ADAR2 regulates RNA stability by modifying access of decay-promoting RNA-binding proteins

Abstract

Adenosine deaminases acting on RNA (ADARs) catalyze the editing of adenosine residues to inosine (A-to-I) within RNA sequences, mostly in the introns and UTRs (un-translated regions). The significance of editing within non-coding regions of RNA is poorly understood. Here, we demonstrate that association of ADAR2 with RNA stabilizes a subset of transcripts. ADAR2 interacts with and edits the 3΄UTR of nuclear-retained Cat2 transcribed nuclear RNA (Ctn RNA). In absence of ADAR2, the abundance and half-life of Ctn RNA are significantly reduced. Furthermore, ADAR2-mediated stabilization of Ctn RNA occurred in an editing-independent manner. Unedited Ctn RNA shows enhanced interaction with the RNA-binding proteins HuR and PARN [Poly(A) specific ribonuclease deadenylase]. HuR and PARN destabilize Ctn RNA in absence of ADAR2, indicating that ADAR2 stabilizes Ctn RNA by antagonizing its degradation by PARN and HuR. Transcriptomic analysis identified other RNAs that are regulated by a similar mechanism. In summary, we identify a regulatory mechanism whereby ADAR2 enhances target RNA stability by limiting the interaction of RNA-destabilizing proteins with their cognate substrates.

Figure 1.

ADAR2 edits SINE elements in the 3΄UTR of Ctn RNA. (A and D) Schematic of transcript organization of Ctn RNA, indicating edited adenosines (red) in (A) forward repeat (FwR) and (D) inverted repeat 2 (IR2) respectively. Numbers above each Adenosine indicate its position in the sequence. (B and E) Relative % of unedited versus edited adenosines at each site in (B) FwR and (E) IR2 in WT MEFs and Adar2-KO MEFs. (C and F) Relative % of unedited versus edited adenosines at each site in the (C) FwR and (F) IR2 of transiently expressed Ctn RNA along with ADAR2 (24 h after transfection) in HEK293 cells (in cells where only Ctn RNA was transfected, no editing was observed in majority of sites). X-axis shows the relative position of individual adenosines within the FwR or IR2. Error bars in (B and C) and (E and F) represent the means ± SD of three independent experiments (biological replicates).

Figure 2.

ADAR2 promotes the stability of Ctn RNA. (A) Schematic of mCat2 and Ctn RNA. 3΄UTR-1 primer pair is specific to Ctn RNA, as it has been designed from a region in between IR2 and IR3, which lies downstream the predicted poly(A) site of Cat2 (Please see Supplementary Figure S2 for details of primer location). Arrows indicate approximate primer positions. (B) Relative levels of Ctn RNA in WT and Adar2-KO MEFs, determined by RT-qPCR using 3΄UTR-1 primer. (C) Relative Ctn RNA levels in Ctrl and Adar2 siRNA treated (48 h) transformed MEFs determined by RT-qPCR using 3΄UTR-1 primer. (D) Western blot analysis of ADAR2 in Ctrl and Adar2 siRNA treated (48 h) transformed MEFs respectively. Tubulin is used as a loading control. Measurement of the stability of (Ea and Fa) Ctn RNA and (Eb and Fb) Actin mRNA by RT-qPCR in WT, Adar2-KO MEFs (E) and Adar2 siRNA-treated transformed MEFs (F) in presence of the transcriptional inhibitor Actinomycin D at indicated time points. Half-life of Ctn RNA (t_1/2) in both cells has been indicated. Please note that Actin mRNA does not show significant degradation in the duration of the experiment. Measurement of stability of (Ga) exogenously expressed Ctn RNA in HEK293 cells co-expressed with vector or human Adar2-WT or E396A deaminase-dead mutant of rat Adar2 (38) or RNA binding dead mutant of ADAR2-EAA or Staufen1 and (Gb) Actin mRNA was determined by Actinomycin D chase (after 48 h of transfection) followed by RT-qPCR. To normalize for transfection efficiency, Ctn RNA levels were normalized to GFP mRNA from GFP plasmid, which is co-transfected along with Ctn RNA in all of the experiments. In all RT-qPCR experiments Gapdh mRNA (GAPDH mRNA in HEK293 cells) was used as a normalization control as it did not show gene expression changes with any treatment. Error bars in (B, C, E, F and G) represent mean ± SD of three independent experiments (biological replicates). *P < 0.05, ****P < 0.0001, using Student's t-test.

Figure 3.

