Fig 1: ZCCHC8 is required for its 3' end maturation and telomerase function. (A) Compound heterozygous frameshift (fs) mutations introduced using CRISPR/Cas9 in HCT116 pseudodiploid cells. (B) Immunoblot for ZCCHC8 in HCT116-edited cells. (C) Scheme summarizing TR 3' rapid amplification of cDNA ends sequencing (3'RACE-seq). TR 3' ends were generally divided into mature (451 bp) and extended (>451 bp) where extensions are denoted by gray N's. (D) Summary of TR 3'RACE-seq fractions in isogenic ZCCHC8+/+ and ZCCHC8-/- cells. Color-coded key shows four categories of TR forms: mature (451 nt), adenylated (A)n, short genomically extended (g)n (<465 nt), and long genomically extended (>465 nt). Data are mean of three independent 3'RACE-seq analyses from three RNA isolations each from a different aliquot of a single clone. (E) qRT-PCR of extended TR forms beyond the 451 mature end (>20, >51, >784 nt). Data are mean of three independent RNA isolations similar to D. (F) TR Northern blot of edited ZCCHC8+/+ and ZCCHC8-/- cells. (G) Total TR levels by Northern bot (six blots from three RNA isolations). (H) Telomerase activity measured by telomere repeat amplification protocol (TRAP) assay in ZCCHC8+/+, ZCCHC8-/-, and NAF1S329/S329 HCT116 cell extracts. Activity was quantified on serially diluted extracts (1, 1/5, 1/25, and 1/125) against a PCR-amplified internal control (IC). RNase-treated wild-type extract and no template PCR reaction are included as negative controls. (I) Mean TRAP activity of 1/5× diluted extracts (three independent TRAP assays, each from a different lysate). (J) Summary of 3'RACE-seq of TR forms from control and proband's primary skin fibroblasts with speciation as in D. (K) qRT-PCR values of extended TR forms in primary skin fibroblasts as in E, mean of three technical replicates). (L) Amplified TR from input and Myc-ZCCHC8 immunprecipitated fractions (293FT cells) using primers falling within the mature TR sequence. (M) qRT-PCR of extended TR (>51 nt extended beyond the 3' mature TR end) after transfection of tagged ZCCHC8, DIS3, EXOSC10/RRP6, and PARN into HCT116 ZCCHC8-/- cells (three to four independent transfections/experiment). Data are expressed as mean ± SEM (*) P < 0.05; (**) P < 0.01 (Student's t-test, two-sided).
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