miR-34c-3p Regulates Protein Kinase A Activity Independent of cAMP by Dicing prkar2b Transcripts in Theileria annulata-Infected Leukocytes

Abstract

MicroRNAs (miRNAs) are small noncoding RNAs that can play critical roles in regulating various cellular processes, including during many parasitic infections. Here, we report a regulatory role for miR-34c-3p in cAMP-independent regulation of host cell protein kinase A (PKA) activity in Theileria annulata-infected bovine leukocytes. We identified prkar2b (cAMP-dependent protein kinase A type II-beta regulatory subunit) as a novel miR-34c-3p target gene and demonstrate how infection-induced upregulation of miR-34c-3p repressed PRKAR2B expression to increase PKA activity. As a result, the disseminating tumorlike phenotype of T. annulata-transformed macrophages is enhanced. Finally, we extend our observations to Plasmodium falciparum-parasitized red blood cells, where infection-induced augmentation in miR-34c-3p levels led to a drop in the amount of prkar2b mRNA and increased PKA activity. Collectively, our findings represent a novel cAMP-independent way of regulating host cell PKA activity in infections by Theileria and Plasmodium parasites. IMPORTANCE Small microRNA levels are altered in many diseases, including those caused by parasites. Here, we describe how infection by two important animal and human parasites, Theileria annulata and Plasmodium falciparum, induce changes in infected host cell miR-34c-3p levels to regulate host cell PKA kinase activity by targeting mammalian prkar2b. Infection-induced changes in miR-34c-3p levels provide a novel epigenetic mechanism for regulating host cell PKA activity independent of fluxes in cAMP to both aggravate tumor dissemination and improve parasite fitness.

Keywords: PKA regulatory subunit; PRKAR2B; Plasmodium falciparum; Theileria; miR-34c-3p; microRNA.

FIG 1

miR-34c-3p is differentially expressed in T. annulata-infected leukocytes. (A) miR-34c-3p levels in uninfected B lymphocytes (BL20) increase when BL20 are infected with T. annulata (TBL20). (B) Levels of miR-34c-3p are high in virulent macrophages and decrease in attenuated macrophages. (C) miR-34c-3p levels in attenuated macrophages increase following their transfection with miR-34c-3p mimic. (D) Endogenous levels of miR-30f are low in virulent (V) macrophages and high in attenuated (A) macrophages. Transfection of attenuated macrophages with the miR-34c-3p mimic (Am) did not significantly change miR-30f levels, demonstrating the specificity of the miR-34c-3p mimic. Data are represented as mean ± SEM. n = 3. ***, P < 0.001 compared to virulent macrophages; ###, P < 0.001 compared to attenuated macrophages. miR-34c-3p levels were estimated using qPCR.

FIG 2

Changes in miR-34c-3p levels impact the dissemination potential of T. annulata-transformed macrophages. The ability of virulent (V) macrophages to traverse Matrigel is greater than that of attenuated (A) macrophages. Transfection of attenuated macrophages with the miR-34c-3p mimic (Am) restored Matrigel traversal to virulent macrophage levels. Data are represented as mean ± SEM. ***, P < 0.001 compared to virulent macrophages; ###, P < 0.001 compared to attenuated macrophages.

FIG 3

prkar2b is a direct miR-34c-3p-target gene. (A) Attenuated macrophages transfected with the miR-34c-3p mimic (Am) downregulate the amount of prkar2b transcript. (B) PRKAR2B protein levels also decrease in attenuated macrophages transfected with the miR-34c-3p mimic. (C) Reflecting greater prkar2b expression in attenuated macrophages, prkar2b-luciferase activity is higher in attenuated (A) than virulent (V) macrophages. Luciferase activity decreases when attenuated macrophages are transfected with the miR-34c-3p mimic (Am). (D) prkar2b mRNA levels were estimated using qRT-PCR in virulent macrophages transfected with either WT or mutant PRKAR2B expression plasmids. Mutation of potential seed sites rendered prkar2b transcripts resistant to miR-34c dicing.

FIG 4

miR-34c-3p-induced drop in PRKAR2B amounts upregulates the PKA kinase activity of T. annulata-infected macrophages. (A) T. annulata-infected virulent (V) macrophages have higher PKA activity than attenuated (A) macrophages, and transfection with the miR-34c-mimic (Am) restores PKA activity in attenuated macrophages. (B) CRE-driven luciferase activity is higher in virulent than in attenuated macrophages. Transfection of attenuated macrophages with the miR-34c-mimic increases CREB transactivation of luciferase. Data are represented as mean ± SEM. n = 3. ***, P < 0.001 compared to virulent macrophages; ###, P < 0.001 compared to attenuated macrophages.

FIG 5

miR-34c-3p targets prkar2b to regulate P. falciparum-infected erythrocyte PKA activity independently of fluxes in cAMP. (A) miR-34c-3p levels drastically increase in infected red blood cells (iRBCs) compared to noninfected red blood cells (RBCs). (B) The level of prkar2b transcripts estimated by qRT-PCR is higher in noninfected RBCs compared to that in P. falciparum-infected RBCs. (C) Infected RBCs (dark gray column) display higher PKA activity than noninfected RBCs (black column). Transfection of noninfected RBCs with the miR-34c mimic (RBCm) leads to an increase in PKA activity (light gray column). (D) Inhibition of miR-34c-3p binding to prkar2b in infected RBCs (iRBCi) decreases PKA activity. Data are represented as mean ± SEM. n = 3. **, P < 0.005; ***, P < 0.001.

FIG 6

Model proposing how miR-34c-3p alters PKA kinase activity by targeting PRKAR2B, independent of fluxes in cAMP. PKA is a heterodimer composed of two catalytic subunits bound to two regulatory subunits. Classical activation of PKA occurs when cAMP binds to regulatory subunits, causing a conformational change that liberates catalytic subunits from an inactive R2/C2 complex. miR-34c-3p can regulate PKA catalytic activity independently of cAMP by dicing prkar2b, leading to reduced PRKAR2B expression and greater PKA catalytic activity toward target proteins in the nucleus and cytosol.