Journal: Nucleic acids research

Article Title: Domain specific mutations in dyskerin disrupt 3' end processing of scaRNA13.

doi: 10.1093/nar/gkac706

Figure Lengend Snippet: Figure 3. CRISPR-Cas9-mediated mutagenesis of the dyskerin N-terminal extension (NTE) disrupts 3′ end maturation of scaRNA13. (A) Schematic depicting mutagenesis of the NTE domain (amino acids 35–48) of dyskerin via CRISPR-Cas9 RNP targeting of DKC1 in normal male iPSCs, and the isolated clones under study: unmodified DKC1 locus (no indel; clones 1 and 2), in-frame deletion / mutation of one or more amino acids (clones 3–4 and 6–8), or loss of exon 3 with a large heterologous in-frame insertion (clone 5). (B) scaRNA13 RNA levels in DKC1 NTE-mutant iPSC clones as in (A) by northern blot. 18S rRNA is shown as loading control.n = 3 independent experiments, one representative image is shown. (C) Quantification of relative scaRNA13 levels in DKC1 NTE-mutant iPSC clones as in (B) compared to WT iPSCs, normalized to 18S rRNA. Mean ± S.D. is shown for n = 3 biological replicates. Statistics: ****P < 0.0001, one-way ANOVA. (D) scaRNA13 3′ end profiles in DKC1 NTE-mutant iPSC clones as in (A) using 3′ RACE. n = 3 independent experiments, one representative image is shown. (E) scaRNA13 3′ RACE PCR products from three iPSC clones [1 (DKC1 NTE-no indel), 5 and 6 (DKC1 NTE-mutant)] were subjected to deep sequencing and aligned to the scaRNA13 gene. The relative proportions of mature scaRNA13 and extended scaRNA13 RNA species are depicted. Oligo(A) additions of any length (n) are in solid red. (F) Quantitation of cumulative scaRNA13 oligo- adenylated species of clone 5 and clone 6 as representative of mutant-NTE dyskerin versus clone 1 (no indel) as control. (G) scaRNA13 3′ end profiles by RACE (left) and RNA levels by northern blot (right) in WT iPSCs with transient shRNA-mediated knock-down of DKC1 versus control. n = 3 independent experiments, one representative image is shown. (H) scaRNA13 3′ end profiles by RACE (left) and RNA levels by northern blot (right) in del37L patient iPSCs, comparing original iPSCs (Parent) to those in which the DKC1 locus underwent CRISPR-Cas9-mediated homology-directed repair (Repair). n = 3 independent experiments, one representative image is shown.

Article Snippet: Detection of PAPD5 and DKC1 was done using anti-PAPD5 antibodies (Atlas antibodies, HPA042968, 1:1000) and anti-DKC1 antibodies (SantaCruz Biotechnology, sc-48794), respectively.

Techniques: CRISPR, Mutagenesis, Isolation, Clone Assay, Northern Blot, Control, Sequencing, Quantitation Assay, shRNA, Knockdown

Journal: Nucleic acids research

Article Title: Domain specific mutations in dyskerin disrupt 3' end processing of scaRNA13.

doi: 10.1093/nar/gkac706

Figure Lengend Snippet: Figure 4. PAPD5 inactivation augments scaRNA13 3′ end processing in DKC1 NTE-mutant iPSCs. (A) scaRNA13 RNA levels by northern blot in three DKC1-mutant DC patients and their respective CRISPR PAPD5 knockout clones (KO). Normal iPSCs (WT) shown as a baseline. Inverted image of ethidium bromide staining of 18S rRNA is shown as a loading control. (B) Quantification of relative scaRNA13 levels in response to PAPD5 KO in patient iPSCs in (A), normalized to 18S rRNA. Mean ± S.D. is shown for n = 3 biological replicates. Statistics: ns- not significant, **P < 0.005, 2way ANOVA. (C) Pairwise comparisons of scaRNA13 3′ end profiles in control vs CRISPR PAPD5 knockout clones of three DC patients with DKC1 mutations in different domains of dyskerin (see Figure 2A), using 3′ RACE. n = 3 independent experiments, one representative image is shown. (D) scaRNA13 3′ RACE profiles in five DKC1 NTE-engineered iPSC clones (see Figure 3A), after seven days of RG7834 (+) versus DMSO (–) treatment. n = 3 independent experiments, one representative image is shown. (E) scaRNA13 3′ RACE PCR products from iPSCs as in (C) and (D) were subjected to deep sequencing and aligned to the scaRNA13 gene. The relative proportions of mature scaRNA13 and extended scaRNA13 RNA species were determined. Graph shows quantitation of cumulative scaRNA13 oligo-adenylated species in seven different DKC1 NTE-mutant iPSCs treated with and without RG7834, compared to three iPSCs with intact DKC1 NTE as controls. Statistics: one-way ANOVA ns: not significant, **P < 0.05, ***P < 0.001. (F) scaRNA13 RNA levels by northern blot in DKC1 NTE-mutant iPSC clones after seven days of RG7834 (+) versus DMSO (–) treatment. Clone 2 with unmodified (no indel) DKC1 NTE represents a baseline (Ctrl). (G) Quantification of relative scaRNA13 levels normalized to 18S rRNA in different DKC1 NTE-mutant iPSC clones in response to PAPD5 inhibitor treatment, compared to control clone 2 (DKC1 NTE no indel (Ctrl)) as shown in (F). Mean ± S.D. is shown for five independent DKC1 NTE-mutant clones. Statistics: ns = not significant, **P < 0.005, ****P < 0.0001, one-way ANOVA.

Article Snippet: Detection of PAPD5 and DKC1 was done using anti-PAPD5 antibodies (Atlas antibodies, HPA042968, 1:1000) and anti-DKC1 antibodies (SantaCruz Biotechnology, sc-48794), respectively.

