Skip to main content
. 2016 May 20;6:26202. doi: 10.1038/srep26202

Figure 2. Primer extension assays to test for Pus1p activity.

Figure 2

Panel (A) shows the sequence and predicted secondary structure67 of mouse cytoplasmic tRNAIle(AAU) and mouse mitochondrial tRNAHis(GUG). The known Ψs at positions 27 and 30 in tRNAIle67 and predicted Ψs at positions 27 and 28 in tRNAHis are indicated by boxes. The additional modified nucleotides shown on tRNAIle are: D, dihydrouridine; I, inosine; m1A, 1-methyladenine; m5C, 5-methylcytidine; m5U, 5-methyluridine; m7G, 7-methylguanidine; m2,2G, N2,N2-dimethylguanidine; m2G, N2-methylguanidine; t6A, N6-threonylcarbamoyladenine71. In panels (B,C) a fluorescently-labeled primer specific for mouse tRNAIle (B) and a primer specific for mouse tRNAHis (C) were was used in primer extension reactions to determine the location of Ψ in samples of mouse kidney total RNA as described in Materials and Methods. In panels (B,C) lanes 5–7 contain wild-type control RNA (0) or RNA samples reacted with 0.042 or 0.167M CMCT (solid triangle), whereas lanes 8–10 are the result when RNA from Pus1−/− mice is used in the reactions. In panel (B) the sequence of tRNAIle in this region is shown in Lanes 1–4. In panel (C) the sequence of tRNAHis is shown in Lanes 1–4. For panels (B,C) the stops to RT that indicate Ψ in the primer extension reactions are shown on the right and positions of the modified uridines are indicated on the left, in the sequences. The band seen even in lanes not treated with CMCT, just below G26 in the sequence (lanes 5–10, panel B), is most likely due to the presence of N2,N2-dimethylguanidine (m2,2G, Fig. 2A) at position 26 in mouse cytoplasmic tRNAIle(AAU)71. This nucleotide modification has been observed by others to be a stop to RT even in untreated RNA72.