Stimulation of reverse transcriptase generated cDNAs with specific indels by template RNA structure: retrotransposon, dNTP balance, RT-reagent usage

Penno, Christophe; Kumari, Romika; Baranov, Pavel V.; van Sinderen, Douwe; Atkins, John F.

The first set of experiments was with WTsl-A7 and mutSL-A7constructs. Reactions were performed using three dNTP concentration conditions. RT reactions were performed with 3 dNTP ratio conditions for the substrates: To determine the potential importance of the identity of the RNA template base, C, 5 adjacent to the A7 motif in wtSL-A7 ( = wtSL/C-A7) and MUTsl-A7 ( = MUTsl/C-A7), the C was substituted by G to give the constructs 'wtSL/G-A7' and 'MUTsl/G-A7'. Also in the WT construct a compensatory base substitution was made in the sequence specifying the 5 base of the 5 side of the stem to maintain base pairing ( Figure 4A ). In the MUT construct a corresponding substitution to preclude base pairing was not necessary as its potential partner is already G ( Figure 4B ). The second set of constructs 'wtSL/U-A7' and 'MUTsl/U-A7' is as the first set except for the base adjacent to the motif being C with corresponding compensatory base substitutions (A and U respectively) to maintain (wt), or to abolish (MUT), stem-loop structure formation ( Figure 4C and D). RT reactions were performed using three specific dNTP concentration ratios (1:1, 100:1 and 1:100) for the dNTP substrate specified by the slippage motif and the RNA base adjacent to the motif. LPE analysis showed a similar result as obtained with the WT and mut 'stem-loop' model structure where the last base of the sequence specifying the 3 side of its stem has the base C 5 adjacent to the A7 motif. The RT slippage followed the dNTP ratio 'rules' where higher substrate concentration specified by the slippage motif stimulates base addition ( Figure 4E, lanes 2, 5, 8 and 11 ), and where higher substrate concentration specified by the template base 5 adjacent to the motif, stimulates base omission ( Figure 4E, lanes 3, 6, 9 and 12) . The RT slippage also followed the rules of slippage directionality involving potential formation of the RNA structure 5 of the motif. With the wtSL constructs, base addition is stimulated, and with the MUTsl constructs it is inhibited ( Figure 4E , compare lane sets 1-2 with 7-8, and 4-5 with 10-11). In contrast, slippage omission of at least one base is favored in the ab- PCR product template ('PT7 DNA'). Constructs contain a wild type (wt) G-rich sequence from eIF4A mRNA 5 to the A7 motif. The nt spacing distance (from 0 to 3 nt) is between the last (3 ) G of the G-rich sequence and the A7 motif (bottom). Combination of the G-rich sequence with different spacer lengths was tested as indicated in (B). LPE analysis of the RT-PCR product derived from the cDNA template, and of the PCR product derived from the 'PT7 DNA' template. Standard LPE products (whose synthesis did not involve slippage and reflect the original length of the motif in the chemically synthesized template) are indicated by an orange arrowhead. sence of potential for RNA template stem-loop formation ( Figure 4E , compare lane 9 with 3, and lane 12 with 6). In conclusion, the above result shows that the realignment for the TGIRT enzyme is independent of identity of the base located 5 adjacent to the motif.