Systemic RNA Interference Deficiency-1 (SID-1) Extracellular Domain Selectively Binds Long Double-stranded RNA and Is Required for RNA Transport by SID-1

• Published in: The Journal of biological chemistry Vol. 290; no. 31; pp. 18904 - 18913
• Main Authors: Li, Weiqiang, Koutmou, Kristin S., Leahy, Daniel J., Li, Min
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 31.07.2015; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Animals; Binding, Competitive; Caenorhabditis elegans - metabolism; Caenorhabditis elegans Proteins - chemistry; Caenorhabditis elegans Proteins - genetics; Caenorhabditis elegans Proteins - metabolism; DNA - chemistry; Membrane Proteins - chemistry; Membrane Proteins - genetics; Membrane Proteins - metabolism; Mice; Molecular Sequence Data; Protein Binding; Protein Structure, Tertiary; RNA; RNA Interference; RNA interference (RNAi); RNA Transport; RNA, Double-Stranded - chemistry; RNA, Double-Stranded - metabolism; RNA-binding protein; RNA-protein interaction; SID-1; SIDT1; SIDT2; SID-1; RNA-binding protein; RNA interference (RNAi); RNA; RNA transport; SIDT1; RNA-protein interaction; SIDT2
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M115.658864

During systemic RNA interference (RNAi) in Caenorhabditis elegans, RNA spreads across different cells and tissues in a process that requires the systemic RNA interference deficient-1 (sid-1) gene, which encodes an integral membrane protein. SID-1 acts cell-autonomously and is required for cellular import of interfering RNAs. Heterologous expression of SID-1 in Drosophila Schneider 2 cells enables passive uptake of dsRNA and subsequent soaking RNAi. Previous studies have suggested that SID-1 may serve as an RNA channel, but its precise molecular role remains unclear. To test the hypothesis that SID-1 mediates a direct biochemical recognition of RNA molecule and subsequent permeation, we expressed the extracellular domain (ECD) of SID-1 and purified it to near homogeneity. Recombinant purified SID-1 ECD selectively binds dsRNA but not dsDNA in a length-dependent and sequence-independent manner. Genetic missense mutations in SID-1 ECD causal for deficient systemic RNAi resulted in significant reduction in its affinity for dsRNA. Furthermore, full-length proteins with these mutations decrease SID-1-mediated RNA transport efficiency, providing evidence that dsRNA binding to SID-1 ECD is related to RNA transport. To examine the functional similarity of mammalian homologs of SID-1 (SIDT1 and SIDT2), we expressed and purified mouse SIDT1 and SIDT2 ECDs. We show that they bind long dsRNA in vitro, supportive of dsRNA recognition. In summary, our study illustrates the functional importance of SID-1 ECD as a dsRNA binding domain that contributes to RNA transport. Systemic RNA interference deficiency-1 (SID-1) is a membrane protein required for cellular uptake of RNA in C. elegans. SID-1 extracellular domain selectively binds long dsRNA. Binding affinity between SID-1 ECD and dsRNA is related to RNA transport efficiency by SID-1. This defines SID-1 ECD as a functional domain for dsRNA recognition.