Localized Inhibition of Protein Phosphatase 1 by NUAK1 Promotes Spliceosome Activity and Reveals a MYC-Sensitive Feedback Control of Transcription
Giacomo CossaIsabelle RoeschertFlorian PrinzApoorva BaluapuriRaphael VidalChristina Schülein‐VölkYun-Chien ChangCarsten P. AdeGuido MastrobuoniCyrille GirardAmit KumarLars WortmannSusanne WalzReinhard LührmannStefan KempaBernhard KüsterElmar WolfDominik MumbergMartin Eilers
50
Citation
98
Reference
10
Related Paper
Citation Trend
Abstract:
Deregulated expression of MYC induces a dependence on the NUAK1 kinase, but the molecular mechanisms underlying this dependence have not been fully clarified. Here, we show that NUAK1 is a predominantly nuclear protein that associates with a network of nuclear protein phosphatase 1 (PP1) interactors and that PNUTS, a nuclear regulatory subunit of PP1, is phosphorylated by NUAK1. Both NUAK1 and PNUTS associate with the splicing machinery. Inhibition of NUAK1 abolishes chromatin association of PNUTS, reduces spliceosome activity, and suppresses nascent RNA synthesis. Activation of MYC does not bypass the requirement for NUAK1 for spliceosome activity but significantly attenuates transcription inhibition. Consequently, NUAK1 inhibition in MYC-transformed cells induces global accumulation of RNAPII both at the pause site and at the first exon-intron boundary but does not increase mRNA synthesis. We suggest that NUAK1 inhibition in the presence of deregulated MYC traps non-productive RNAPII because of the absence of correctly assembled spliceosomes.Keywords:
Spliceosome
RNA polymerase II
Transcription
Protein phosphatase 1
The spliceosome is a dynamic RNA‐protein macromolecular machine that is responsible for the splicing of intronic sequences from pre‐mRNA. The spliceosome undergoes major structural changes during the splicing reaction and its components must be recycled for each new round of splicing. Although the spliceosome cycle has been extensively studied at the molecular level, little is known about the dynamics of splicing and spliceosome assembly in living cells. We have been using Photobleaching and Photoactivation microscopy techniques to study how splicing factors are recruited to nascent transcripts in the living cell nucleus. Currently, we are developing tools to visualize in real‐time the dynamics of RNA Pol II transcription, splicing and spliceosome assembly.
Spliceosome
Minor spliceosome
Polypyrimidine tract
Splicing factor
Transcription
Exonic splicing enhancer
Precursor mRNA
Cite
Citations (0)
The Prp19-associated complex (NTC) is essential for pre-mRNA splicing and is associated with the spliceosome during spliceosome activation. NTC is required for specifying interactions of U5 and U6 with pre-mRNA to stabilize their association with the spliceosome after dissociation of U4. Here, we show that a novel splicing factor, Yju2, is associated with components of NTC, and that it is required for pre-mRNA splicing both in vivo and in vitro. During spliceosome assembly, Yju2 is associated with the spliceosome at nearly the same time as NTC but is destabilized after the first catalytic reaction, whereas other NTC components remain associated until the reaction is complete. Extracts depleted of Yju2 could be complemented by recombinant Yju2, suggesting that Yju2 and NTC are not entirely in association with each other. Yju2 is not required for the binding of NTC to the spliceosome or for NTC-mediated spliceosome activation. Complementation analysis of the affinity-isolated spliceosome formed in Yju2-depleted extracts demonstrated that Yju2 acts in concert with an unidentified heat-resistant factor(s) in an ATP-independent manner to promote the first catalytic reaction of pre-mRNA splicing after Prp2-mediated structural rearrangement of the spliceosome.
