displayed no unwinding activity. It became apparent that a helicase, as defined by sequence, was not necessarily a helicase as defined by enzymatic function.

Much as certain DNA "helicases" do not actually unwind duplexes, many RNA helicases may not primarily or perhaps not at all function to unwind RNA duplexes in the cell. Although RNA helicases generally unwind RNA duplexes in vitro, provided...