Dissection of the factor requirements for spliceosome disassembly and the elucidation of its dissociation products using a purified splicing system

Abstract

The spliceosome is a single-turnover enzyme that needs to be dismantled after catalysis to both release the mRNA and recycle small nuclear ribonucleoproteins (snRNPs) for subsequent rounds of pre-mRNA splicing. The RNP remodeling events occurring during spliceosome disassembly are poorly understood, and the composition of the released snRNPs are only roughly known. Using purified components in vitro, we generated post-catalytic spliceosomes that can be dissociated into mRNA and the intron-lariat spliceosome (ILS) by addition of the RNA helicase Prp22 plus ATP and without requiring the step 2 proteins Slu7 and Prp18. Incubation of the isolated ILS with the RNA helicase Prp43 plus Ntr1/Ntr2 and ATP generates defined spliceosomal dissociation products: the intron-lariat, U6 snRNA, a 20-25S U2 snRNP containing SF3a/b, an 18S U5 snRNP, and the ''nineteen complex'' associated with both the released U2 snRNP and intron-lariat RNA. Our system reproduces the entire ordered disassembly phase of the spliceosome with purified components, which defines the minimum set of agents required for this process. It enabled us to characterize the proteins of the ILS by mass spectrometry and identify the ATPase action of Prp43 as necessary and sufficient for dissociation of the ILS without the involvement of Brr2 ATPase.[Keywords: intron-lariat spliceosome; disassembly; Prp22; Prp43; Brr2] Supplemental material is available for this article. Pre-mRNA splicing proceeds by way of two phosphoester transfer reactions and is catalyzed by the spliceosome, which consists of the U1, U2, U4/U6, and U5 small nuclear ribonucleoproteins (snRNPs) and numerous nonsnRNP proteins (Will and Lü hrmann 2011). The snRNPs are involved in recognizing short conserved sequences of the pre-mRNA, including the 59 and 39 splice sites (SS) and the branch site (BS), and in positioning the reactive nucleotides for catalysis. The spliceosome is a dynamic molecular machine, undergoing several major structural rearrangements during its functional cycle. These events are mediated by at least eight conserved DExD/H-box NTPases (hereafter termed RNA helicases) that act at discrete stages of the splicing pathway (Staley and Guthrie 1998;Cordin et al. 2012).Spliceosome assembly is initiated by the binding of U1 and U2 snRNPs to the 59 and the BS, respectively. This is followed by the recruitment of the U4/U6.U5 tri-snRNP, forming the precatalytic B complex. Catalytic activation of the spliceosome (leading to complex B act ) involves the displacement of U1 and U4 snRNAs from the spliceosome and the formation of new base pair interactions between the U6 and U2 snRNAs and the 59 SS (Staley and Guthrie 1998). The resulting RNA structure plays a central role in catalyzing both steps of splicing (Nilsen 1998).These RNA-RNA rearrangements are remodeled by the combined actions of the RNA helicases Prp28 and Brr2, which disrupt the base-pairing between U1 snRNA and the 59 SS and between the U4 and U6 snRNAs, respectively (Laggerbauer et al. 1998;Raghunathan and Guthrie 1998;Staley...

Dramatically reduced spliceosome in Cyanidioschyzon merolae

“…Fourmann et al (63) recently showed that Prp22 activity is associated with the disappearance of the RES complex (Bud13, Pml1, and Ist3), Cwc21, and Cwc22 from the postcatalytic spliceosome. We have not found any of these proteins in C. merolae, suggesting that Prp22-catalyzed protein dissociation is only incidental to its role in mRNA release.…”

Functions and regulation of the Brr2 RNA helicase during splicing

“…50,116 Prp43 is recruited in complex with the Ntr1 and Ntr2 proteins during C complex formation, before the second step of splicing. 16 As Brr2 can interact with the Ntr2 subunit of this complex, 117 it again may support recruitment of the Prp43 machinery for intron-lariat spliceosome disassembly.…”

“…46,50 Brr2 is located on one side of a triangular central body of the spliceosomal B act complex, close to the DEAH/RHA helicase Prp2 (Fig. 5A).…”

“…46 Furthermore, spliceosome disassembly can proceed in the presence of nucleotide tri-phosphates other than ATP, which Brr2 does not utilize. 50 Taken together, molecular mechanisms must be at work, through which Brr2 can be up-or down-regulated repeatedly during snRNP assembly and splicing (Fig. 1).…”

Investigating splicing in two disparate organisms: a highly reduced spliceosome in Cyandioschyzon Merolae and additional functionality in Saccharomyces Cerevisiae's PRP24

“…To this end, Prp22 has been implicated in the release of the mature mRNA and the disassembly of the RES complex (Bud13, Pml1, and Ist3), Cwc21, and Cwc22 from the postcatalytic spliceosome (Fourmann et al 2013). However, as a consequence of finding none of the proteins removed from the spliceosome by Prp22, it appears that this function of Prp22 is incidental to its role in mRNA release (Table 2).…”