Jonathan P. Staley

Mechanical Devices of the Spliceosome: Motors, Clocks, Springs, and Things

We thank J. Thorner, J. Lorsch, and D. Herschlag for provocative discussions; J. Abelson, R. Luhrmann, T. Nilsen, and R. Reed for preprints; and G. Chanfreau, C. Collins, A. Frankel, A. Kistler, K. Lynch, S. Rader, B. Raumann, D. Ryan, C. Siebel, J. Wagner, Y. Wang, J. Wilhelm, and members of the Guthrie laboratory for critical reading of the manuscript. We are indebted to S. Korolev and G. Waksman (Washington University School of Medicine) and P. Weber (Schering-Plough Research Institute) for contributing Figure 8Figure 8. We apologize to our colleagues for work that could not be cited due to a limitation on references. Cited work from this laboratory is supported by NIH grant GM21119 to C. G. J. P. S. is supported by a California Division-American Cancer Society Fellowship #1–59–97B. C. G. is an American Cancer Society Research Professor of Molecular Genetics.

RNA catalyses nuclear pre-mRNA splicing

In nuclear pre-messenger RNA splicing, introns are excised by the spliceosome, a dynamic machine composed of both proteins and small nuclear RNAs (snRNAs). Over thirty years ago, after the discovery of self-splicing group II intron RNAs, the snRNAs were proposed to catalyse splicing. However, no definitive evidence for a role of either RNA or protein in catalysis by the spliceosome has been reported so far. By using metal rescue strategies in spliceosomes from budding yeast, here we show that the U6 snRNA catalyses both of the two splicing reactions by positioning divalent metals that stabilize the leaving groups during each reaction. Notably, all of the U6 catalytic metal ligands we identified correspond to the ligands observed to position catalytic, divalent metals in crystal structures of a group II intron RNA. These findings indicate that group II introns and the spliceosome share common catalytic mechanisms and probably common evolutionary origins. Our results demonstrate that RNA mediates catalysis within the spliceosome.

Assembly of ribosomes and spliceosomes: complex ribonucleoprotein machines.

Ribosomes and spliceosomes are ribonucleoprotein nanomachines that catalyze translation of mRNA to synthesize proteins and splicing of introns from pre-mRNAs, respectively. Assembly of ribosomes involves more than 300 proteins and RNAs, and that of spliceosomes over 100 proteins and RNAs. Construction of these enormous ribonucleoprotein particles (RNPs) is a dynamic process, in which the nascent RNPs undergo numerous ordered rearrangements of RNA-RNA, RNA-protein, and protein-protein interactions. Here we outline similar principles that have emerged from studies of ribosome and spliceosome assembly. Constituents of both RNPs form subassembly complexes, which can simplify the task of assembly and segregate functions of assembly factors. Reorganization of RNP topology, and proofreading of proper assembly, are catalyzed by protein- or RNA-dependent ATPases or GTPases. Dynamics of intermolecular interactions may be facilitated or regulated by cycles of post-translational modifications. Despite this repertoire of tools, mistakes occur in RNP assembly or in processing of RNA substrates. Quality control mechanisms recognize and turnover misassembled RNPs and reject improper substrates.

Exon ligation is proofread by the DexD/H-box ATPase Prp22p

To produce messenger RNA, the spliceosome excises introns from precursor (pre)-mRNA and splices the flanking exons. To establish fidelity, the spliceosome discriminates against aberrant introns, but current understanding of such fidelity mechanisms is limited. Here we show that an ATP-dependent activity represses formation of mRNA from aberrant intermediates having mutations in any of the intronic consensus sequences. This proofreading activity is disabled by mutations that impair the ATPase or RNA unwindase activity of Prp22p, a conserved spliceosomal DExD/H-box ATPase. Further, cold-sensitive prp22 mutants permit aberrant mRNA formation from a mutated 3′ splice-site intermediate in vivo. We conclude that Prp22p generally represses splicing of aberrant intermediates, in addition to its known ATP-dependent role in promoting release of genuine mRNA. This dual function for Prp22p validates a general model in which fidelity can be enhanced by a DExD/H-box ATPase.

