Characterization of Transcription Termination-Associated RNAs: New Insights into their Biogenesis, Tailing, and Expression in Primary Tumors

Figure 2

TTSa-RNA biogenesis is independent of both DICER and AGO2. (a) Size distribution of AGO2-immunoprecipitated TTSa-RNAs from HCT116 and HCT116 Dicer^Ex5 (b) nuclear lysate. (c) Coverage of AGO2-bound TTSa-RNAs around GENCODE v25 annotated human TTSs in parental HCT116 (in red) and HCT116 Dicer^Ex5 subclone (in blue). (d) qPCR analysis of selected TTSa-RNAs in parental HCT116 and HCT116 Dicer^Ex5 subclone (). RNU44 was used for normalization. (e) Microarray analysis (GEO dataset GSE6427) of the CDKNA1, PSMB1, WEE1, BUB3, and YWHAG mRNA expression in parental HCT116 and HCT116 Dicer^Ex5 subclone. (g) Abundance (RPM) of TTSa-RNAs in sRNA libraries generated from whole cell extract of parental HelaS3 and AGO2KO cells. (h) AGO2 has been genetically knocked out (AGO2KO) in HeLaS3 cells by using specific Zinc Finger Nucleases. In these cells, AGO2 is undetectable at protein level (upper panel) and strongly reduced at mRNA level (lower panel). (f) qPCR analysis of selected TTSa-RNAs in parental HeLaS3 and HeLaS3 AGO2 KO subclone (). RNU44 was used for normalization. qPCR data are expressed as mean ± SEM. ( value ≤ 0.05; paired t test).