Toll-Like Receptor 9 Alternatively Spliced Isoform Negatively Regulates TLR9 Signaling in Teleost Fish

Frank Fang-Yao Lee; Hsiang-Chieh Chuang; Nai-Yu Chen; Govindarajulu Nagarajan; Pinwen Peter Chiou

doi:10.1371/journal.pone.0126388

Toll-Like Receptor 9 Alternatively Spliced Isoform Negatively Regulates TLR9 Signaling in Teleost Fish

PLoS One. 2015 May 8;10(5):e0126388. doi: 10.1371/journal.pone.0126388. eCollection 2015.

Authors

Frank Fang-Yao Lee¹, Hsiang-Chieh Chuang², Nai-Yu Chen³, Govindarajulu Nagarajan³, Pinwen Peter Chiou³

Affiliations

¹ Marine Research Station, Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan; Molecular and Biological Agricultural Sciences, Taiwan International Graduate Program, Academia Sinica, Taipei, Taiwan; Biotechnology Center, National Chung-Hsing University, Taichung, Taiwan.
² Marine Research Station, Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan; Department of Life Science, National Taiwan University, Taipei, Taiwan.
³ Marine Research Station, Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan.

Abstract

Toll-like receptor 9 (TLR9) recognizes and binds unmethylated CpG motifs in DNA, which are found in the genomes of bacteria and DNA viruses. In fish, Tlr9 is highly diverse, with the number of introns ranging from 0 to 4. A fish Tlr9 gene containing two introns has been reported to express two alternatively spliced isoforms, namely gTLR9A (full-length) and gTLR9B (with a truncated C'-terminal signal transducing domain), whose regulation and function remain unclear. Here, we report a unique regulatory mechanism of gTLR9 signaling in orange-spotted grouper (Epinephelus coioides), whose gTlr9 sequence also contains two introns. We demonstrated that the grouper gTlr9 gene indeed has the capacity to produce two gTLR9 isoforms via alternative RNA splicing. We found that gTLR9B could function as a negative regulator to suppress gTLR9 signaling as demonstrated by the suppression of downstream gene expression. Following stimulation with CpG oligodeoxynucleotide (ODN), gTLR9A and gTLR9B were observed to translocate into endosomes and co-localize with ODN and the adaptor protein gMyD88. Both gTLR9A and gTLR9B could interact with gMyD88; however, gTLR9B could not interact with downstream IRAK4 and TRAF6. Further analysis of the expression profile of gTlr9A and gTlr9B upon immune-stimulation revealed that the two isoforms were differentially regulated in a time-dependent manner. Overall, these data suggest that fish TLR9B functions as a negative regulator, and that its temporal expression is mediated by alternative RNA splicing. This has not been observed in mammalian TLR9s and might have been acquired relatively recently in the evolution of fish.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Alternative Splicing*
Animals
Fish Proteins / genetics
Fish Proteins / metabolism*
Perciformes / genetics
Perciformes / metabolism*
Protein Isoforms / analysis
Protein Isoforms / genetics
Protein Isoforms / metabolism
Signal Transduction*
TNF Receptor-Associated Factor 6 / analysis
TNF Receptor-Associated Factor 6 / genetics
TNF Receptor-Associated Factor 6 / metabolism
Toll-Like Receptor 9 / genetics*

Substances

Fish Proteins
Protein Isoforms
TNF Receptor-Associated Factor 6
Toll-Like Receptor 9

Grants and funding

This work was supported by National Science Council (NSC97-2313-B-001-003-MY3; NSC100-2313-B-001-005-MY3) and an Academia Sinica intramural funding to PP Chiou. The National Science Council has been restructured to Ministry of Science and Technology (MOST) since 2014. MOST website: http://www.most.gov.tw. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.