An ATP-independent role for Prp16 in promoting aberrant splicing.

The spliceosome is assembled through a step-wise process of binding and release of its components to and from the pre-mRNA. The remodeling process is facilitated by eight DExD/H-box RNA helicases, some of which have also been implicated in splicing fidelity control. In this study, we unveil a contrasting role for the prototypic splicing proofreader, Prp16, in promoting the utilization of aberrant 5' splice sites and mutated branchpoints.

Arresting Spliceosome Intermediates at Various Stages of the Splicing Pathway.

The spliceosome is a dynamic ribonucleoprotein particle and is assembled via sequential binding of five snRNAs and numerous protein factors. To understand the molecular mechanism of the splicing reaction, it is necessary to dissect the spliceosome pathway and isolate spliceosome intermediates in various stages of the pathway for biochemical and structural analysis. Here, we describe protocols for preparing intron-containing transcripts, cell-free splicing extracts, and in vitro splicing reactions, as well as procedures to arrest the spliceosome at different stages of the pathway for characterization of specific splicing complexes from the budding yeast Saccharomyces cerevisiae.

Evidence for complex dynamics during U2 snRNP selection of the intron branchpoint.

Splicing of pre-mRNA is initiated by binding of U1 to the 5' splice site and of Msl5-Mud2 heterodimer to the branch site (BS). Subsequent binding of U2 displaces Msl5-Mud2 from the BS to form the prespliceosome, a step governing branchpoint selection and hence 3' splice site choice, and linking splicing to myelodysplasia and many cancers in human. Two DEAD-box proteins, Prp5 and Sub2, are required for this step, but neither is stably associated with the pre-mRNA during the reaction.

Functional analysis of Cwc24 ZF-domain in 5' splice site selection.

The essential splicing factor Cwc24 contains a zinc-finger (ZF) domain required for its function in splicing. Cwc24 binds over the 5' splice site after the spliceosome is activated, and its binding prior to Prp2-mediated spliceosome remodeling is important for proper interactions of U5 and U6 with the 5' splice site sequence and selection of the 5' splice site. Here, we show that Cwc24 transiently interacts with the 5' splice site in formation of the functional RNA catalytic core during spliceosome remodeling, and the ZF-motif is required for specific interaction of Cwc24 with the 5' splice site.

Dynamic protein-RNA interactions in mediating splicing catalysis.

The spliceosome is assembled via sequential interactions of pre-mRNA with five small nuclear RNAs and many proteins. Recent determination of cryo-EM structures for several spliceosomal complexes has provided deep insights into interactions between spliceosomal components and structural changes of the spliceosome between steps, but information on how the proteins interact with pre-mRNA to mediate the reaction is scarce. By systematic analysis of proteins interacting with the splice sites (SSs), we have identified many previously unknown interactions of spliceosomal components with the pre-mRNA.

Cwc23 is a component of the NTR complex and functions to stabilize Ntr1 and facilitate disassembly of spliceosome intermediates.

Cwc23 is a member of the J protein family, and has been shown to interact with Ntr1, a scaffold protein that interacts with Ntr2 and Prp43 to form the NTR complex that mediates spliceosome disassembly. We show that Cwc23 is also an intrinsic component of the NTR complex, and that it interacts with the carboxyl terminus of Ntr1. Metabolic depletion of Cwc23 concurrently depleted Ntr1 and Ntr2, suggesting a role for Cwc23 in stabilizing these two proteins.

A central role of Cwc25 in spliceosome dynamics during the catalytic phase of pre-mRNA splicing.

Splicing of precursor mRNA occurs via two consecutive steps of transesterification reaction; both require ATP and several proteins. Despite the energy requirement in the catalytic phase, incubation of the purified spliceosome under proper ionic conditions can elicit competitive reversible transesterification, debranching, and spliced-exon-reopening reactions without the necessity for ATP or other factors, suggesting that small changes in the conformational state of the spliceosome can lead to disparate chemical consequences for the substrate. We show here that Cwc25 plays a central role in modulating the conformational state of the catalytic spliceosome during normal splicing reactions.

