Prp8 retinitis pigmentosa mutants cause defects in the transition between the catalytic steps of splicing

Abstract

Pre-mRNA splicing must occur with high fidelity and efficiency for proper gene expression. The spliceosome uses DExD/H box helicases to promote on-pathway interactions while simultaneously minimizing errors. Prp8 and Snu114, an EF2-like GTPase, regulate the activity of the Brr2 helicase, promoting RNA unwinding by Brr2 at appropriate points in the splicing cycle and repressing it at others. Mutations linked to retinitis pigmentosa (RP), a disease that causes blindness in humans, map to the Brr2 regulatory region of Prp8. Previous in vitro studies of homologous mutations in Saccharomyces cerevisiae show that Prp8-RP mutants cause defects in spliceosome activation. Here we show that a subset of RP mutations in Prp8 also causes defects in the transition between the first and second catalytic steps of splicing. Though Prp8-RP mutants do not cause defects in splicing fidelity, they result in an overall decrease in splicing efficiency. Furthermore, genetic analyses link Snu114 GTP/GDP occupancy to Prp8-dependent regulation of Brr2. Our results implicate the transition between the first and second catalytic steps as a critical place in the splicing cycle where Prp8-RP mutants influence splicing efficiency. The location of the Prp8-RP mutants, at the "hinge" that links the Prp8 Jab1-MPN regulatory "tail" to the globular portion of the domain, suggests that these Prp8-RP mutants inhibit regulated movement of the Prp8 Jab1/MPN domain into the Brr2 RNA binding channel to transiently inhibit Brr2. Therefore, in Prp8-linked RP, disease likely results not only from defects in spliceosome assembly and activation, but also because of defects in splicing catalysis.

Brr2 is a splicing fidelity factor

“…cDNA was created using dN9 primers and Superscript III Reverse Transcriptase using the manufacturers protocols. Methods and primers for RT-qPCR of Act-Cup reporter cDNA are described in (MacRae et al 2018), methods and primers for RT-qPCR of endogenous introncontaining transcripts are described in (Mayerle and Guthrie 2016). Prp16 primers are noted in the legend of Figure 4.…”

“…D. Heat map showing results of Act-Cup assays. Values shown were calculated by log2([Cumax Prp8-RP]/[Cumax Prp8-WT]) (Mayerle and Guthrie 2016). Blue indicates reduced growth in coppercontaining media (hyperaccuracy) of brr2-RP relative to wild type with the same reporter, while yellow indicates improved growth (error-prone splicing).…”

“…Spliceosomal protein Brr2 is a DEIH box ATPase and the most highly processive spliceosomal helicase (Raghunathan and Guthrie 1998). Initial Brr2 characterizations focused on its role in unwinding the snRNA duplexes during spliceosome activation (Lauber et al 1996;Raghunathan and Guthrie 1998), however Brr2 has since been implicated in multiple steps of the splicing cycle: during the transition between the 1 st and 2 nd catalytic steps (Hahn et al 2012;Mayerle and Guthrie 2016), during spliceosome disassembly (Small et al 2006), and in pre-mRNA discard from the spliceosome (Chen et al 2013). Brr2's helicase and ATPase activities are highly regulated, via both intramolecular and extramolecular mechanisms, to ensure that Brr2 acts only at appropriate points in the splicing cycle (Absmeier et al 2017).…”

Functions and regulation of the Brr2 RNA helicase during splicing

“…Brr2, in contrast, stays associated with the spliceosome after recruitment of the tri-snRNP during the remaining phases of splicing. Moreover, in addition to spliceosome activation, Brr2 has been found to be important for the retention of U5 and U6 snRNAs during spliceosome activation 44,45 and to play a role during splicing catalysis 46,47 and spliceosome disassembly. 48 However, not all of these additional functions apparently rely on the enzyme's ATPase and helicase activities.…”

“…1), which would be in line with the Prp8 Jab1 domain being important for Brr2 function during splicing catalysis. 47 This organization would leave the Brr2 NTR available to engage in contacts with and possibly regulate the activity of Prp16. 54 However, the limited local resolution in the corresponding region of the Prp16-containing C complex structure does not unequivocally reveal the state of the NTR or the Jab1 domain.…”