A role for ubiquitin in the spliceosome assembly pathway

The spliceosome uses numerous strategies to regulate its function in mRNA maturation. Ubiquitin regulates many cellular processes, but its potential roles during splicing are unknown. We have developed a new strategy that reveals a direct role for ubiquitin in the dynamics of splicing complexes. A ubiquitin mutant (I44A) that can enter the conjugation pathway but is compromised in downstream functions diminishes splicing activity by reducing the levels of the U4/U6-U5 small nuclear ribonucleoprotein (snRNP). Similarly, an inhibitor of ubiquitins protein-protein interactions, ubistatin A, reduces U4/U6-U5 triple snRNP levels in vitro. When ubiquitin interactions are blocked, ATP-dependent disassembly of purified U4/U6-U5 particles is accelerated, indicating a direct role for ubiquitin in repressing U4/U6 unwinding. Finally, we show that the conserved splicing factor Prp8 is ubiquitinated within purified triple snRNPs. These results reveal a previously unknown ubiquitin-dependent mechanism for controlling the pre-mRNA splicing pathway.

The splicing factor Prp43p, a DEAH box ATPase, functions in ribosome biogenesis.

ABSTRACT Biogenesis of the small and large ribosomal subunits requires modification, processing, and folding of pre-rRNA to yield mature rRNA. Here, we report that efficient biogenesis of both small- and large-subunit rRNAs requires the DEAH box ATPase Prp43p, a pre-mRNA splicing factor. By steady-state analysis, a cold-sensitive prp43 mutant accumulates 35S pre-rRNA and depletes 20S, 27S, and 7S pre-rRNAs, precursors to the small- and large-subunit rRNAs. By pulse-chase analysis, the prp43 mutant is defective in the formation of 20S and 27S pre-rRNAs and in the accumulation of 18S and 25S mature rRNAs. Wild-type Prp43p immunoprecipitates pre-rRNAs and mature rRNAs, indicating a direct role in ribosome biogenesis. The Prp43p-Q423N mutant immunoprecipitates 27SA2 pre-rRNA threefold more efficiently than the wild type, suggesting a critical role for Prp43p at the earliest stages of large-subunit biogenesis. Consistent with an early role for Prp43p in ribosome biogenesis, Prp43p immunoprecipitates the majority of snoRNAs; further, compared to the wild type, the prp43 mutant generally immunoprecipitates the snoRNAs more efficiently. In the prp43 mutant, the snoRNA snR64 fails to methylate residue C2337 in 27S pre-rRNA, suggesting a role in snoRNA function. We propose that Prp43p promotes recycling of snoRNAs and biogenesis factors during pre-rRNA processing, similar to its recycling role in pre-mRNA splicing. The dual function for Prp43p in the cell raises the possibility that ribosome biogenesis and pre-mRNA splicing may be coordinately regulated.

Spliceosome discards intermediates via the DEAH box ATPase Prp43p

To promote fidelity in nuclear pre-mRNA splicing, the spliceosome rejects and discards suboptimal substrates that have engaged the spliceosome. Whereas DExD/H box ATPases have been implicated in rejecting suboptimal substrates, the mechanism for discarding suboptimal substrates has remained obscure. Corroborating evidence that suboptimal, mutated lariat intermediates can be exported to the cytoplasm for turnover, we have found that the ribosome can translate mutated lariat intermediates. By glycerol gradient analysis, we have found that the spliceosome can dissociate mutated lariat intermediates in vivo in a manner that requires the DEAH box ATPase Prp43p. Through an in vitro assay, we demonstrate that Prp43p promotes the discard of suboptimal and optimal 5′ exon and lariat intermediates indiscriminately. Finally, we demonstrate a requirement for Prp43p in repressing splicing at a cryptic splice site. We propose a model for the fidelity of exon ligation in which the DEAH box ATPase Prp22p slows the flow of suboptimal intermediates through exon ligation and Prp43p generally promotes discard of intermediates, thereby establishing a pathway for turnover of stalled intermediates. Because Prp43p also promotes spliceosome disassembly after exon ligation, this work establishes a parallel between the discard of suboptimal intermediates and the dissociation of a genuine excised intron product.

