RLI

RLI is an essential and highly conserved protein that is required for both eukaryotic translation initiation as well as ribosome biogenesis. The most studied homologues are Rli1p in yeast and Pixie in Drosophila.

Structure
RLI is a 68 kDa cytoplasmic protein found in most eukaryota and archae. Since the crystal structure for RLI has not yet been determined, all that is known has been inferred from protein sequencing. The protein sequences between species is very well conserved, for example Pixie and yeast Rli1p are 66% identical, and Rli1p and human RLI are 67% identical.

RLI belongs to the ABCE family of ATP-binding cassette (ABC) proteins. ABC proteins typically also contain a transmembrane region, and utilize ATP to transport substrates across a membrane, however RLI is unique in that it is a soluble protein that contains ABC domains. RLI has two C-terminal ABC domains; upon binding ATP they form a characteristic “ATP-sandwich,” with two ATP molecules sandwiched between the two dimerized ABC domains. Hydrolysis of ATP allows the dimer to dissociate in a fully reversible process. Incubation of the protein with a non-hydrolyzable ATP analogue or a mutation of the ABC domain causes a complete loss of protein function.

RLI also has a cysteine-rich N-terminal region that is predicted to tightly bind two [4Fe-4S] clusters. Mutation of this region, or depletion of available Fe/S clusters, renders the protein unable to function, and loss of cell viability, making RLI the only known essential cytoplasmic protein dependent on Fe/S cluster biosynthesis in the mitochondria. The function of the Fe/S clusters is unknown, although it has been suggested that they regulate the ABC domains in response to a change in the redox environment, for example in the presence of reactive oxygen species.

Function
RLI and its homologues in yeast and Drosophila have two major identified functions: transcription initiation and ribosome biogenesis. In addition, human RLI is a known inhibitor of RNAse L. This was the fist activity identified and the source of its name (RNAse L Inhibitor).

Translation Initiation
Translation initiation is an essential process required for proper protein expression and cell viability. Rli1p has been found to co-purify with eukaryotic initiation factors, specifically eIF2, eIF5, and eIF3, as well as the 40S subunit of the ribosome. These initiation factors must associate with the ribosome in stoichiometric proportions, while Rli1p is required in catalytic amounts. The following mechanism for the process has been proposed: One ABC domain binds the 40S subunit, while the other binds an initiation factor. Binding of ATP allows for dimerization, which subsequently brings the initiation factor and ribosomal subunit in close enough contact to associate. ATP hydrolysis releases the two substrates and allows the cycle to begin again. This model is similar to one that has been proposed for DNA repair enzymes with ABC domains, in which each domain binds either side of a broken piece of DNA, with hydrolysis allowing the pieces to be brought together and subsequently repaired.

Ribosome Biogenesis
RLI and its homologues are also thought to play a role in ribosome biogenesis, nuclear export, or both. They have been found in the nucleus associated with the 40S and 60S subunits, as well as Hcr1p, a protein required for rRNA processing. It has been shown that the Fe/S clusters are necessary for ribosome biogenesis and/or nuclear export, although the exact mechanism is unknown.

RNAse inhibitor
Human RLI was first identified because of its ability to inhibit RNAse L, which plays a crucial role in antiviral activity in mammals. This cannot account for the conservation of the protein in all other organisms, since only mammals have the RNAse L system. It has been suggested that RLI in lower eukaryotes functions by inhibiting RNAses involved in ribosomal biosynthesis, thereby regulating the process.

Role of Mitochondria
The mitochondria’s energetic and metabolic functions have been established to be non-essential for yeast cell viability. The only function that has been implicated in being necessary for survival is the biosynthesis of Fe/S clusters. RLI is the only known essential cytoplasmic Fe/S protein that is absolutely dependent on the mitochondrial Fe/S synthesis and export system for proper maturation. Rli1p is therefore a novel link between the mitochondria and ribosome function and biosynthesis, and therefore the viability of the cell.