UNDERSTANDING THE ROLE OF ATP HYDROLYSIS IN THE SPLICEOSOME

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2 UNDERSTANDING THE ROLE OF ATP HYDROLYSIS IN THE SPLICEOSOME RNA Splicing Lecture 2, Biological Regulatory Mechanisms, H. Madhani Dept. of Biochemistry and Biophysics MAJOR MESSAGES Eight essential ATPases are required for pre-mrna splicing. All appear to act by disrupting RNA-RNA and RNA-protein interactions. Prp28 and Brr2 (which both arrive as a part of the triple snrnp) unwind the U1-5 splice site duplex and the U4- duplexes, respecitvely. These rearrangements are required for the -5 splice site interaction and the formation of the U2- catalytic core. The Brr2 helicase is regulated by other U5 snrnp proteins. The IEP-like protien Prp8 plays a key role in regulation as well as in stabilizing the catalytic core: Prp8 s C-terminal Jab/MPN domain directly activates the helicase activity of Brr2. Prp2, Prp16, and Prp22 are related subfamily DEAH-box RNA-dependent ATPases whose actions are required before, between and after the two chemical steps of splicing, respectively. Each associates with the spliceosome transiently. The action of the Prp2 ATPase is required after the major RNA rearrangements, but prior to catalysis. A pair of non-snrnp proteins, Yju2 and Cwc25, activate the step 1 spliceosome. A different pair of nonsnrnp proteins, Slu7 and Prp18, activate the step 2 spliceosome. Prp16 and Prp22 inactivate the catalytic conformations of the spliceosome, likely by disrupting interactions that stabilize the step 1 and step 2 conformations, respectively. Catalytically slow spliceosomes are also inactivated by Prp16 and Prp22. The inactive form of the spliceosome is the substrate for the spliceosomal disassemblase, Prp43-Ntr1. Disassembly serves as part an editing mechanism that disassembles spliceosomes that were successfully assembled, yet are now stalled at either chemical step. Mutations that reduce ATP hydrolysis rates enable suboptimal substrates to proceed through the catalytic steps by providing time for catalysis to occur before the active site is inactivated.

3 THE QUESTION: Splicing does not involve a change in the number of phosphodiester bonds in the substrate. Why then are ATP and 8 essential RNA-dependent ATPases of the DEAD/DEAH box family required for the process (n.b. there is no evidence that Group II intron removal requires ATP)? Hypothesis #1: ATP is used as energy to drive mechanical work on RNA-containing substrates. Hypothesis #2: The rate of ATP hydrolysis is used to as a molecular timer to evaluate the rate of a particular step. ATPases that promote RNA rearrangments: Brr2 and Prp28 Below we will focus on two ATPases that disrupt RNA-RNA duplexes thereby assisting in the RNA rearragnements that characterize spliceosome assembly. Brr2 -- a helicase required for the ejection of U4. brr2-1 was identified and cloned one of nine complementation groups identified by a Northen blot/primer-extension screen of 340 cold-sensitive lethal mutants (brr stands for bad response to refrigeration ). Brr2 is a component of the U4/U5/ triple snrnp. Incubation of a splicing extract with ATP results in release of U4 from the triple snrnp, while incubation of the brr2-1 mutant does not result in U4 release.

4 Purified Brr2 has a 3-5 helicase activity capable of unwinding the U4- complex in vitro; this activity is strongly stimulated by a C-terminal domain of Prp8. The high resolution cryoem structure of the S. cerevisiae triple snrnp shows Brr2 docked to U4 snrna just downstream of where U4 base-pairs with (it occupies a similar position in the cryoem structures of the B complex). Thus, Brr2 is almost certainly the helicase that unwinds the U4- RNA duplexes (stem I and stem II) during the transition from the B to Bact spliceosomal complexes.

5 Prp28 -- a putative helicase required for the ejection of U1. Like brr2-1, prp28-1 is a cold-sensitive mutation that blocks splicing in vivo. Prp28 encodes a member of the DEAD/DEAD box family of RNAdependent ATPases, which includes bona fide RNA helicases. Mutant prp28-1 extracts block spliceosome assembly at a stage prior to U1 and U4 dissociation (A complex). Strikingly, the PRP28 gene, which is normally essential for S. cerevisiae viability can be deleted in strains harboring mutations in U1 snrna residues or U1C snrnp protein involved in stabilizing the U1-5 splice site interaction.

