Figure 2: hTR is transcribed as an extended molecule that accumulates in PARN-KO cells.

A. Nascent RNAend-Seq of hTR in HeLa and hES cells with tail profiles for nascent (N) and steady-state (SS) transcripts. Cells are pulsed for 1 hour (1H) or 4 hours (4H). Grey shading indicates 95% CI intervals from 2 biologic replicates. Percent of hTR reads composed of oligo-A transcripts plotted for nascent (N) and steady state (SS) HeLa and hES hTR transcripts. Error bars are S.E.M. hTR Reads≥12k.

B. Nascent RNAend-Seq of H/ACA snoRNAs U17a and U68 with tail profiles for nascent and steady-state transcripts. Gray shading indicates 95% CI derived from 2 biologic replicates. Diagram of U17a and U68 intronic loci shows distance to nearest exon. Percent of snoRNA reads composed of oligo-A transcripts is plotted for nascent (N) and steady state (SS) U68 and U17a transcripts. U68 reads≥5k, U17a reads≥7k

C. Western blot (WB) of PARN CRISPR-KO clones and rescued cells. Parental (WT) cells and PARN-KO clones (4,2,6) and PARN-KO clone 4 transduced with lentiviral Flag-GFP (FL-GFP) or Flag-PARN (FL-PARN).

D. Telomere Repeat Amplification Protocol (TRAP) in parental (WT) and PARN-KO (clones 2,4,6) HeLa extract as well as TCAB1-KO extract. Each sample has 1x and 4x dilutions. Buffer indicates no extract, +RNAse indicates RNAse A added to WT extract. Presence of internal control (I.C) indicates successful PCR. TRAP activity relative to WT is quantified (right) and represents the results of 3 experiments. Error bars are S.E.M.

E. qRT-PCR of hTR relative to parental (WT) and normalized to GAPDH in PARN-KO cells (clones 2,4,6) and clone 4 with either GFP or PARN expressed. Error bars are S.E.M.

F. 3’ RACE-Seq of hTR in parental (black), PARN-KO+GFP (green), PARN-KO+PARN cells (grey). Percent of oligo-A hTR reads are plotted. hTR Reads≥160k

G. W.B. of HeLa or PARN-KO clone 4 HeLa cells stably transduced with FL-GFP or FL-PARN cDNA.

H. qRT-PCR of hTR relative to parental (WT+GFP) and normalized to GAPDH in PARN-KO cells with GFP, PARN WT or PARN-mutant cDNA stably expressed.

I. qRT-PCR of extended hTR relative to parental (WT+GFP) and normalized to total hTR in PARN-KO cells with GFP, PARN-WT or PARN-mutant cDNA stably expressed. Position of reverse primers is shown