Techniques: Mutagenesis, Northern Blot, CRISPR, Knock-Out, Clone Assay, Staining, Control, Sequencing, Quantitation Assay

Journal: Nucleic acids research

Article Title: Domain specific mutations in dyskerin disrupt 3' end processing of scaRNA13.

doi: 10.1093/nar/gkac706

Figure Lengend Snippet: Figure 5. The NTE domain is implicated in interactions between dyskerin molecules and the 3′ end of bound RNAs in H/ACA RNPs. (A) Dyskerin-TERC interface within the H/ACA RNP lobe of the human telomerase cryo-EM structure shown in a cartoon representation. 3′ terminal nucleotides 450 and 451 of TERC, which are not resolved in the cryo-EM structure, are represented with a dashed line (red). Left: Anti-parallel like interaction surface formed between the N-terminal regions of 5′ dyskerin (cyan; F35-T48 shown in sticks) and 3′ dyskerin (green; subset of residues within V64-P71 shown in sticks) that protects the 3′ end of TERC is highlighted. Right: Magnified views of the interaction between the N-termini of the two dyskerin molecules where residues mutated in DKC1 NTE-mutant iPSC clone 3 (see Figure 3A) are shown as red sticks. (B) scaRNA13 3′ end profiles in DKC1 p.T49M mutant patient iPSCs compared to normal (WT). n = 3 independent experiments, one representative image is shown. (C) scaRNA13 RNA levels by northern blot of samples in (B). Inverted image of ethidium bromide staining of 18S rRNA is shown as a loading control. n = 3 independent experiments, one representative image is shown. (D) TERC levels by northern blot in CRISPR DKC1 iPSC clone 2 (NTE-no indel) and clone 5 (NTE-mutant), comparing cells in which TERC has been stably overexpressed versus empty vector (Ctrl). n = 2 independent experiments, one representative image is shown. (E) scaRNA13 levels by northern blot in DKC1 NTE-mutant iPSC clone 5, where scaRNA13 has been stably overexpressed using a transposon-based expression vector (scaRNA13) versus control (Ctrl), followed by seven days of RG7834 (+) versus DMSO (–) treatment. DKC1DKC1iPSC clone 2 (NTE-no indel) is shown as a control. (F) Quantification and comparison of relative normalized scaRNA13 levels in (E). Mean ± S.D. is shown for two DKC1 NTE-mutant clones. Statistics: ns- not significant, *P < 0.05, ****P < 0.0001, one-way ANOVA. (G) Schematics of scaRNA13 predicted secondary structures after genome-engineering. First: Full-length scaRNA13 secondary structure showing tandem, duplex hinge-hairpin-hinge box H and ACA domains. Second: Schematic of predicted mature 3′ half of scaRNA13 (dubbed 5′del-scaRNA13). Third: Schematic of the predicted mature 5′ half of scaRNA13 (dubbed 3′del-scaRNA13). Fourth: Schematic of box ACA1 mutant scaRNA13 (dubbed ACA1-scaRNA13). (H) scaRNA13 3′ end profiles by RACE in WT and del37L patient iPSCs clones carrying CRISPR-Cas9 engineered mutations in the endogenous scaRNA13 locus (predicted product in parentheses): Ctrl (full-length scaRNA13), 5′del (3′ half scaRNA13); 3′del (5′ half scaRNA13), KO (null; biallelic deletion of scaRNA13 residues 21–273), 5′+3′trans (compound heterozygous; 5′del- and 3′del-scaRNA13), ΔACA1 (3′ half scaRNA13, due to disruption of 5′ box ACA1). n = 3 independent experiments, one representative image is shown. (I) Northern blot showing transcript levels and sizes using a probe targeting the 3′ half of scaRNA13 (5’del-scaRNA13), in engineered clones in (H). n = 2 independent experiments, one representative image is shown.

Article Snippet: Detection of PAPD5 and DKC1 was done using anti-PAPD5 antibodies (Atlas antibodies, HPA042968, 1:1000) and anti-DKC1 antibodies (SantaCruz Biotechnology, sc-48794), respectively.

Techniques: Cryo-EM Sample Prep, Mutagenesis, Northern Blot, Staining, Control, CRISPR, Stable Transfection, Plasmid Preparation, Expressing, Comparison, Clone Assay, Disruption

Journal: American Journal of Cancer Research

Article Title: Comprehensive analysis identifies long non-coding RNA RNASEH1-AS1 as a potential prognostic biomarker and oncogenic target in hepatocellular carcinoma

doi: 10.62347/JPHF4071

Figure Lengend Snippet: RNASEH1-AS1 is regulated by DKC1 and mediates its cancer-promoting effects. (A) The effects of RNASEH1-AS1 knockdown on the expression of four hub genes. (B) The effects of knockdown of four hub genes on the expression of RNASEH1-AS1. Knockdown of RNASEH1-AS1 attenuates the promoting effects of DKC1 overexpression on HCC cell proliferation (C, D), migration (E) and invasion (F). (G) RIP assay showed that RNASEH1-AS1 can be significantly enriched by the antibody of DKC1. (H) RNA pull-down assay showed that DKC1 can be pulled down by the biotin-labeled RNASEH1-AS1 but not its antisense probe. (I) RNA stability assay revealed that the degradation rate of RNASEH1-AS1 was accelerated after DKC1 knockdown. Scale bar =75 μm. *P<0.05, **P<0.01.

Article Snippet: The DKC1 was detected via western blot using anti-DKC1 antibody (1:1000; Cloud-Clone, PAC446Hu01).

Techniques: Expressing, Over Expression, Migration, Pull Down Assay, Labeling, Stability Assay