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Concerted action of RNase T2 and PLD exonucleases in RNA processing for TLR7 activation

ABSTRACT: Toll-like receptor 7 (TLR7) plays a key role in the recognition of RNA viruses and the subsequent induction of an innate immune response. TLR7 contains two ligand binding pockets that recognize distinct RNA degradation products: Pocket 1 recognizes guanosine nucleotides and pocket 2 coordinates short, pyrimidine-rich RNA fragments, while the engagement of both pockets is required to activate TLR7. How these ligands are generated within the endolysosomal compartment is currently unknown. Using a pDC cell line, we here show that the endonuclease RNase T2 and the 5' exonucleases PLD3 and PLD4 act in concert to produce these ligands. On the one hand, RNase T2 generates 2',3'-cyclophosphate-guanosine terminated RNA fragments upon which PLD exonuclease activity acts to release the terminal 2',3'-cyclic GMP. This molecule serves as the physiological ligand for the first binding pocket. On the other hand, PLD activity is also required to generate short RNA fragments that occupy the second binding pocket of TLR7. While PLD3 and PLD4 exert comparable catalytic activities, the almost exclusive expression of PLD4 in pDCs suggests that PLD4 is the relevant exonuclease for pDC activation in the human system. Biochemical and structural studies of PLD3 and PLD4 show that these enzymes form homodimers with two ligand binding sites. Dimer formation is critical for catalytic activity, and previously identified disease-associated mutants fail to form stable dimers. In conclusion, our data provide a mechanistic basis for the recognition of RNA fragments by TLR7.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Jan Bernd Stöckl

LAB HEAD: Thomas Fröhlich

PROVIDER: PXD045912 | Pride | 2024-06-19

REPOSITORIES: Pride

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Project description:A time course of the effect of overexpressing GPI-PLD on global gene expression in HepG2 cells was compared to control virus. Experiment Overall Design: This experiment compares the global gene expression in HepG2 cells that were either transduced with an adenovirus expressing GPI-phospholipase D or an adenovirus expressing beta galactosidase as a control. Treatment was for 6 or 12 hrs. Four replicates for each condition were used. 10 micrograms of total RNA was assayed per genechip using standard Affymetrix protocols. CONTEXT: Recent studies demonstrated that de novo lipogenesis is increased in patients with nonalcoholic fatty liver disease (NAFLD). Patients with NAFLD also have plasma lipid abnormalities. These lipid abnormalities may in part be related to insulin resistance, which is common in patients with NAFLD. Insulin resistance is associated with alterations in proteins involved in lipid metabolism including glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD), which is involved in triglyceride metabolism. OBJECTIVE: The objective of the study was to determine whether alterations in serum and hepatic levels of GPI-PLD occur in patients with NAFLD. DESIGN AND PATIENTS: We examined the following: 1) levels of serum GPI-PLD in nondiabetics with nonalcoholic steatohepatitis, compared with matched controls; 2) hepatic expression of GPI-PLD mRNA in patients with normal liver or NAFLD; and 3) effect of overexpressing GPI-PLD vs. beta-galactosidase (control) on global gene expression in a human hepatoma cell line. RESULTS: The serum levels of GPI-PLD were significantly higher in patients with nonalcoholic steatohepatitis than in matched controls (119 +/- 24 vs.105 +/- 15 microg/ml, P = 0.047). The hepatic expression of GPI-PLD mRNA was increased nearly 3-fold in NAFLD patients, compared with patients with normal liver (3.1 +/- 2.6 vs. 1.1 +/- 1.0 arbitrary units per microgram total RNA, P = 0.026). Finally, overexpressing GPI-PLD was associated with an increase in de novo lipogenesis genes. CONCLUSIONS: Patients with NAFLD have elevated serum levels and hepatic expression of GPI-PLD, and its overexpression in vitro is associated with increased expression of de novo lipogenesis genes. These results suggest that GPI-PLD may play a role in the pathogenesis of NAFLD and/or its metabolic features and warrants further investigation.

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Concerted action of RNase T2 and PLD exonucleases in RNA processing for TLR7 activation

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