Spliceosome
Splicing factor
Minor spliceosome
Polypyrimidine tract
Cite
Citations (78)
The spliceosome catalyzes pre-mRNA splicing in two steps. After catalytic step I, a major remodeling of the spliceosome occurs to establish the active site for step II. Here, we report the isolation of a cDNA encoding hSlu7, the human homolog of the yeast second step splicing factor Slu7. We show that hSlu7 associates with the spliceosome late in the splicing pathway, but at a stage prior to recognition of the 3' splice site for step II. In the absence of hSlu7, splicing is stalled between the catalytic steps in a novel complex, the CDeltahSlu7 complex. We provide evidence that this complex differs significantly in structure from the known spliceosomal complexes, yet is a functional intermediate between the catalytic steps of splicing. Together, our observations indicate that hSlu7 is required for a structural alteration of the spliceosome prior to the establishment of the catalytically active spliceosome for step II.
Spliceosome
Splicing factor
Polypyrimidine tract
Exonic splicing enhancer
Minor spliceosome
Prp24
Cite
Citations (55)
Spliceosome
Minor spliceosome
Polypyrimidine tract
Splicing factor
Precursor mRNA
Exonic splicing enhancer
Cite
Citations (360)
Pre-messenger RNA splicing involves multi-step assembly of the large spliceosome complexes that catalyse the two consecutive trans-esterification reactions, resulting in intron removal. There is evidence that proof-reading mechanisms monitor the fidelity of this complex process. Transcripts that fail these fidelity tests are thought to be directed to degradation pathways, permitting the splicing factors to be recycled. While studying the roles of splicing factors in vivo, in budding yeast, we performed targeted depletion of individual proteins, and analysed the effect on co-transcriptional spliceosome assembly and splicing efficiency. Unexpectedly, depleting factors such as Prp16 or Prp22, that are known to function at the second catalytic step or later in the splicing pathway, resulted in a defect in the first step of splicing, and accumulation of arrested spliceosomes. Through a kinetic analysis of newly synthesized RNA, we observed that a second step splicing defect (the primary defect) was rapidly followed by the first step of splicing defect. Our results show that knocking down a splicing factor can quickly lead to a recycling defect with splicing factors sequestered in stalled complexes, thereby limiting new rounds of splicing. We demonstrate that this 'feed-back' effect can be minimized by depleting the target protein more gradually or only partially, allowing a better separation between primary and secondary effects. Our findings indicate that splicing surveillance mechanisms may not always cope with spliceosome assembly defects, and suggest that work involving knock-down of splicing factors or components of other large complexes should be carefully monitored to avoid potentially misleading conclusions.
Spliceosome
Exonic splicing enhancer
Splicing factor
Protein splicing
Prp24
Polypyrimidine tract
Precursor mRNA
Cite
Citations (13)
Small molecule inhibitors that target components of the spliceosome have great potential as tools to probe splicing mechanism and dissect splicing regulatory networks in cells. These compounds also hold promise as drug leads for diseases in which splicing regulation plays a critical role, including many cancers. Because the spliceosome is a complicated and dynamic macromolecular machine comprised of many RNA and protein components, a variety of compounds that interfere with different aspects of spliceosome assembly is needed to probe its function. By screening chemical libraries with high-throughput splicing assays, several labs have added to the collection of splicing inhibitors, although the mechanistic insight into splicing yielded from the initial compound hits is somewhat limited so far. In contrast, SF3B1 inhibitors stand out as a great example of what can be accomplished with small molecule tools. This group of compounds were first discovered as natural products that are cytotoxic to cancer cells, and then later shown to target the core spliceosome protein SF3B1. The inhibitors have since been used to uncover details of SF3B1 mechanism in the spliceosome and its impact on gene expression in cells. Continuing structure activity relationship analysis of the compounds is also making progress in identifying chemical features key to their function, which is critical in understanding the mechanism of SF3B1 inhibition. The knowledge is also important for the design of analogs with new and useful features for both splicing researchers and clinicians hoping to exploit splicing as pressure point to target in cancer therapy. WIREs RNA 2017, 8:e1381. doi: 10.1002/wrna.1381 For further resources related to this article, please visit the WIREs website.
