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.

A bioinformatic analysis of LTR retrotransposons revealed several that may utilize recoding in synthesis of their Gag-Pol, with some being candidates for utilization of transcription slippage (51) . We selected three of these candidates for in vitro testing of TGIRT enzyme slippage during reverse transcription of cassettes. In the two Drosophila melanogaster candidates tested pol was in the -1 frame with respect to gag, whereas in the third candidate, which was from maize (Zea mays), its pol was in the +1 frame with respect to gag. Drosophila candidate Dme1 ChrX 2630566 has the motif 5 -AU6-3 and was tested with a chemically synthetized RNA containing 22 nt 5 and 26 nt 3 to the motif ( Figure 5, A) . Candidate Dme1 Chr3 26087113 has the motif 5 -GA4U4-3 and the chemically synthetized RNA to test it contained 18 nt 5 and 32 nt 3 of the motif (Supplementary Figure S7A and C). To more closely resemble physiological conditions, in these reactions the TGIRT-mediated reverse transcription was performed at room temperature and in low salt buffer (this differed from the 60 • C and higher salt conditions utilized in the switching template experiment above). The RT reaction was performed with a sequence specific primer for each test candidate cassette. Each candidate was tested with 3 specific dNTP ratios (1:1, 100:1 and 1:100) for the dNTP substrate specified by the slippage motif and the RNA base 5 adjacent to the motif. LPE analy-sis showed slippage for the candidate Dme1 ChrX 2630566 having the U6 tract in the RNA template: efficiency and distribution follow the dNTP rule for slippage ( Figure 5B ). Candidate Dme1 Chr3 26087113 showed no slippage (Supplementary Figure S7C ).