Structural toggle in the RNaseH domain of Prp8 helps balance splicing fidelity and catalytic efficiency

Abstract

Pre-mRNA splicing is an essential step of eukaryotic gene expression that requires both high efficiency and high fidelity. Prp8 has long been considered the "master regulator" of the spliceosome, the molecular machine that executes pre-mRNA splicing. Cross-linking and structural studies place the RNaseH domain (RH) of Prp8 near the spliceosome's catalytic core and demonstrate that alleles that map to a 17-aa extension in RH stabilize it in one of two mutually exclusive structures, the biological relevance of which are unknown. We performed an extensive characterization of alleles that map to this extension and, using in vitro and in vivo reporter assays, show they fall into two functional classes associated with the two structures: those that promote error-prone/efficient splicing and those that promote hyperaccurate/inefficient splicing. Identification of global locations of endogenous splice-site activation by lariat sequencing confirms the fidelity effects seen in our reporter assays. Furthermore, we show that error-prone/efficient RH alleles suppress a mutant deficient at promoting the first catalytic step of splicing, whereas hyperaccurate/inefficient RH alleles exhibit synthetic sickness. Together our data indicate that RH alleles link splicing fidelity with catalytic efficiency by biasing the relative stabilities of distinct spliceosome conformations. We hypothesize that the spliceosome "toggles" between such error-prone/efficient and hyperaccurate/inefficient conformations during the splicing cycle to regulate splicing fidelity.

Prp8 impacts cryptic but not alternative splicing frequency

“…4 A and B). Such dynamics are required to position pre-mRNA for catalysis, and spliceosome dynamics may be linked to high-fidelity splicing (6,13,14,25).…”

“…During assembly, the spliceosome undergoes considerable conformational rearrangements (1). Prp8 is the largest and most highly conserved protein in the spliceosome and has been directly implicated in splicing fidelity (6). The bulk of Prp8 surrounds and stabilizes the spliceosome's catalytic core, contacting the catalytic U6 snRNA and pre-mRNA substrate (7,8).…”

“…In catalytic core closure, Prp8's N-terminal and large domains come into close proximity, helping to organize and support the snRNAs and pre-mRNA for catalysis (9)(10)(11)(12)(13). There is evidence that the spliceosome's catalytic core opens and closes repeatedly during a typical splicing cycle, and, importantly, "open" and "closed" forms of the spliceo-some have been linked to changes in splicing fidelity (6,14,15). Cryptic splicing could result from local movements of the pre-mRNA within the catalytic core of the spliceosome while it is in an open form.…”

RNase H As Gene Modifier, Driver of Evolution and Antiviral Defense

“…A conserved RNA recognition motif (RRM) in the RT domain is likely involved in pre-mRNA and/or small nuclear snRNA binding ( Grainger and Beggs, 2005 ). The inactive RNase H domain may be essential for balancing accuracy and efficiency during the splicing process ( Will and Lührmann, 2011 ; Mayerle et al, 2017 ). Interestingly, the RT-like domain of Prp8 likely originates from a prokaryotic group II intron ( Dlakić and Mushegian, 2011 ), and pre-mRNA splicing probably evolved from a group II intron ribozyme ( Abelson, 2013 ).…”

High Precision Detection of Rare Splice Isoforms Using Multiplexed Primer Extension Sequencing

“…Locations of branchpoints (Supplementary Information Table 7) were determined by consolidating the most used branchpoint from lariat sequencing data 28 and previously described branch locations based on sequence motif searches 29 .…”