Fig 1: A physical and functional connection between the NEXT and HUSH complexes(A) Schematic representation of the HUSH complex.(B)WB analysis of FLAG IPs from chromatin lysates of WT and MTR4–3F cells. Chromatin input and IP samples were probed with antibodies against FLAG, ZFC3H1, MTR4, MPP8, and H3 (input loading control).(C) As in (B) but using ZCCHC8–3F cells. Membranes were probed with antibodies against FLAG, MPP8, MTR4, and H3 (input loading control).(D) WB analysis of MPP8 IPs from lysates of WT and TASOR-3F cells. IgG IPs were included as a negative control. Lysates from each cell line were split into two, with input samples loaded for each IP. Membranes were probed with antibodies against MPP8, FLAG, ZFC3H1, MTR4, ZCCHC8, and RPLP0 (input loading control).(E) WB analysis of MPP8 IPs from lysates of MPP8–3F-mAID or TASOR-3F-mAID cells either mock or IAA treated (8 h). Input and IP samples were probed with antibodies against MPP8, FLAG, ZCCHC8, MTR4, and RPLP0 (input loading control).(F) WB analysis of MPP8 IPs from lysates of TASOR-3F cells. Lysate extractions and IPs were carried out in increasing NaCl concentrations (0.1–1.0 M) as indicated. Membranes were probed with antibodies against MPP8, ZFC3H1, MTR4, ZCCHC8, FLAG, and Actin (ACTB, input loading control).(G) WB analysis of MPP8 IPs from WT lysates following mock or Benzonase treatment before final elution from beads. IgG IPs serve as a negative control. Lysates were split into two for either MPP8 or IgG IPs, with input samples loaded for each IP. Input and IP samples were probed with antibodies against MPP8, ZCCHC8, MTR4, TASOR, and TUBULIN (input loading control).(H) Metagene (upper) and heatmap (lower) profiles of unique mapped RNA-seq reads from WT, Zcchc8-/-, and Zfc3h1-/- datasets within a 10 kb window centered on MPP8 ChIP peaks. Heatmap rows are sorted by MPP8 peak signal intensities. Coverage of uniquely mapped reads are displayed for + and - strands.(I) Genome browser views of five MPP8 target loci. Displayed tracks include input and MPP8 ChIP-seq data from two replicate experiments as well as RNA-seq data from two replicates of WT, Zcchc8-/-, and Zfc3h1-/- samples. Strand directions (+/-) are noted along with genomic coordinates. TE hosting genes are indicated in parentheses.
Fig 2: MID1 is connected to the mTOR-dependent translation initiation pathwaya Identification of the MID1-interactome. MID1-FLAG was expressed in HEK293T cells and MID1-complexes were purified by immunoprecipitation. MID1-binding proteins were identified by mass spectrometry. The mTOR-dependent translation initiation pathway is shown and the number of proteins identified belonging either to the eukaryotic translation initiation factor complex (eIF complex) or the ribosome are indicated. b Validation of the mass spectrometry results shown in a and Table 1. MID1-FLAG was expressed in HEK293T cells and MID1-complexes were purified by immunoprecipitation (IP FLAG). As negative control, unspecific IgG agarose beads were used (IgG). Immunoprecipitates were analyzed on western blots using specific antibodies to detect MID1-FLAG, eIF3A, eIF4G, RPLP0, RPL5, RPS3. c Effect of ribosome disassembly on the composition of the MID1-complex. MID1-FLAG was expressed in HEK293T cells and immunopurified (IP FLAG) in the presence or absence of high concentrations of EDTA. Immunoprecipitates were analyzed on western blots using specific antibodies for MID1-FLAG, eIF3A, RPLP0, RPL5, RPS3. d To analyze the MID1-complex composition and its dependency on RNA, MID1-FLAG was expressed in HEK293T cells and immunopurified (IP FLAG) in the presence or absence of RNAse. As negative control, unspecific IgG agarose beads were used (IgG). Immunoprecipitates were analyzed on western blots using specific antibodies for MID1-FLAG, eIF3A, eIF4G, RPLP0, RPL5, RPS3. e, f mTOR regulates translation of APP. e In vitro translation of in vitro transcribed APP-mRNA tagged to luciferase in the presence or absence of the mTOR-inhibitor temsirolimus. The level of translated luciferase reporter was measured in a luciferase assay. Columns represent mean values ± SEM. n = 3. *p < 0.01. f Primary neurons were treated with the mTOR-inhibitor temsirolimus. Protein extracts were analyzed on western blots, detecting APP and ß-actin as loading control. Graph shows quantification of western blots, mean values ± SEM. n = 3. *p < 0.01. APP amyloid precursor protein
Fig 3: NEXT and HUSH suppress non-polyadenylated and polyadenylated TE RNAs, respectively(A) qRT-PCR analysis of L1 LINE transcripts from total RNA harvested from OsTIR1-HA, Zcchc8–3F-mAID, Mpp8-3F-mAID, or Zcchc8-/- Mpp8-3F-mAID cell lines either mock or IAA treated (72 h). Data representation as in Figure 1H.(B) WB analysis of lysates from three biological WT replicates and three Zcchc8-/- clonal cell lines. Membranes were probed with antibodies against ZCCHC8, L1ORF1 and Actin (ACTB, loading control).(C) Quantification of L1ORF1 protein levels from the WB in (B). Data show the average value from three replicates, normalized to ACTB levels and plotted as the fold change relative to WT samples. Statistical significance was assessed as in Figure 4C.(D) WB analysis of Mpp8-3F-mAID, Zcchc8-/-, and Zcchc8-/- Mpp8-3F-mAID cell extracts following either mock or IAA treatment (72 h). Membranes were probed with antibodies against ZCCHC8, MPP8, L1ORF1, and RPLP0 (loading control).(E) Metagene profiles of 3' end-seq signals from pA+ and pA+,- 3' end-seq libraries of WT or Zcchc8-/- cells and displayed within a 10 kb window centered on MPP8 ChIP-seq peaks. Forward and reverse strands are plotted independently with replicates plotted separately as indicated in the legend.(F) Genome browser tracks of example upregulated L1 LINEs and LTR RNAs from RNA-seq data generated upon MPP8 or ZCCHC8 depletion. Data from Mpp8-mAID samples, either mock or IAA treated (48 h), are from pA+ selected, un-stranded libraries. Data from WT and Zcchc8-/- cells are from rRNA-depleted, stranded libraries with the relevant strand data represented here. Annotations are displayed as in Figure 1F.(G) qRT-PCR analysis of L1Md_F transcripts from total RNA harvested from samples described in (A). Amplicons were designed to amplify either 5', center, or 3' regions of the L1Md_F2 LINE transcript as indicated in the schematics(H) As in (G) but for L1Md_T transcripts.
Supplier Page from Abcam for Anti-RPLP0 antibody [EP15646]