Structural Biochemistry/Nucleic Acid/RNA/RNA Helicase

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Discovered in the 1980s, RNA helicases are enzymes that use ATP to bind and remodel RNA and ribonucleoprotein complexes (RNPs). Mostly all helicases work and interact with many other proteins inside a multi-component assembly. While it is it unknown how RNA helicases exactly locate their binding sites on the complexes, experiments show that they most likely either bind to cofactors, which then guides them to the complex, or the helicases themselves find the binding sites according to a complex code of features on the RNAs. RNA helicases also play an important role in eukaryotic RNA metabolism and are found in all kingdoms of life. But little is known about them and how they work in the cell. RNA helicases are similar to DNA helicases and share similar functions.

RNA Helicase Classifications[edit]

RNA helicases can also be classified into six superfamilies (SFs). SFs 1 and 2 are comprised of helicases that are non-ring forming. All eukaryotic RNA helicases belong to these superfamilies. SFs 3 to 6 are helicases that can form rings and can be found in bacteria and viruses.

SFs 1 and 2 can be broken down into well-defined helicase families. Each family has distinct structural and functional properties. Six of the families have RNA helicases while the rest consist of DNA helicases. Helicases in SF 1 and 2 have a core made of two similar helicase domains and have at least 12 characteristic sequence motifs at positions in the helicase core. Not all helicases in one family will have the same motifs but they have high sequence conservation. In other families, sequence conservation is low. Across superfamilies, sequence conservation is even lower. This suggests differentiation between DNA and RNA helicases was not an evolutionary force in the classification of helicase families.

The helicase core is also surrounded on either side by C- and N-terminal domains. The terminal domains are essential to the helicase’s cellular specificity because they assist specific complexes in recruiting proteins. They accomplish this through their interactions with other proteins or by recognizing specific nucleic acid sections. Unlike the core’s sequence motifs, the C- and N-terminal domains are not conserved between families. Certain families in SF1 and SF2 are also identifiable by their characteristic beta-hairpin in between the VA and VI motifs of the helicase core. The helicase families who show this beta-hairpin are the Ski2-like, DHeAH/RHA and NS3/NPH-II families. Other families, such the Upf1-like and RIG-I-like families, have noticeable inserts between or within the helicase core domains.

NPH-II helicase, found in vaccinia virus, and NS3 helicase from the hepatitis C virus are two RNA helicases that are essential for viral replication, and have had extensive studies. Both of these helicases load on a 3' single strand of RNA and moves toward the 5' end of the strand. These helicases begin to unwind the RNA through bursts and pauses, beginning at the junction of the single and double strand. During pauses, the helicase could be either preparing to continue unwinding, but it could also dissociate from the RNA. As of the present, there is still little known about the fundamental characteristics of the helicases acting on the RNA.
Source: Li PTX, Vieregg J, Tinoco I Jr. How RNA Unfolds and Refolds. Annu Rev Biochem. 2008;77:77-100.

RNA Helicase Mechanisms[edit]

RNA helicases employ two mechanisms for unwinding: canonical and by local strand separation. Both methods are ATP-dependent because ATP binding is needed not only for the helicase to bind to the duplex but to also keep the two helicase domains together. In both canonical and local strand unwinding, the helicase domains surround the nucleic acid in similar directions and make contact with the RNA’s sugar-phosphate backbone. This allows for complete attachment of the RNA helicase and movement along the RNA by 1nt per ATP consumed. Many translocating helicases can move in bursts of up to 18 nt steps before they perform a rate limiting step, allowing for quick unwinding of the RNA duplex. In local strand unwinding, the bound RNA strand often show bends in its backbone due to the presence of ATP analogs while in canonical unwinding no such bend is exhibited. The bends decrease preference for the duplex structure and most likely represent the RNA conformation of the two strands after the duplex is unwound.

Canonical Duplex Unwinding[edit]

Canonincal Unwinding Mechanism for RNA Helicase

When RNA helicases unwind RNA strands canonically, the RNA helicase attaches itself on the single-stranded region of the RNA strand and then translocates along the bound strand. It has defined direction and can either go 3’ to 5’ or 5’ to 3’ as it displaces the complementary strand. Each translocating step has multiple processes, including ATP binding and hydrolysis. ATP binding and hydrolysis drives the process forward. This type of winding requires strands to have single-stranded regions in a defined polarity with respect to the duplex. The RNA helicases families who are known to perform this mechanism are Upf1-like, Ski2-like, RIG-I-like and DEAH/RHA.

Local Strand Separation[edit]

Duplex Unwinding by Local Strand Separation

Local strand separation occurs when a RNA helicase loads itself directly on a duplex region of the RNA, and uses ATP to separate the strands. Unlike the canonical method, this type of unwinding does not require a single-stranded region with specific orientation nor ATP hydrolysis. ATP binding is sufficient for duplex unwinding to occur, ATP hydrolysis however is needed to successfully detach the helicase from the RNA. Sometimes the enzyme will dissociate before the strands have completely separated because of the strands quickly re-annealing. As the RNA strand gets longer, however, this type of unwinding is unfavorable and inefficient. The DEAD-box family unwinds duplexes this way and can only handle duplexes with 10 to 12 basepairs.

Other Functions[edit]

RNA helicases also have other functions aside from unwinding duplexes. It can also displace proteins on RNAs. This is called RNP remodeling. RNP remodeling appears to be important in how RNA helicase functions since RNAs are usually attached to a protein in vivo. RNP remodeling, however, is not essential in unwinding but also works for helicases that unwind canonically and for DEAD-box proteins. Some helicases can only remove certain proteins, while others can remove a wider variety of proteins.

RNA helicases have also shown to help in the RNA folding process. An example are the RNA helicases who facilitate and regulate RNA folding in fungal mitochondria as RNA chaperones. They should not be confused with protein chaperones which also help in RNA folding. RNA chaperones guide the RNA through the series of folding steps while continually proofreading. It determines if the substrates formed are correct or incorrect. If correct, the process is continued but if incorrect, the substrate is disregarded and the RNA chaperone opens up a new reaction path for RNA folding. Protein chaperones on the other hand catalyze the steps of the folding pathway and help stabilize the subsequent RNA structure.

Other helicase families show activities in regards to the innate immune system. The RIG-I RNA helicase translocates to a RNA duplex but instead of unwinding it, the helicase acts as a pattern recognition receptor and determines if a viral RNA present in the cytoplasm. It detects the viral RNA based upon the long double stranded RNA’s it creates during viral replication. Only viral RNA's are detected because a majority of RNA’s in eukaryotic cells form only short RNA duplexes, which is ideal for the local strand unwinding process.

DEAH/RHA helicases also help in both the separation of a spliced mRNA from the spliceosome and the ligation of the exon to the mRNA. It separates the spliced mRNA from the spliceosome by attaching to the mRNA and moving from the 3’ to 5’ direction, breaking the RNA-RNA and RNA-protein along the way.

References[edit]

Jankowsky, Eckhard. "RNA helicases at work: binding and rearranging." Trends in Biochemical Sciences. xx (2010): 1-11. Web.