HuR destabilizes Ctn RNA in absence of ADAR2. (A) RIP using HuR antibody followed by RT-qPCR to detect Ctn RNA in WT and Adar2-KO MEFs. Normalization of RIP results was carried out by quantifying in parallel the relative levels of Gapdh mRNA in each IP sample. Stability of (Ba) Ctn RNA and (Bb) Actin mRNA in sh-Ctrl and sh-HuR expressed Adar2-KO MEFs. Stability of (Ca) Ctn RNA and (Cb) Actin mRNA in sh-Ctrl and sh-HuR expressed WT MEFs. In all RT-qPCR experiments Gapdh mRNA was used as a normalization control as it did not show changes in its levels with any treatment. Error bars represent mean ± SD of six (A) and three (B and C) independent experiments (biological replicates). Student's t-test was performed for determining statistical significance.

Figure 4.

HuR facilitates the interaction between PARN and Ctn RNA in absence of ADAR2. (A) PARN RIP followed by Ctn RNA RT-qPCR in WT and Adar2-KO MEFs. Normalization of RIP results was carried out by quantifying in parallel the relative levels of Gapdh mRNA in each RIP sample. Decay curves of (Ba) Ctn RNA and (Bb) Actin mRNA in Ctrl and Parn-depleted Adar2-KO MEFs. Decay curves of (Ca) Ctn RNA and (Cb) Actin mRNA in Ctrl and Parn-depleted WT MEFs. (D) PARN RIP followed by RT-qPCR of Ctn RNA in sh-Ctrl and sh-HuR transfected Adar2-KO MEFs. Normalization of RIP results was carried out by quantifying in parallel the relative levels of Gapdh mRNA in each RIP sample. (E) Co-IP of FLAG-HuR and endogenous PARN in MEFs in presence and absence of RNase A. (F) % Ctn RNA levels measured in the PolyA+ and PolyA- fractions of WT and Adar2-KO MEFs by RT-qPCR using 3΄UTR-1 primer. In all RT-qPCR experiments Gapdh mRNA was used as a normalization control as it did not show gene expression changes with any treatment. Error bars represent mean ± SD of three (A–E) and five (F) independent experiments (biological replicates). *P <0.05, **P <0.01 using Student's t-test.

Figure 5.

ADAR2 regulates the stability of various RNAs, including lncRNA Hottip. (A) Heat map of PolyA+ RNA-sequencing of WT and Adar2-KO showing differential gene expression (>2-fold difference). (B) RT-qPCR analysis of representative transcripts downregulated in Adar2-KO MEFs. (C) Plot comparing RNA levels, including that of edited transcripts between WT and Adar2-KO MEFs. The position of Slc7a2 (encodes Ctn RNA and mCat2 mRNA) has been marked in green and indicated by an arrow. (D) Levels of Hottip RNA measured by RNA-seq in WT and Adar2-KO MEFs. (E) Decay rates of Hottip in WT and Adar2-KO MEFs. (F) ADAR2 RIP followed by RT-qPCR to detect interaction between ADAR2 and Hottip in WT MEFs. Normalization of RIP results was carried out by quantifying in parallel the relative levels of Gapdh mRNA in each RIP sample. (G) Levels of Hoxa13 (Hottip target gene) measured by RNA-seq (upper panel) and RT-qPCR (lower panel) in WT and Adar2-KO MEFs (H) Pie chart showing percentage of ADAR2-interacting and non-interacting RNAs (based on ADAR2 RIP in mouse brain cells) (32) that are downregulated in Adar2-KO MEFs. (I–N) ADAR2 RIP followed by RT-qPCR of potential ADAR2-interacting mRNAs in transformed WT MEFs. Normalization of RIP results was carried out by quantifying in parallel the relative levels of Gapdh mRNA in each RIP sample. In all RT-qPCR experiments Gapdh was used as a normalization control as it did not show gene expression changes with any treatment. Error bars represent mean ± SD of three independent experiments (biological replicates). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant using Student's t-test.

Figure 6.

ADAR2 stabilizes RNAs by limiting the binding of RNA destabilizing proteins—HuR and PARN. (A–C) HuR RIP or (D) PARN RIP followed by RT-qPCR to measure the interaction of HuR with Hottip, Agl and Car5b and (D) PARN with Hottip RNAs in WT and Adar2–KO MEFs. Normalization of RIP results was carried out by quantifying in parallel the relative levels of Gapdh mRNA in each RIP sample. (E) RT-qPCR to detect Hottip levels in Ctrl and Parn-siRNA treated Adar2-KO MEFs. (F) Decay curve of Hottip RNA in Ctrl and HuR-depleted Adar2-KO MEFs. (G) Model depicting potential role of ADAR2 in stabilizing a sub-set of RNA by preventing the association of RNA-destabilizing factors to these RNAs. In all RT-qPCR experiments Gapdh was used as a normalization control as it did not show gene expression changes with any treatment. Error bars represent mean ± SD of three independent experiments (biological replicates). Statistical significance was determined by Student's t-test.