Determination of Sequence Requirements for Pseudouridylation of RPL11a and TEF1 mRNAs.

<p>A. 55 nt RNA constructs containing 55 nt (R55), 20 nt (R20), 10 nt (R10), or 6 nt (R6) of sequence corresponding to the sequence around the site of modification in RPL11a mRNA were <i>in vitro</i> transcribed and modified with purified Pus1p. The RNA was then used for the CMCT-RT stop experiment, and the data were plotted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110799#pone-0110799-g003" target="_blank">figure 3A</a>. B. For these modification experiments, the proportion of reverse transcription that stopped at the site of pseudouridylation was plotted for both +CMCT (blue) and mock (red) conditions as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110799#pone-0110799-g003" target="_blank">figure 3B</a>. C. The sequences of the constructs used. For R20, R10, and R6, the sequences conserved from RPL11a are in blue. D. 55 nt constructs of containing the 55 nt of sequence surrounding the site of modification in RPL11a mRNA were <i>in vitro</i> transcribed with the following mutants: no mutation (R55), site of pseudouridylation (U68) to C (R55_0C), U69A (R55_1A), UCU(65-67) to AGC (R55_AGC) or both U69A and UCU->AGC (R55_AGC1A). The RNA was then used for the CMCT-RT stop experiment, and the data were plotted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110799#pone-0110799-g003" target="_blank">figure 3A</a>. E. For these modification experiments, the proportion of reverse transcription that stopped at the site of pseudouridylation was plotted for both +CMCT (blue) and mock (red) conditions as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110799#pone-0110799-g003" target="_blank">figure 3B</a>. F. The sequences of the constructs used. The mutated nucleotides are in red. G-L. Same as A-F, for the following TEF1 constructs: 55 nt around the site of pseudouridylation in TEF1 (T55), 20 nt around the site (T20), 6 nt around the site (T6), 6 nt around the site in a 5 nt stem loop (T6-ds), T6ds with the U that is modified mutated to C (T6_ds_0C), the C 1 nt downstream mutated to U (T6_ds_1U), the G 2 nt upstream mutated to A (T6_ds_-2A) or both those mutations (T6_ds_1U-2A). For the sequence of T6-ds, the complementary nucleotides comprising the 5 nt stem are underlined.