The DExD/H-box ATPase Prp2p destabilizes and proofreads the catalytic RNA core of the spliceosome

After undergoing massive RNA and protein rearrangements during assembly, the spliceosome undergoes a final, more subtle, ATP-dependent rearrangement that is essential for catalysis. This rearrangement requires the DEAH-box protein Prp2p, an RNA-dependent ATPase. Prp2p has been implicated in destabilizing interactions between the spliceosome and the protein complexes SF3 and RES, but a role for Prp2p in destabilizing RNA–RNA interactions has not been explored. Using directed molecular genetics in budding yeast, we have found that a cold-sensitive prp2 mutation is suppressed not only by mutations in SF3 and RES components but also by a range of mutations that disrupt the spliceosomal catalytic core element U2/U6 helix I, which is implicated in juxtaposing the 5′ splice site and branch site and in positioning metal ions for catalysis within the context of a putative catalytic triplex; indeed, mutations in this putative catalytic triplex also suppressed a prp2 mutation. Remarkably, we also found that prp2 mutations rescue lethal mutations in U2/U6 helix I. These data provide evidence that RNA elements that comprise the catalytic core are already formed at the Prp2p stage and that Prp2p destabilizes these elements, directly or indirectly, both to proofread spliceosome activation and to promote reconfiguration of the spliceosome to a fully competent, catalytic conformation.     

Saccharomyces cerevisiae NineTeen complex (NTC)-associated factor Bud31/Ycr063w assembles on precatalytic spliceosomes and improves first and second step pre-mRNA splicing efficiency

Pre-mRNA splicing occurs in spliceosomes whose assembly and activation are critical for splice site selection and catalysis. The highly conserved NineTeen complex protein complex stabilizes various snRNA and protein interactions early in the spliceosome assembly pathway. Among several NineTeen complex-associated proteins is the nonessential protein Bud31/Ycr063w, which is also a component of the Cef1p subcomplex. A role for Bud31 in pre-mRNA splicing is implicated by virtue of its association with splicing factors, but its specific functions and spliceosome interactions are uncharacterized. Here, using in vitro splicing assays with extracts from a strain lacking Bud31, we illustrate its role in efficient progression to the first catalytic step and its requirement for the second catalytic step in reactions at higher temperatures. Immunoprecipitation of functional epitope-tagged Bud31 from in vitro reactions showed that its earliest association is with precatalytic B complex and that the interaction continues in catalytically active complexes with stably bound U2, U5, and U6 small nuclear ribonucleoproteins. In complementary experiments, wherein precatalytic spliceosomes are selected from splicing reactions, we detect the occurrence of Bud31. Cross-linking of proteins to pre-mRNAs with a site-specific 4-thio uridine residue at the -3 position of exon 1 was tested in reactions with WT and bud31 null extracts. The data suggest an altered interaction between a ～25-kDa protein and this exonic residue of pre-mRNAs in the arrested bud31 null spliceosomes. These results demonstrate the early spliceosomal association of Bud31 and provide plausible functions for this factor in stabilizing protein interactions with the pre-mRNA.     

Autoregulated splicing of muscleblind-like 1 (MBNL1) Pre-mRNA

Muscleblind-like 1 (MBNL1) is a splicing factor whose improper cellular localization is a central component of myotonic dystrophy. In myotonic dystrophy, the lack of properly localized MBNL1 leads to missplicing of many pre-mRNAs. One of these events is the aberrant inclusion of exon 5 within the MBNL1 pre-mRNA. The region of the MBNL1 gene that includes exon 5 and flanking intronic sequence is highly conserved in vertebrate genomes. The 3'-end of intron 4 is non-canonical in that it contains a predicted branch point that is 141 nucleotides from the 3'-splice site and an AAG 3'-splice site. Using a minigene that includes exon 4, intron 4, exon 5, intron 5, and exon 6 of MBNL1, we showed that MBNL1 regulates inclusion of exon 5. Mapping of the intron 4 branch point confirmed that branching occurs primarily at the predicted distant branch point. Structure probing and footprinting revealed that the highly conserved region between the branch point and 3'-splice site is primarily unstructured and that MBNL1 binds within this region of the pre-mRNA. Deletion of the MBNL1 response element eliminated MBNL1 splicing regulation and led to complete inclusion of exon 5, which is consistent with the suppressive effect of MBNL1 on splicing.     

DExD/H-box Prp5 protein is in the spliceosome during most of the splicing cycle

The DExD/H-box Prp5 protein (Prp5p) is an essential, RNA-dependent ATPase required for pre-spliceosome formation during nuclear pre-mRNA splicing. In order to understand how this protein functions, we used in vitro, biochemical assays to examine its association with the spliceosome from Saccharomyces cerevisiae. GST-Prp5p in splicing assays pulls down radiolabeled pre-mRNA as well as splicing intermediates and lariat product, but reduced amounts of spliced mRNA. It cosediments with active spliceosomes isolated by glycerol gradient centrifugation. In ATP-depleted extracts, GST-Prp5p associates with pre-mRNA even in the absence of spliceosomal snRNAs. Maximal selection in either the presence or absence of ATP requires a pre-mRNA with a functional intron. Prp5p is present in the commitment complex and functions in subsequent pre-spliceosome formation. Reduced Prp5p levels decrease levels of commitment, pre-spliceosomal and spliceosomal complexes. Thus Prp5p is most likely an integral component of the spliceosome, being among the first splicing factors associating with pre-mRNA and remaining until spliceosome disassembly. The results suggest a model in which Prp5p recruits the U2 snRNP to pre-mRNA in the commitment complex and then hydrolyzes ATP to promote stable association of U2 in the pre-spliceosome. They also suggest that Prp5p could have multiple ATP-independent and ATP-dependent functions at several stages of the splicing cycle.     

GPKOW is essential for pre-mRNA splicing in?vitro and suppresses splicing defect caused by dominant-negative DHX16 mutation in?vivo

Human GPKOW [G-patch (glycine-rich) domain and KOW (Kyrpides, Ouzounis and Woese) domain] protein contains a G-patch domain and two KOW domains, and is a homologue of Arabidopsis MOS2 and Saccharomyces Spp2 protein. GPKOW is found in the human spliceosome, but its role in pre-mRNA splicing remains to be elucidated. In this report, we showed that GPKOW interacted directly with the DHX16/hPRP2 and with RNA. Immuno-depletion of GPKOW from HeLa nuclear extracts resulted in an inactive spliceosome that still bound DHX16. Adding back recombinant GPKOW restored splicing to the depleted extract. In vivo, overexpression of GPKOW partially suppressed the splicing defect observed in dominant-negative DHX16 mutant expressing cells. Mutations at the G-patch domain greatly diminished the GPKOW–DHX16 interaction; however, the mutant was active in splicing and was able to suppress splicing defect. Mutations at the KOW1 domain slightly altered the GPKOW–RNA interaction, but the mutant was less functional in vitro and in vivo. Our results indicated that GPKOW can functionally impact DHX16 but that interaction between the proteins is not required for this activity.     

Regulation of alternative RNA splicing by exon definition and exon sequences in viral and mammalian gene expression

Intron removal from a pre-mRNA by RNA splicing was once thought to be controlled mainly by intron splicing signals. However, viral and other eukaryotic RNA exon sequences have recently been found to regulate RNA splicing, polyadenylation, export, and nonsense-mediated RNA decay in addition to their coding function. Regulation of alternative RNA splicing by exon sequences is largely attributable to the presence of two majorcis-acting elements in the regulated exons, the exonic splicing enhancer (ESE) and the suppressor or silencer (ESS). Two types of ESEs have been verified from more than 50 genes or exons: purine-rich ESEs, which are the more common, and non-purine-rich ESEs. In contrast, the sequences of ESSs identified in approximately 20 genes or exons are highly diverse and show little similarity to each other. Through interactions with cellular splicing factors, an ESE or ESS determines whether or not a regulated splice site, usually an upstream 3 splice site, will be used for RNA splicing. However, how these elements function precisely in selecting a regulated splice site is only partially understood. The balance between positive and negative regulation of splice site selection likely depends on thecis-element's identity and changes in cellular splicing factors under physiological or pathological conditions.     

DEAD-box helicases as integrators of RNA,nucleotide and protein binding

DEAD-box helicases perform diverse cellular functions in virtually all steps of RNA metabolism from Bacteria to Humans. Although DEAD-box helicases share a highly conserved core domain, the enzymes catalyze a wide range of biochemical reactions. In addition to the well established RNA unwinding and corresponding ATPase activities, DEAD-box helicases promote duplex formation and displace proteins from RNA. They can also function as assembly platforms for larger ribonucleoprotein complexes, and as metabolite sensors. This review aims to provide a perspective on the diverse biochemical features of DEAD-box helicases and connections to structural information. We discuss these data in the context of a model that views the enzymes as integrators of RNA, nucleotide, and protein binding. This article is part of a Special Issue entitled: The Biology of RNA helicases — Modulation for life.     

The PNPase,exosome and RNA helicases as the building components of evolutionarily-conserved RNA degradation machines

The structure and function of polynucleotide phosphorylase (PNPase) and the exosome, as well as their associated RNA-helicases proteins, are described in the light of recent studies. The picture raised is of an evolutionarily conserved RNA-degradation machine which exonucleolytically degrades RNA from 3′ to 5′. In prokaryotes and in eukaryotic organelles, a trimeric complex of PNPase forms a circular doughnut-shaped structure, in which the phosphorolysis catalytic sites are buried inside the barrel-shaped complex, while the RNA binding domains create a pore where RNA enters, reminiscent of the protein degrading complex, the proteasome. In some archaea and in the eukaryotes, several different proteins form a similar circle-shaped complex, the exosome, that is responsible for 3′ to 5′ exonucleolytic degradation of RNA as part of the processing, quality control, and general RNA degradation process. Both PNPase in prokaryotes and the exosome in eukaryotes are found in association with protein complexes that notably include RNA helicase.     

Yeast and human RNA helicases involved in ribosome biogenesis: Current status and perspectives

Ribosome biogenesis is a fundamental process that is conserved in eukaryotes. Although spectacular progress has been made in understanding mammalian ribosome synthesis in recent years, by far, this process has still been best characterised in the yeast Saccharomyces cerevisiae. In yeast, besides the rRNAs, the ribosomal proteins and the 75 small nucleolar RNAs, more than 250 non-ribosomal proteins, generally referred to as trans-acting factors, are involved in ribosome biogenesis. These factors include nucleases, RNA modifying enzymes, ATPases, GTPases, kinases and RNA helicases. Altogether, they likely confer speed, accuracy and directionality to the ribosome synthesis process, however, the precise functions for most of them are still largely unknown. This review summarises our current knowledge on eukaryotic RNA helicases involved in ribosome biogenesis, particularly focusing on the most recent advances with respect to the molecular roles of these enzymes and their co-factors in yeast and human cells. This article is part of a Special Issue entitled: The Biology of RNA helicases—Modulation for life.     

The Ded1/DDX3 subfamily of DEAD-box RNA helicases

Abstract

In eukaryotic organisms, the orthologs of the DEAD-box RNA helicase Ded1p from yeast and DDX3 from human form a well-defined subfamily that is characterized by high sequence conservation in their helicase core and their N- and C- termini. Individual members of this Ded1/DDX3 subfamily perform multiple functions in RNA metabolism in both nucleus and cytoplasm. Ded1/DDX3 subfamily members have also been implicated in cellular signaling pathways and are targeted by diverse viruses. In this review, we discuss the considerable body of work on the biochemistry and biology of these proteins, including the recently discovered link of human DDX3 to tumorigenesis.     

Evidence that U2/U6 helix I promotes both catalytic steps of pre-mRNA splicing and rearranges in between these steps

During pre-mRNA splicing, the spliceosome must configure the substrate, catalyze 5′ splice site cleavage, reposition the substrate, and catalyze exon ligation. The highly conserved U2/U6 helix I, which adjoins sequences that define the reactive sites, has been proposed to configure the substrate for 5′ splice site cleavage and promote catalysis. However, a role for this helix at either catalytic step has not been tested rigorously and previous observations question its role at the catalytic steps. Through a comprehensive molecular genetic study of U2/U6 helix I, we found that weakening U2/U6 helix I, but not mutually exclusive structures, compromised splicing of a substrate limited at the catalytic step of 5′ splice site cleavage, providing the first compelling evidence that this helix indeed configures the substrate during 5′ splice site cleavage. Further, mutations that we proved weaken only U2/U6 helix I suppressed a mutation in PRP16, a DEAH-box ATPase required after 5′ splice site cleavage, providing persuasive evidence that helix I is destabilized by Prp16p and suggesting that this structure is unwound between the catalytic steps. Lastly, weakening U2/U6 helix I also compromised splicing of a substrate limited at the catalytic step of exon ligation, providing evidence that U2/U6 helix I reforms and functions during exon ligation. Thus, our data provide evidence for a fundamental and apparently dynamic role for U2/U6 helix I during the catalytic stages of splicing.     

An ancient anion‐binding structural module in RNA and DNA helicases

RNA and DNA helicases manipulate or translocate along single strands of nucleic acids by grasping them using a conserved structural motif. We have examined the available crystal structures of helicases of the two principal superfamilies, SF1 and SF2, and observed that the most conserved interactions with the nucleic acid occur between the phosphosugar backbone of a trinucleotide and the three strand‐helix loops within a (β‐strand/α‐helix)₃ structural module. At the first and third loops is a conserved hydrogen‐bonded feature called a thr‐motif, often seen at α‐helical N‐termini, with the threonine as the N‐cap residue. These loops can be aligned with few insertions or deletions, and their main chain atoms are structurally congruent amongst the family members and between the two modules found as tandem pairs in all SF1 and SF2 proteins. The other highly conserved interactions with nucleic acid involve mainchain NH groups, often at the helical N‐termini, interacting with phosphate groups. We comment on how the sequence motifs that are commonly used to identify helicases map to locations on the module and discuss the implications of the conserved orientation of nucleic acid on the surface of the module for directional stepping along DNA or RNA. Proteins 2010. © 2010 Wiley‐Liss, Inc.