Article Figures & Data
Figures
- Figure 1. CSDE1 interacts with different miRISC in animals.
(A) Western blot analysis of miRNA pull-downs using 2′-O-methyl–biotinylated oligonucleotides complementary to listed miRNAs from the lysate of mESCs (V6.5), NIH3T3, and HEK293T cells. Representative immunoblots of four independent experiments are shown. (B) Left panel: 2′-O-methyl pull-down of multiple miRNAs from Drosophila embryos extracts followed by Western blot detection of UNR and Drosophila AGO1 (dAGO1). Right panel: co-immunoprecipitation of UNR and dAGO1. The data are representative of three independent experiments.
- Figure 2. CSDE1 interacts with AGO2 through its N-terminal domains.
(A) Immunoprecipitation of endogenous AGO2 using anti-AGO2 antibody (left) and miR-20a-5p miRNA pull-down (PD) from the lysate of NIH3T3 cells, before (−) and after (+) RNases treatment. The samples were run on the SDS–PAGE gel and probed with the antibodies indicated. Same cell extracts were used to perform both immunoprecipitation (IP) of AGO2 and miR-20a-5p pull-down (PD). Representative data of two independent experiments are shown. (B) Schematics of the deletion mutants of CSDE1 used in the study. (C) Immunoprecipitation of CSDE1 using anti-FLAG antibody from the lysate of HEK293T cells transiently expressing FLAG-tagged wild-type (WT) and deletion mutants of CSDE1. The immunoprecipitates were run on the SDS–PAGE gel and the proteins indicated were probed. β-actin was used as a loading control. The data are representative of four independent experiments. Migration of the molecular weight marker is indicated (kD). The star (*) denotes nonspecific band.
- Figure 3. CSDE1 influences luciferase activity of miRNA reporter constructs.
(A) Upper panel: relative luciferase levels (Renilla/firefly) of miRNA reporter constructs with “0” (left) and “8” (right) let-7 binding sites detected upon Control and CSDE1 knockdown conditions in NIH3T3 cells. Lower panel: Western blot confirming the expression of CSDE1 in Control (siCtrl) versus CSDE1 knockdown (siCSDE1) conditions in NIH3T3 cells. (B) Upper panel: relative luciferase levels (Renilla/firefly) of miRNA reporter construct with “8” binding sites in shCSDE1 NIH3T3 cells transiently expressing the deletion mutants of CSDE1 as indicated. Lower panel: representative figure for the Western blot confirming the expression of FLAG-tagged CSDE1 and its mutants in shCSDE1 NIH3T3 cells used for the luciferase assays. (C) Western blot confirming the expression of CCND1 in shCSDE1 HEK293T cells transiently expressing the deletion mutants of CSDE1 as indicated. β-actin was used as a loading control. Immunoblots representative of two independent experiments are shown. In (A, B), data are presented as mean ± SEM. (*P < 0.03, **P < 0.01 from three independent experiments; two tailed t test).
- Figure S1. CSDE1 does not influence reporter expression in tethering assays.
(A, B) Top: schematics of reporter constructs with (A) or without (B) poly(A) signal at the 3′ end of the mRNA. Bottom: bar diagrams representing the relative luciferase values (Renilla/firefly) of respective constructs in HeLa cells transiently expressing lambda N-HA–tagged CSDE1, LacZ (negative control), silencing domain of TNRC6 (SD_TNRC6), and TTP (positive controls). (C) Representative Western blot confirming the expression of proteins indicated in HeLa cells used for the tethering assays. α-tubulin was used as the loading control. * denotes the band for respective protein expression. (D) Bar diagram representing the relative luciferase values (Renilla/firefly) of the construct with terminal poly (A) tail in shCtrl and shCSDE1 HEK293T cells transiently expressing lambda N-HA–tagged LacZ and AGO2. (E) Representative Western blot confirming the expression of proteins indicated in shCtrl and shCSDE1 HEK293T cells used for the tethering assays. β-actin was used as a loading control. Data in (A, B, C, D) are presented as mean ± SEM. (*P < 0.03, n.s. P > 0.05 from three independent experiments; two tailed t test).
- Figure S2. CSDE1 is dispensable for the assembly of miRISC.