6 The fully assembled splceosome (the so-called Bact complex) is *still* not active CryoEM studies have shown that at this stage of spliceosome assembly, the branchpoint helix is prevented from being juxtaposted with the 5 splice site by U2-associated proteins Prp11 and the SF3b

7 complex. Bact -- reactants are separated by 50 Å assembled spliceosome P I P C LI C P=precursor LI=lariat-intermediate L=excised lariat C=catalytically active spliceosome I=catalytically inactive spliceosome Prp2 --the final ATPase to act before step 1 catalysis After spliceosome assembly an ejection of U1 and U4, the spliceosome is still not catalytically active. An ATP-dependent step (which involves ejection of multiple proteins) mediated by the Prp2-Spp2 com- Prp2 Yju2-Cwc25 plex is required to make the spliceosome almost compent for catalysis. Spp2 is a member of a family proteins called G-patch proteins that a subset of DEAD/DEAH box proteins require for their action. Prp2 causes the displacement of multiple proteins including Prp11 and the cancer-associated SF3b complex (harboring Hsh155, Rds3 and Cus1 subunits in yeast). Yju2 and Cwc25 are sufficient to activate the spliceosome after the Prp2+ATP step The action of Prp2 enables the spliceosome to receive two proteins, Cwc25 and Yju2 whose addition is sufficient to trigger spliceosomal step 1catalysis.

8 The active step 1 spliceosome is like a sandwich make of protein bread and an RNA filling CryoEM studies have shown that the catalytic RNA core of the spliceosome is held in the hand of Prp8 (and associated proteins). The binding of Cwc25-Yju2 encases the RNA core into an enzynmatically active form of the step 1 spliceosome. P c aka the C complex

9 Prp16 inactivates the step 1 spliceosome assembled spliceosome Prp2 Yju2-Cwc25 PI PC LIC Prp16 LII Several lines of evidence indicate Prp16 inactivates the catalytic core of the spliceosome and while promoting the ejection of Cwc25 and Yju2. assembled spliceosome Prp2 I P Yju2-Cwc25 C P C LI Prp16 Slu7-Prp18 C LII LI LC Two proteins are sufficient to activate the spliceosome for step 2 after the Prp16 step The action of Prp16 enables a two-protein complex, Slu7-Prp18, to bind to the spliceosome that together are sufficient stabilze the catalytic conformation for step 2. assembled spliceosome Prp2 P I Yju2-Cwc25 PC LIC Prp16 LIc aka the C* complex Slu7-Prp18 I C LI LI C L Prp22 I L

10 Prp22 inactivates the step 2 spliceosome After the second catalytic step, the spliceosome is again inactivated, this time by the Prp22 ATPase. This results in mrna release from the spliceosome and ejection of the step 2 activators Slu7 and Prp18. The spliceosome now contains the excised intron lariat. This particle is recognized by the Prp43 helicase complexed with the Ntr1 protein, a targetting factor. Prp43 removes the snrnps from the lariatintron, which is then debranched by the Dbr1 enzyme and degraded by the nuclear exosome, the major 3 -->5 exonuclease in the eukaryotic nucleus. Prp16 mutants that suppress a branchpoint adenosine mutation display low ATPase activity The first mutation in a splicing component isolated as a suppressor of intron mutation was prp16-1.it was isolated as a suppressor of the A-->C mutation in the branchpoint adenosine nucleophile.mutations in Prp16 that suppress branchpoint mutants invariably have lowered ATPase activity. The key question: how can a mutation that reduces the activity of an ATPase required for splicing enable suboptimal substrates to successfully undergo the chemical step of splicing? Moreover, how can alteration of Prp16, a factor required for the second chemical step of splicing, but not the first, suppress a mutation that blocks the first step of splicing?

11 The proofreading model One model is that Prp16 (and Prp22) can inactivate spliceosomes either before or after splicing catalyisis, depending on the rate of chemistry. If chemistry is slow, then Prp16 inactivates the step 1 spliceosome (e.g. by displacing Cwc25 and Yju2), placing it back to where it was after the Prp2 step. If we further propose that any assembled, but inactivated spliceosome is a substrate for Prp43, and realize that splicing is chemically reversible, then we arrive at this framework for understanding the spliceosome: Yju2-Cwc25 Prp16 Slu7-Prp18 Prp22 P I P C LI C LI I LI C L C L I Prp16 Prp22 Prp43 Prp43 Prp43 P free φ φ As can be seen from the model, slowing down Prp16 enables suboptimal substrates to undergo chemistry, increasing the probability that they will undergo the second chemical step. This framework predicts that Prp22 mutants should suppress mutants defective in step 2 catalysis. Indeed, this is the case. ATP-dependent discard of mutant substrates has been demonstrated in S. cerevisiae extracts, providing direct evidence for the model. However, it is important to note that whether Prp16 and Prp22 prevent errors in vivo on normal, endogenous substrates it not clear. Also, S. cerevisiae is unusual in being intron-poor (~3% of genes have introns) and displaying unusually stringent match-to-consensus for splicing relative to other fungi (e.g. Cryptococcus neoformans: 99.5% of genes have introns). S. cerevisiae also appears to have lost numerous spliceosomal proteins during its evolution that are conserved between fungi and mammals. Thus, other proofreading mechanisms/steps may be at play that have not been discovered through studies of S. cerevisiae.