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

Figure 1

TTSa-RNAs are associated with nuclear AGO1 and AGO2. (b) Coverage of AGO1- and AGO2-immunoprecipitated TTSa-RNAs (HeLa S3) and FLAG:AGO2-immunoprecipitated TTSa-RNAs (HEK293) around GENCODE v25 annotated TTSs. Red and blue represent sRNAs in the sense and antisense orientation with respect to gene transcription, respectively. (c) Overlap of genes giving rise to AGO1- and AGO2-associated TTSa-RNAs. (d) Purified nuclei from HeLaS3 cells were extracted with a forcing urea/detergent buffer to yield a soluble nuclear extract (SNE) and chromatin pellet extract (CPE). SNE and CPE were analyzed by Western blot for the presence of chromatin-associated proteins (BAF-155 and H3) and AGO2. (e) RNA was isolated from HeLaS3 SNE and CPE and analyzed by qRT-PCR for the detection of ACTB mRNA (as a non-chromatin-associated RNA), MALAT-1 (as a chromatin-associated RNA), miR-21, RNU44, and 5 selected TTSa-RNAs (). Synthetic ath-miR159a was added to SNE and CPE fractions before RNA isolation and subsequently used for qRT-PCR normalization.