(A) Top: relative miRNA levels of miR-20a-5p in total RNA and IPed RNA of AGO2 immunoprecipitation from P19 stem cell lysate. Trizol extraction method was used to isolate both the RNAs and RT-qPCR were used to quantify the miRNA levels. Bottom: Western blot confirming the knockdown of CSDE1 and efficiency of AGO2 immunoprecipitation. β-actin was used as a loading control. The data are presented as mean ± SEM (n.s. P > 0.05 from three independent experiments; two tailed t test). (B) Immunoprecipitation of CSDE1 was performed using anti-CSDE1 antibody with 2–4 mg of protein extracts prepared from HEK293T cells while IgG was used as a control. The immunoprecipitates were run on the SDS–PAGE gel and probed for the indicated proteins. * denotes the band for AGO2 protein. (C) miRNA pull-down (PD) using 2′-O-methyl–biotinylated oligonucleotides complementary to miR-20a-5p and immunoprecipitation of AGO2 using anti-AGO2 antibody were performed from the lysate of P19 stem cells under Control (siCtrl) and CSDE1 knockdown (siCSDE1) conditions. The samples were run on the SDS–PAGE gel and probed with the antibodies indicated. Both miRNA PD and IP of AGO2 were performed from the same cell extracts but run on different gels. The data in (B, C) are representative of two independent experiments.
- Figure 4. CSDE1 mediates interaction between AGO2 and LSM14A.
(A) Immunoprecipitation of AGO2 and CSDE1 using respective antibodies from NIH3T3 cells while IgG was used as a control. The immunoprecipitates were run on the SDS–PAGE gel and probed for the indicated proteins. (B) Immunoprecipitation (IP) of AGO2 using anti-AGO2 antibody was performed from the lysate of P19 stem cells under Control (siCtrl) and CSDE1 knockdown (siCSDE1) conditions. The samples were run on the SDS–PAGE gel and probed with the antibodies indicated. α-tubulin was used as a loading control. The IP and input data were generated from the same cell extracts. (A, B) The data are representative of two independent experiments. (C) Immunoprecipitation (IP) of CSDE1 was performed using anti-FLAG antibody from the lysate of HEK293T cells transiently expressing FLAG-tagged wild-type (WT) and deletion mutants of CSDE1. The immunoprecipitates were run on the SDS–PAGE gel and the proteins indicated were probed. Migration of the molecular weight marker is indicated (kD). β-actin was used as a loading control. The data are representative of four independent experiments.
- Figure S3. LSM14A is dispensable for AGO2 interaction with CSDE1.
Immunoprecipitation of endogenous CSDE1 from the lysate of NIH3T3 cells of Control (siCtrl) and LSM14A (siLSM14A) knockdown conditions. The immunoprecipitates were then run on the SDS–PAGE gel and probed for the indicated proteins. The IP and input data were generated from the same cell extracts.
- Figure S4. CSDE1 interaction with CCR4–NOT and decapping factors.
(A) Immunoprecipitation of HA-AGO2 using anti-HA antibody from the lysate of shCtrl and shCSDE1 HEK293T cells transiently expressing HA-AGO2 and V5-CCR4. The immunoprecipitates were run on the SDS–PAGE gel and probed for the indicated proteins. The IP and input data were generated from the same cell extracts. (B) Immunoprecipitation of endogenous CNOT1 from the lysate of P19 stem cells of Control (siCtrl) and CSDE1 (siCSDE1) knockdown conditions. The immunoprecipitates were run on the SDS–PAGE gel and probed for the indicated proteins. The IP and input data were generated from the same cell extracts. The data in (A, B) are representative of two independent experiments. (C) Immunoprecipitation of CSDE1 was performed using anti-CSDE1 antibody from NIH3T3 cells while IgG was used as a control. The immunoprecipitates were run on the SDS–PAGE gel and probed for the indicated proteins.
- Figure 5. CSDE1 associates with mRNA decapping machinery.
(A) Immunoprecipitation of endogenous CSDE1 using anti-CSDE1 antibody from the lysate of HEK293T cells, before (−) and after (+) RNases treatment. The samples were run on the SDS–PAGE gel and probed with the antibodies indicated. The data representative of two independent experiments are shown. (B) Immunoprecipitation of CSDE1 was performed using anti-FLAG antibody from the lysate prepared from shCSDE1 HEK293T cells transiently expressing FLAG-tagged wild-type (WT) and deletion mutants of CSDE1. The immunoprecipitates were run on the SDS–PAGE gels and the proteins indicated were probed. β-actin was used as a loading control. The data are representative of four independent experiments.
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