Figure 4.

Recapitulation of the formation of tsRNA and circRNA isoforms with IRAlus across their flanking introns. (A) Visualization of one identified TS-circRNA event in the human POLR2A locus from the UCSC genome browser. Green and red arrows indicate the direction of POLR2A transcription and polarity of the three Alu elements in the flanking introns, respectively. (B) The RPM values of POLR2A tsRNA and circRNA isoforms (measured by poly(A)- and non-poly(A)-selected data from the seven examined cell lines). (C) qRT-PCR analyses (similar to Figure 2F and G) of the expression fold changes for the POLR2A TS-circRNA event before and after RNase R treatments (top) and oligo-dT pull down and oligo-dT pull down with RNase R treatments (bottom). The statistical significance was evaluated using the two-tailed t-test. **P < 0.01 and ***P < 0.001. NS, no signal. (D) RTase-free validation of the tsRNA and circRNA isoforms for POLR2A TS-circRNA event by RPA. Total RNA from HeLa was treated by polyA pull-down (tsRNA isoform) and RNase R (circRNA isoform), respectively. RPA was performed by hybridizing ³²P labeled RNA probe in excess to total RNA from HeLa or in vitro transcript containing chimeric junction (size standard). Negative control: probe only. Positive control: the probe hybridized with 100 ng synthesized complementary strand. The arrow indicates the size (295 bp) of the fully protected fragments. The lower band shown in the figure is partially protected probe. (E) Detection of expanded length of POLR2A tsRNA isoform by nanopore long reads. Of note, this sequencing is of the endogenous locus. Blue and orange arrows indicate two different pairs of primers of POLR2A TS-circRNA event. Empty rectangles (E8 and E11) represent the exons located outside the predicted circles of exonic circRNAs (the blue solid rectangles, i.e. E9 and E10). E, exon. (F) Schematic diagrams of egfp expression vectors with various genomic sequences for POLR2A NCL (T1–T7) (41). T1 represents the genomic region for POLR2A NCL RNA (i.e. exons 9 and 10) with its wild-type flanking introns; T2-T7 represent a series of Alu deletions (gray crosses) inserted into the pZW1 expression vector. The green solid rectangles indicate half egfp sequences from the expression vector backbone. EV, empty vector. (G) qRT-PCR analysis of expression fold changes relative to T1 circRNA expression (with RNase R treatment, left) and T1 tsRNA expression (with oligo-dT pull down, right). The expression levels of T1-T7 circRNAs (or tsRNAs) were normalized by egfp expression before RNase R treatments (or oligo-dT pull down). qRT-PCR experiments were performed in triplicate and repeated twice. Error bars represent the mean values ± one standard deviation. Black arrows indicate the PCR primers for spliced RNAs. (H) Analysis of the recapitulated circRNA (exon skipping; the far left panel), tsRNA (including the transcript fragment of E9-E10-E9-E10; the middle panel), and co-linear (including the transcript fragment of E9–E10; the far right panel) isoform expression by nanopore long reads. The numbers of mapped nanopore reads for T1–T7 are illustrated in the far right panel.