Role of Cwc24 in the First Catalytic Step of Splicing and Fidelity of 5' Splice Site Selection.

Cwc24 is an essential splicing factor but only transiently associates with the spliceosome, with an unknown function. The protein contains a RING finger and a zinc finger domain in the carboxyl terminus. The human ortholog of Cwc24, RNF113A, has been associated with the disorder trichothiodystrophy.

Functional roles of DExD/H-box RNA helicases in Pre-mRNA splicing.

Splicing of precursor mRNA takes place via two consecutive steps of transesterification catalyzed by a large ribonucleoprotein complex called the spliceosome. The spliceosome is assembled through ordered binding to the pre-mRNA of five small nuclear RNAs and numerous protein factors, and is disassembled after completion of the reaction to recycle all components. Throughout the splicing cycle, the spliceosome changes its structure, rearranging RNA-RNA, RNA-protein and protein-protein interactions, for positioning and repositioning of splice sites.

A novel mechanism for Prp5 function in prespliceosome formation and proofreading the branch site sequence.

The DEAD-box RNA helicase Prp5 is required for the formation of the prespliceosome through an ATP-dependent function to remodel U2 small nuclear ribonucleoprotein particles (snRNPs) and an ATP-independent function of unknown mechanism. Prp5 has also been implicated in proofreading the branch site sequence, but the molecular mechanism has not been well characterized. Using actin precursor mRNA (pre-mRNA) carrying branch site mutations, we identified a Prp5-containing prespliceosome with Prp5 directly bound to U2 small nuclear RNA (snRNA).

Structural requirement of Ntc77 for spliceosome activation and first catalytic step.

The Prp19-associated complex is required for spliceosome activation by stabilizing the binding of U5 and U6 on the spliceosome after the release of U4. The complex comprises at least eight proteins, among which Ntc90 and Ntc77 contain multiple tetratricopeptide repeat (TPR) elements. We have previously shown that Ntc90 is not involved in spliceosome activation, but is required for the recruitment of essential first-step factor Yju2 to the spliceosome.

Sad1 counteracts Brr2-mediated dissociation of U4/U6.U5 in tri-snRNP homeostasis.

The yeast Sad1 protein was previously identified in a screen for factors involved in the assembly of the U4/U6 di-snRNP particle. Sad1 is required for pre-mRNA splicing both in vivo and in vitro, and its human orthologue has been shown to associate with U4/U6.U5 tri-snRNP.

The spliceosome catalyzes debranching in competition with reverse of the first chemical reaction.

Splicing of nuclear pre-mRNA occurs via two steps of the transesterification reaction, forming a lariat intermediate and product. The reactions are catalyzed by the spliceosome, a large ribonucleoprotein complex composed of five small nuclear RNAs and numerous protein factors. The spliceosome shares a similar catalytic core structure with that of fungal group II introns, which can self-splice using the same chemical mechanism.

A weak spliceosome-binding domain of Yju2 functions in the first step and bypasses Prp16 in the second step of splicing.

Yju2 is an essential splicing factor required for the first catalytic step after the action of Prp2. We dissected the structure of Yju2 and found that the amino (Yju2-N) and carboxyl (Yju2-C) halves of the protein can be separated and reconstituted for Yju2 function both in vivo and in vitro. Yju2-N has a weak affinity for the spliceosome but functions in promoting the first reaction, with the second reaction being severely impeded.

Link of NTR-mediated spliceosome disassembly with DEAH-box ATPases Prp2, Prp16, and Prp22.

The DEAH-box ATPase Prp43 is required for disassembly of the spliceosome after the completion of splicing or after the discard of the spliceosome due to a splicing defect. Prp43 associates with Ntr1 and Ntr2 to form the NTR complex and is recruited to the spliceosome via the interaction of Ntr2 and U5 component Brr2. Ntr2 alone can bind to U5 and to the spliceosome.

The interaction of Prp2 with a defined region of the intron is required for the first splicing reaction.

