Article Title: Metazoan tRNA introns generate stable circular RNAs in vivo
Figure Lengend Snippet: In vivo expression of stable, heterologous tricRNAs in human cells. ( A ) tricRNA vectors used for circular RNA expression, showing the tricRNA scaffold. The gray arrows represent the A and B boxes of the intragenic tRNA promoters. External 5S and U6 promoter constructs were also generated. ( B ) pTRIC31905 (tRNA:Tyr GUA ) and pTRIC31143 (tRNA:Leu CAA ) native intron vectors were transfected into HeLa cells for 24 h and their expression was measured by qRT-PCR. β-actin mRNA was used as normalization standard. U1 snRNA was used as a negative control (Control). tric31905-c and tric31143-c are convergent primer pairs, whereas tric31905-d and tric31143-d are divergent primer pairs used to measure expression of tric31905 and tric31143, respectively. Standard deviations were calculated from three biological replicates. ( C ) Secondary structures of RNAs expressed from pTRIC-Y and pTRIC-L scaffolds, highlighting the TSEN cleavage sites (arrows), anticodons (blue) and NotI–SacII restriction sites (gray). ( D ) Vectors for two circular Spinach2 constructs, pTRIC-Y:Sp2 (blue) and pTRIC-L:Sp2 (red), were transfected into HeLa cells and expression was determined using qRT-PCR. A control pTRIC31905 native intron vector was also used (black). The three experiments were analyzed using convergent (Spinach2-c) and divergent (Spinach2-d) primer pairs. β-actin mRNA was used as normalization standard and U1 snRNA was used as a negative PCR control (Control). Standard deviations were calculated from three biological replicates. ( E ) In-gel detection of Broccoli-tagged RNAs expressed from three different pTRIC-Y constructs bearing tRNA, U6 sRNA, or 5S rRNA promoters. HEK293T cells were transfected and total RNA was run on a 6% TBE-urea gel. The gel was washed to remove the urea, renatured in buffer, and stained with DFHBI-1T to reveal Broccoli ( right panel), then stained with SYBR Gold to reveal total RNA ( left panel). (RnR) RNase R treatment prior to gel electrophoresis. In vitro transcribed pre-tRNA-Broccoli (5 ng) was used as a positive control (first lane). In the experimental lanes, the upper bands (bracket) correspond to the precursor tRNAs; the lower band (arrow) corresponds to the mature circular Broccoli tricRNA. ( F ) Capping of the pre-tRNA improves production of circular RNAs. Two different U6 snRNA promoter constructs were tested, one that includes the first 4 nt of human U6 snRNA (U6) and a construct that includes the first 27 nt (U6*). HEK293T cells were transfected for the indicated times prior to harvesting total RNA. Electrophoresis, gel-detection, and band labels as described in E .
Article Snippet: RNA samples were electrophoresed through 6% or 10% TBE-urea gels (Life Technologies).
Techniques: In Vivo, Expressing, RNA Expression, Construct, Generated, Cellular Antioxidant Activity Assay, Transfection, Quantitative RT-PCR, Negative Control, Plasmid Preparation, Polymerase Chain Reaction, Staining, Nucleic Acid Electrophoresis, In Vitro, Positive Control, Electrophoresis