Spliceosome
Minor spliceosome
Cite
Citations (158)
The NineTeen Complex (NTC) of proteins associates with the spliceosome during pre-mRNA splicing and is essential for both steps of intron removal. The NTC and other NTC-associated proteins are recruited to the spliceosome where they participate in regulating the formation and progression of essential spliceosome conformations required for the two steps of splicing. It is now clear that the NTC is an integral component of active spliceosomes from yeast to humans and provides essential support for the spliceosomal snRNPs (small nuclear ribonucleoproteins). In the present article, we discuss the identification and characterization of the yeast NTC and review recent work in yeast that supports the essential role for this complex in the regulation and fidelity of splicing.
Spliceosome
snRNP
Minor spliceosome
Splicing factor
Small nuclear ribonucleoprotein
Cite
Citations (105)
The spliceosome is the macromolecular machine responsible for pre-mRNA splicing, an essential step in eukaryotic gene expression. During splicing, myriad subunits join and leave the spliceosome as it works on the pre-mRNA substrate. Strikingly, there are very few small molecules known to interact with the spliceosome. Splicing inhibitors are needed to capture transient spliceosome conformations and probe important functional components. Such compounds may also have chemotherapeutic applications, as links between splicing and cancer are increasingly uncovered. To identify new splicing inhibitors, we developed a high-throughput assay for in vitro splicing using a reverse transcription followed by quantitative PCR readout. In a pilot screen of 3080 compounds, we identified three small molecules that inhibit splicing in HeLa extract by interfering with different stages of human spliceosome assembly. Two of the compounds similarly affect spliceosomes in yeast extracts, suggesting selective targeting of conserved components. By examining related molecules, we identified chemical features required for the activity of two of the splicing inhibitors. In addition to verifying our assay procedure and paving the way to larger screens, these studies establish new compounds as chemical probes for investigating the splicing machinery.
Spliceosome
Prp24
Precursor mRNA
Cite
Citations (34)
Abstract Human pre-catalytic spliceosomes contain several proteins that associate transiently just prior to spliceosome activation and are absent in yeast, suggesting that this critical step is more complex in higher eukaryotes. We demonstrate via RNAi coupled with RNA-Seq that two of these human-specific proteins, Smu1 and RED, function both as alternative splicing regulators and as general splicing factors and are required predominantly for efficient splicing of short introns. In vitro splicing assays reveal that Smu1 and RED promote spliceosome activation, and are essential for this step when the distance between the pre-mRNA’s 5′ splice site (SS) and branch site (BS) is sufficiently short. This Smu1-RED requirement can be bypassed when the 5′ and 3′ regions of short introns are physically separated. Our observations suggest that Smu1 and RED relieve physical constraints arising from a short 5′SS-BS distance, thereby enabling spliceosomes to overcome structural challenges associated with the splicing of short introns.
Spliceosome
Minor spliceosome
Group II intron
Splicing factor
Exonic splicing enhancer
Cite
Citations (32)
Removal of introns from pre-mRNA is an essential step of gene expression. The splicing reaction is catalyzed in a large complex termed the spliceosome. Introns are recognized during the early steps of spliceosome assembly with the formation of commitment complexes. Intron recognition is mediated by the interaction of splicing factors with conserved sequences present in the pre-mRNA. BBP/SF1 participates in this recognition by interacting with the pre-mRNA branch point in both yeast and mammals. This protein, which is essential in yeast, also interacts with the U2AF65/Mud2 splicing factor. However, its precise role in splicing complex formation is still unclear. We have now analyzed the presence of BBP and Mud2 in yeast splicing complexes using supershift and coprecipitation assays. We found that BBP is present together with Mud2 in commitment complex 2 (CC2), but is not detectable in commitment complex 1 (CC1). Furthermore, genetic and biochemical depletion of BBP demonstrated that it is required for CC2 formation. In addition we observed that BBP and Mud2 are not detectable in pre-spliceosomes. These are the first commitment complex components that are shown to be released during or immediately after pre-spliceosome formation. Interestingly, depletion of BBP or disruption of MUD2 had no significant effect on pre-spliceosome formation and splicing in vitro but led to a transient accumulation of CC1. These observations support a model in which BBP and Mud2 are recycled during transition from CC2 to pre-spliceosome.
Spliceosome
Splicing factor
Precursor mRNA
Cite
Citations (95)