Characterization of Cwc2, U6 snRNA, and Prp8 interactions destabilized by Prp16 ATPase at the transition between the first and second steps of splicing
Description
To better understand rearrangements within the S. cerevisiae catalytic center, we characterize factors that modulate function of Prp16: Cwc2, N-terminal domain of Prp8, and U6-41AACAAU46 region. Alleles of these factors were identified through genetic screens for mutants that correct cs defects of prp16-302 allele. Several of the identified U6, cwc2, and prp8 alleles are located in close proximity of each other in cryo-EM structures of the spliceosomal catalytic conformations. Cwc2 and U6 interact with the intron sequences in the first step, but they do not seem to contribute to the stability of the second step catalytic center. On the other hand, the N-terminal segment of Prp8 not only affects intron positioning for the first step, but it also makes important contacts in the proximity of the active site for both the first and the second steps of splicing.
Files
Steps to reproduce
Plasmid-shuffle growth assays Cell cultures were adjusted to OD600=1 and 10-fold serial dilutions were spotted on plates containing 5-fluoroorotic acid (5FOA), grown at indicated temperatures, and photographed after 3-6 days. Copper growth assays yMK20, yMK79, or JU75 strains carrying indicated alleles were transformed with LEU-marked ACT1-CUP1 reporter plasmids (Lesser and Guthrie, 1993a). Cultures were grown in –LEU medium, diluted to A600=1.0 and equal volumes were spotted on –LEU plates containing 0-1.0 mM CuSO4 and grown at 30ºC for 4 days. Data for each experiment, cropped and aligned for clarity, originate from a single plate. Primer extension assays Total RNA was isolated from strains carrying ACT1-CUP1 reporter plasmids using glass beads and a standard phenol:chloroform protocol. Primer complementary to the second exon of ACT1 (5′-GGCACTCATGACCTTC-3′) was kinased at the 5′ end using T4 PNK and -32P ATP (Hartmann, SRP-501). Annealing (containing 3 μg of total yeast RNA and 32P-labeled primer) and extension reactions were carried out as described by Eysmont et al., 2019. Bioscreen C growth measurements Cell cultures were adjusted to OD600=0.1 and transferred in 300 μl triplicates to a 100-well Honeycomb 2 microplate. Cultures were grown at 16ºC or 18 ºC with continuous, double-orbital shaking in the Bioscreen C Pro system. OD600 measurements were taken at regular intervals of 15 minutes over 140 hours. Growth curves were generated using the average of three repeats. Screen for cwc2 suppressors of prp16-302 To increase the analyzed pool of mutants, we carried out the selection in parallel for two regions of Cwc2 (corresponding to aa 1-159, flanked by SnaBI and EcoRI sites, and aa 159-339, flanked by EcoRI and PflMI). In vivo gap repair selection for improvement of prp16-302 cs growth defects was carried out at 16ºC. Error-prone PCR was performed using a buffer containing 0.5 mM MnCl2, 5.5 mM MgCl2 and 0.05 U/µl of Taq DNA polymerase. Screen for prp8 N-terminal domain suppressors of prp16-302 To increase the number of tested mutants, mutagenesis of the N-terminal Prp8 domain was carried out in four regions using NheI, BamHI, SalI, SexAI, and SacI restriction sites, covering nucleotides +1 to +477 or +477 to +1017 of PRP8. In vivo gap repair selection for improvement of prp16-302 cs growth defects was carried out as for cwc2 suppressors.
Categories
Funders
- Foundation for Polish SciencePolandGrant ID: Regenerative Mechanisms for Health – ReMedy” project MAB/2017/2