title: Mechanistic insights into RNase L through use of an MDMX-derived multi-functional protein domain.
abstract: RNase L is part of the innate immune response to viral infection. It is activated by a small oligonucleotide (2-5A) whose synthesis is initiated as part of the interferon response. Binding of 2-5A to the N-terminal regulatory region, the ANK domain, of RNase L activates its ribonuclease activity and results in cleavage of RNA in the cell, which ultimately leads to apoptosis of the infected cell. The mechanism by which 2-5A activates the ribonuclease activity of RNase L is currently unclear but 2-5A has been shown to induce dimerization of RNase L. To investigate the importance of dimerization of RNase L, we developed a 15kDa dimerization-inducing protein domain that was fused to the N-terminus of RNase L. From these studies we provide direct evidence that dimerization of RNase L occurs at physiologically relevant protein concentrations and correlates with activation of ribonuclease activity. We also show that the binding of 2-5A to RNase L promotes dimerization of the ANK domain and suggest how this could transmit a signal to the rest of the protein to activate ribonuclease activity. Finally, we show that the dimerization-inducing domain can be used as a general fusion partner to aid in protein expression and purification.
authors.affiliation: Hoffmann-La Roche Inc., Discovery Technology, Nutley, NJ 07110, USA. cgarvie@broadinstitute.org
authors.forename: Colin W
authors.initials: CW
authors.lastname: Garvie
authors.forename: Keshav
authors.initials: K
authors.lastname: Vasanthavada
authors.forename: Qing
authors.initials: Q
authors.lastname: Xiang
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D017089:Ankyrin Repeat; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004722:Endoribonucleases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism*
chemical_list: D012313:RNA; D000255:Adenosine Triphosphate; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease