Alternative splicing contributes to muscle development, but a complete set of muscle-splicing factors and their combinatorial interactions are unknown. Previous work identified ACUAA ("STAR" motif) as an enriched intron sequence near musclespecific alternative exons such as Capzb exon 9. Mass spectrometry of myoblast proteins selected by the Capzb exon 9 intron via RNA affinity chromatography identifies Quaking (QK), a protein known to regulate mRNA function through ACUAA motifs in 3 ′ UTRs. We find that QK promotes inclusion of Capzb exon 9 in opposition to repression by polypyrimidine tract-binding protein (PTB). QK depletion alters inclusion of 406 cassette exons whose adjacent intron sequences are also enriched in ACUAA motifs. During differentiation of myoblasts to myotubes, QK levels increase two-to threefold, suggesting a mechanism for QK-responsive exon regulation. Combined analysis of the PTB-and QK-splicing regulatory networks during myogenesis suggests that 39% of regulated exons are under the control of one or both of these splicing factors. This work provides the first evidence that QK is a global regulator of splicing during muscle development in vertebrates and shows how overlapping splicing regulatory networks contribute to gene expression programs during differentiation.

Nuclear pre-messenger RNA (pre-mRNA) splicing requires multiple spliceosomal small nuclear RNA (snRNA) and pre-mRNA rearrangements. Here we reveal a new snRNA conformational switch in which successive roles for two competing U2 helices, stem IIa and stem IIc, promote distinct splicing steps. When stem IIa is stabilized by loss of stem IIc, rapid ATP-independent and Cus2p-insensitive prespliceosome formation occurs. In contrast, hyperstabilized stem IIc improves the first splicing step on aberrant branchpoint pre-mRNAs and rescues temperature-sensitive U6-U57C, a U6 mutation that also suppresses first-step splicing defects of branchpoint mutations. A second, later role for stem IIa is revealed by its suppression of a cold-sensitive allele of the second-step splicing factor PRP16. Our data expose a spliceosomal progression cycle of U2 stem IIa formation, disruption by stem IIc, and then reformation of stem IIa before the second catalytic step. We propose that the competing stem IIa and stem IIc helices are key spliceosomal RNA elements that optimize juxtaposition of the proper reactive sites during splicing. Ribonucleoprotein particles (RNPs) present special challenges with respect to their assembly and function. One reason is that structural elements made from folded RNA may need to be rearranged during assembly and function. For example, the RNA that forms the core elements of the ribosome (Noller 2006), telomerase (Collins 2006), and signal recognition particle (Hainzl et al. 2005) must be properly folded to function. The same is true for the spliceosome, which catalyzes pre-messenger RNA (pre-mRNA) splicing in eukaryotes, with the added complexity that assembly of this RNP occurs each functional cycle and requires dramatic changes in RNA structure during its function (Ares and Weiser 1995; Staley and Guthrie 1998).A number of structural changes must occur in both the RNA and protein components as the spliceosome progresses through the splicing cycle. Important landmarks in the splicing cycle are the two cleavage-ligation steps that result in splicing and several structural changes that coincide with the need for ATP and one or another essential DExD/H family protein (Kramer 1996;Brow 2002). The sequential presentation of the premRNA branchpoint to the 5Ј splice site, followed by presentation of the free exon product of the first reaction to the second reactive phosphate center at the 3Ј splice site is expected to require significant substrate rearrangement between the first and second catalytic steps. To position the pre-mRNA reactive sites, the spliceosome undergoes changes in composition and structure, many of which are mediated by intra-and intermolecular small nuclear RNA (snRNA) rearrangements (Staley and Guthrie 1998;Collins and Guthrie 2001;Brow 2002;Konarska et al. 2006). Several landmark changes during the first catalytic step include the disruption of extensive basepairing between U4 and U6 snRNA, the disruption of intra-U2 and U6 snRNA helices to create a new U6-U2 snRNA interaction, and the exchang...

A screen for suppressors of a U2 snRNA mutation identified CUS2, an atypical member of the RNA recognition motif (RRM) family of RNA binding proteins. CUS2 protein is associated with U2 RNA in splicing extracts and interacts with PRP11, a subunit of the conserved splicing factor SF3a. Absence of CUS2 renders certain U2 RNA folding mutants lethal, arguing that a normal activity of CUS2 is to help refold U2 into a structure favorable for its binding to SF3b and SF3a prior to spliceosome assembly. Both CUS2 function in vivo and the in vitro RNA binding activity of CUS2 are disrupted by mutation of the first RRM, suggesting that rescue of misfolded U2 involves the direct binding of CUS2. Human Tat-SF1, reported to stimulate Tat-specific, transactivating region-dependent human immunodeficiency virus transcription in vitro, is structurally similar to CUS2. Anti-Tat-SF1 antibodies coimmunoprecipitate SF3a66 (SAP62), the human homolog of PRP11, suggesting that Tat-SF1 has a parallel function in splicing in human cells.In eukaryotes, the removal of introns from nuclear transcripts requires two transesterification reactions carried out by spliceosomes. Five small nuclear ribonucleoprotein particles (snRNPs), U1, U2, U5, U4, and U6, and many extrinsic protein factors act in concert to build a spliceosome and execute the splicing reactions (38,39,49,50,53). The spliceosome is clearly the most dynamic of the RNP enzymes. Major changes in the secondary structure of U4, U6, and U2 snRNAs during the spliceosome cycle are inferred from genetic and crosslinking studies (reviewed in reference 63). The snRNAs arrive at the assembling spliceosome in a form unlike that necessary for the catalytic activity of splicing and must be rearranged before splicing can proceed (7,45). A less studied corollary of this finding is that these rearrangements must be undone during spliceosome disassembly, and snRNA structure must be regenerated in an appropriate form for another round of spliceosome assembly and splicing (56). Although individual proteins are clearly linked to changes in the composition and organization of splicing complexes at distinct points in the splicing pathway (for a review, see reference 63), it has been difficult to assign responsibility for a specific RNA rearrangement event to any single protein.The first ATP-dependent step during in vitro spliceosome assembly is the stable binding of the U2 snRNP to the branch point region of the intron, an event normally dependent on formation of ATP-independent complexes between the premRNA and other proteins, as well as the U1 snRNP (51). These ATP-independent complexes (called the E complex in mammalian studies and commitment complexes in yeast studies) contain pre-mRNA that has been recognized both at the 5Ј splice site by the U1 snRNP and at the branch point by the homologous mammalian (SF1) or yeast (BBP) branch pointinteracting proteins (2,13,29,38,48). Formation of this complex is expected to specify an exon joining event because the 5Ј splice site and branch point are selected,...

Stable addition of U2 small nuclear ribonucleoprotein (snRNP) to form the prespliceosome is the first ATP-dependent step in splicing, and it requires the DEXD͞H box ATPase Prp5p. However, prespliceosome formation occurs without ATP in extracts lacking the U2 snRNP protein Cus2p. Here we show that Prp5p is required for the ATP-independent prespliceosome assembly that occurs in the absence of Cus2p. Addition of recombinant Cus2p can restore the ATP dependence of prespliceosome assembly, but only if it is added before Prp5p. Prp5p with an altered ATP-binding domain (Prp5-GNTp) can support growth in vivo, but only in a cus2 deletion strain, mirroring the in vitro results. Other Prp5 ATP-binding domain substitutions are lethal, even in the cus2 deletion strain, but can be suppressed by U2 small nuclear RNA mutations that hyperstabilize U2 stem IIa. We infer that the presence of Cus2p and stem IIa-destabilized forms of U2 small nuclear RNA places high demands on the ATP-driven function of Prp5p. Because Prp5p is not dispensable in vitro even in the absence of ATP, we propose that the core Prp5p function in bringing U2 to the branchpoint is not directly ATP-dependent. The positive role of Cus2p in rescuing mutant U2 can be reconciled with its antagonistic effect on Prp5 function in a model whereby Cus2p first helps Prp5p to activate the U2 snRNP for prespliceosome formation but then is displaced by Prp5p before or during the stabilization of U2 at the branchpoint.pre-mRNA splicing ͉ branch site ͉ RNA helicase ͉ commitment complex ͉ mRNA P re-mRNA splicing is a dynamic process, occurring within a large ribonucleoprotein complex called the spliceosome. Multiple ATP-dependent RNA and protein rearrangements take place before, between, and after the two transesterifications required to produce mature mRNAs (for reviews, see refs. 1-5). Important unsolved questions in splicing concern how these rearrangements are catalyzed at each ATP-dependent step. A total of eight members of the DEXD͞H family of ATPdependent RNA helicases have been assigned roles in splicing, each apparently responsible for catalyzing a specific transition in a particular splicing complex in conjunction with ATP hydrolysis (4, 5). In yeast, Prp5p and Sub2p have been implicated in U2 small nuclear ribonucleoprotein (snRNP) recruitment (6-11); Prp28p has been implicated in the addition of the U4͞U6.U5 tri-snRNP (12) and destabilization of U1 snRNP (13); Brr2p has been implicated in the destabilization of U4 snRNP before catalysis (14); Prp2p, Prp16p, and Prp22p have been implicated in the activation of the complex for the cleavage-ligation reactions of the pre-mRNA substrate (15-18); and Prp22p and Prp43p have been implicated in the release of mRNA and disassembly of the splicing complex (17-19). RNA mutations consistent with hyperstabilization or destabilization of specific spliceosomal RNA duplexes cause consistent changes in the demand for DEXD͞H protein function in genetic tests (4), but the molecular nature of these spliceosomal transitions and how the...