Regulation of Prp43-mediated disassembly of spliceosomes by its cofactors Ntr1 and Ntr2

Abstract

The DEAH-box NTPase Prp43 disassembles spliceosomes in co-operation with the cofactors Ntr1/Spp382 and Ntr2, forming the NTR complex. How Prp43 is regulated by its cofactors to discard selectively only intron-lariat spliceosomes (ILS) and defective spliceosomes and to prevent disassembly of earlier and properly assembled/wild-type spliceosomes remains unclear. First, we show that Ntr1΄s G-patch motif (Ntr1GP) can be replaced by the GP motif of Pfa1/Sqs1, a Prp43΄s cofactor in ribosome biogenesis, demonstrating that the specific function of Ntr1GP is to activate Prp43 for spliceosome disassembly and not to guide Prp43 to its binding site in the spliceosome. Furthermore, we show that Ntr1΄s C-terminal domain (CTD) plays a safeguarding role by preventing Prp43 from disrupting wild-type spliceosomes other than the ILS. Ntr1 and Ntr2 can also discriminate between wild-type and defective spliceosomes. In both type of spliceosomes, Ntr1-CTD impedes Prp43-mediated disassembly while the Ntr1GP promotes disassembly. Intriguingly, Ntr2 plays a specific role in defective spliceosomes, likely by stabilizing Ntr1 and allowing Prp43 to enter a productive interaction with the GP motif of Ntr1. Our data indicate that Ntr1 and Ntr2 act as ‘doorkeepers’ and suggest that both cofactors inspect the RNP structure of spliceosomal complexes thereby targeting suboptimal spliceosomes for Prp43-mediated disassembly.

Intrinsically Disordered Protein Ntr2 Modulates the Spliceosomal RNA Helicase Brr2

“…The Ntr complex drives spliceosome disassembly after mRNA release (18)(19)(20). Moreover, Ntr2 seems to target defective spliceosomes for NTR-mediated discard by a yet unknown mechanism (24). Brr2 has previously been attributed a role in spliceosome disassembly (8) and its interaction with Ntr2 now provides a physical link to this process.…”

“…The complex also disassembles various early spliceosome intermediates, such as the precatalytic B and catalytically activated B act complexes (21,22). In contrast, the complete Ntr complex only disassembles intron-lariat spliceosomes and aberrant spliceosome assembly intermediates, with Ntr2 preventing unwanted disassembly of other, correctly formed intermediates by a yet unknown mechanism (23,24). Ntr2 could thus molecularly link Brr2 to spliceosome disassembly and quality control.…”

Crystal Structure of the Escherichia coli DExH-Box NTPase HrpB

“…The final concentrations in the reactions were 0.5 mM protein, 25 nM RNA and 1 mM ATP/MgCl 2 in unwinding buffer (20 mM HEPES-NaOH, pH 8.0, 150 mM NaCl, 0.5 mM MgCl 2 ). As a positive control for unwinding of the RNA bearing a 3'-overhang, yeast Prp43 in complex with an activating G-patch protein, Pfa1, (Fourmann et al, 2017) (kind gift from R. Ficner, Universit€ at Gö ttingen), was used. As a positive control for unwinding of the RNA containing a 5'-overhang, a truncated version of human Upf1, lacking the CH domain, was used (Chakrabarti et al, 2011) (kind gift from S. Chakrabarti, Freie Universit€ at Berlin).…”

Structural insights into the mechanism of the DEAH-box RNA helicase Prp43

“…The first reported function of Prp43 is its involvement in pre-mRNA splicing. In the course of this process, Prp43 acts at the latest stage of the splicing cycle and it is required to dismantle the intron-lariat spliceosome into the excised lariat and the U2•U5•U6 snRNPs (Arenas and Abelson, 1997; Fourmann et al, 2013, 2016a). Recently, the target substrate of Prp43 during this process was revealed which is the RNA network between the U2 snRNP and the branch site of the intron (Fourmann et al, 2016b).…”