Evidence for a group II intron–like catalytic triplex in the spliceosome

To catalyze pre-mRNA splicing, U6 small nuclear RNA positions two metals that interact directly with the scissile phosphates. U6 metal ligands correspond stereospecifically to metal ligands within the catalytic domain V of a group II self-splicing intron. Domain V ligands are organized by base-triple interactions, which also juxtapose the 3′ splice site with the catalytic metals. However, in the spliceosome, the mechanism for organizing catalytic metals and recruiting the substrate has remained unclear. Here we show by genetics, cross-linking and biochemistry in yeast that analogous triples form in U6 and promote catalytic-metal binding and both chemical steps of splicing. Because the triples include an element that defines the 5′ splice site, they also provide a mechanism for juxtaposing the pre-mRNA substrate with the catalytic metals. Our data indicate that U6 adopts a group II intron–like tertiary conformation to catalyze splicing.

Staying on message: ensuring fidelity in pre-mRNA splicing

The faithful expression of genes requires that cellular machinery select substrates with high specificity at each step in gene expression. High specificity is particularly important at the stage of nuclear pre-mRNA splicing, during which the spliceosome selects splice sites and excises intervening introns. With low specificity, the usage of alternative sites would yield insertions, deletions and frame shifts in mRNA. Recently, biochemical, genetic and genome-wide approaches have significantly advanced our understanding of splicing fidelity. In particular, we have learned that DExD/H-box ATPases play a general role in rejecting and discarding suboptimal substrates and that these factors serve as a paradigm for proofreading NTPases in other systems. Recent advances have also defined fundamental questions for future investigations.

Cancer therapies activate RIG-I-like receptor pathway through endogenous non-coding RNAs

Emerging evidence indicates that ionizing radiation (IR) and chemotherapy activate Type I interferon (IFN) signaling in tumor and host cells. However, the mechanism of induction is poorly understood. We identified a novel radioprotective role for the DEXH box RNA helicase LGP2 (DHX58) through its suppression of IR-induced cytotoxic IFN-beta [1]. LGP2 inhibits activation of the RIG-I-like receptor (RLR) pathway upon binding of viral RNA to the cytoplasmic sensors RIG-I (DDX58) and MDA5 (IFIH1) and subsequent IFN signaling via the mitochondrial adaptor protein MAVS (IPS1). Here we show that MAVS is necessary for IFN-beta induction and interferon-stimulated gene expression in the response to IR. Suppression of MAVS conferred radioresistance in normal and cancer cells. Germline deletion of RIG-I, but not MDA5, protected mice from death following total body irradiation, while deletion of LGP2 accelerated the death of irradiated animals. In human tumors depletion of RIG-I conferred resistance to IR and different classes of chemotherapy drugs. Mechanistically, IR stimulated the binding of cytoplasmic RIG-I with small endogenous non-coding RNAs (sncRNAs), which triggered IFN-beta activity. We demonstrate that the small nuclear RNAs U1 and U2 translocate to the cytoplasm after IR treatment, thus stimulating the formation of RIG-I: RNA complexes and initiating downstream signaling events. Taken together, these findings suggest that the physiologic responses to radio-/chemo-therapy converge on an antiviral program in recruitment of the RLR pathway by a sncRNA-dependent activation of RIG-I which commences cytotoxic IFN signaling. Importantly, activation of interferon genes by radiation or chemotherapy is associated with a favorable outcome in patients undergoing treatment for cancer. To our knowledge, this is the first demonstration of a cell-intrinsic response to clinically relevant genotoxic treatments mediated by an RNA-dependent mechanism.