In Saccharomyces cerevisiae, the 3' splice site is not required for the first catalytic reaction of splicing. We have previously reported that at least 24 nucleotides downstream of the branch point is required for the first reaction to take place, but the precatalytic spliceosome forms efficiently on the truncated pre-mRNA with only 5 nucleotides retained downstream of the branch point. The factors that mediate this length-dependent control of the first catalytic step are not known.

Functional roles of protein splicing factors.

RNA splicing is one of the fundamental processes in gene expression in eukaryotes. Splicing of pre-mRNA is catalysed by a large ribonucleoprotein complex called the spliceosome, which consists of five small nuclear RNAs and numerous protein factors. The spliceosome is a highly dynamic structure, assembled by sequential binding and release of the small nuclear RNAs and protein factors.

DEAH-box ATPase Prp16 has dual roles in remodeling of the spliceosome in catalytic steps.

The assembly of the spliceosome involves dynamic rearrangements of interactions between snRNAs, protein components, and the pre-mRNA substrate. DExD/H-box ATPases are required to mediate structural changes of the spliceosome, utilizing the energy of ATP hydrolysis. Two DExD/H-box ATPases are required for the catalytic steps of the splicing pathway, Prp2 for the first step and Prp16 for the second step, both belonging to the DEAH subgroup of the protein family.

Splicing factor Cwc22 is required for the function of Prp2 and for the spliceosome to escape from a futile pathway.

Cwc22 was previously identified to associate with the pre-mRNA splicing factor Cef1/Ntc85, a component of the Prp19-associated complex (nineteen complex [NTC]) involved in spliceosome activation. We show here that Cwc22 is required for pre-mRNA splicing both in vivo and in vitro but is neither tightly associated with the NTC nor required for spliceosome activation. Cwc22 is associated with the spliceosome prior to catalytic steps and remains associated throughout the reaction.

Cwc25 is a novel splicing factor required after Prp2 and Yju2 to facilitate the first catalytic reaction.

Cwc25 has previously been identified to associate with pre-mRNA splicing factor Cef1/Ntc85, a component of the Prp19-associated complex (nineteen complex, or NTC) involved in spliceosome activation. We show here that Cwc25 is neither tightly associated with NTC nor required for spliceosome activation but is required for the first catalytic reaction. The affinity-purified spliceosome formed in Cwc25-depleted extracts contained only pre-mRNA and could be chased into splicing intermediates upon the addition of recombinant Cwc25 in an ATP-independent manner, suggesting that Cwc25 functions in the final step of the first catalytic reaction after the action of Prp2.

Ntc90 is required for recruiting first step factor Yju2 but not for spliceosome activation.

The Prp19-associated complex (NineTeen Complex [NTC]) is required for spliceosome activation by specifying interactions of U5 and U6 with pre-mRNA on the spliceosome after the release of U4. The NTC consists of at least eight protein components, including two tetratricopeptide repeat (TPR)-containing proteins, Ntc90 and Ntc77. Ntc90 has nine copies of the TPR with seven clustered in the carboxy-terminal half of the protein, and interacts with all identified NTC components except for Prp19 and Ntc25.

Both catalytic steps of nuclear pre-mRNA splicing are reversible.

Nuclear pre-messenger RNA (pre-mRNA) splicing is an essential processing step for the production of mature mRNAs from most eukaryotic genes. Splicing is catalyzed by a large ribonucleoprotein complex, the spliceosome, which is composed of five small nuclear RNAs and more than 100 protein factors. Despite the complexity of the spliceosome, the chemistry of the splicing reaction is simple, consisting of two consecutive transesterification reactions.

Dynamic interactions of Ntr1-Ntr2 with Prp43 and with U5 govern the recruitment of Prp43 to mediate spliceosome disassembly.

The Saccharomyces cerevisiae splicing factors Ntr1 (also known as Spp382) and Ntr2 form a stable complex and can further associate with DExD/H-box RNA helicase Prp43 to form a functional complex, termed the NTR complex, which catalyzes spliceosome disassembly. We show that Prp43 interacts with Ntr1-Ntr2 in a dynamic manner. The Ntr1-Ntr2 complex can also bind to the spliceosome first, before recruiting Prp43 to catalyze disassembly.