FIGURE 6: Both the canonical Poly(A) polymerase Pap1p and non-canonical Poly(A) polymerase Trf4p play a vital role in the polyadenylation of the sncRNAs. Scattered/Bar plot revealing the steady-state levels of 5S, 5.8S, U1, and snR10 RNAs estimated from the 2 ng cDNA samples prepared using random hexanucleotide primers (sky blue bars) or oligo-dT₃₀ anchor primer (indigo blue bars) by RT-qPCR from WT strain (yBD-161) and strains carrying mutations in the RRP6 (rrp6-Δ, yBD-162), PAP1 (pap1-1, yBD-163), RRP6 and PAP1 (pap1-1 rrp6-Δ, yBD-179) (panels A-D) or WT (yBD-263), RRP6 (rrp6-Δ) (yBD-265), TRF4 (trf4-Δ) (yBD-306), TRF5 (trf5-Δ) (yBD-334) genes (panels E-H). The pap1-1 and pap1-1 rrp6-Δ strains were pre-grown at 25°C, followed by splitting the culture into two halves. Half of the culture continued to grow at 25°C for 2 hours and a 2-hour shift to 37°C were performed on the other half of the culture before harvesting them. Total RNA and cDNA isolation from them followed by RT-qPCR reaction carried out as described in materials and methods. SCR1 (in the case of Random Primer) and ACT1 mRNA (in the case of oligo dT₃₀ primer) were used as the internal loading control. Normalized values of each of the ncRNAs in the WT yeast strain were set to 1. Three independent cDNA preparations (biological replicates, n = 3) were used to determine the levels of various ncRNAs. The statistical significance of difference reflected in the ranges of P values estimated from Student’s two-tailed t-tests for a given pair of test strains for every message are presented with the following symbols, *<0.05, **<0.005, and ***<0.001; N, not significant.