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TSSC4 is a component of U5 snRNP that promotes tri-snRNP formation.

ABSTRACT: U5 snRNP is a complex particle essential for RNA splicing. U5 snRNPs undergo intricate biogenesis that ensures that only a fully mature particle assembles into a splicing competent U4/U6•U5 tri-snRNP and enters the splicing reaction. During splicing, U5 snRNP is substantially rearranged and leaves as a U5/PRPF19 post-splicing particle, which requires re-generation before the next round of splicing. Here, we show that a previously uncharacterized protein TSSC4 is a component of U5 snRNP that promotes tri-snRNP formation. We provide evidence that TSSC4 associates with U5 snRNP chaperones, U5 snRNP and the U5/PRPF19 particle. Specifically, TSSC4 interacts with U5-specific proteins PRPF8, EFTUD2 and SNRNP200. We also identified TSSC4 domains critical for the interaction with U5 snRNP and the PRPF19 complex, as well as for TSSC4 function in tri-snRNP assembly. TSSC4 emerges as a specific chaperone that acts in U5 snRNP de novo biogenesis as well as post-splicing recycling.

SUBMITTER: Klimesova K

PROVIDER: S-EPMC8206348 | biostudies-literature |

REPOSITORIES: biostudies-literature

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The human U5 snRNP 52K protein (CD2BP2) interacts with U5-102K (hPrp6), a U4/U6.U5 tri-snRNP bridging protein, but dissociates upon tri-snRNP formation.

Project description:The U5 snRNP plays an essential role in both U2- and U12-dependent splicing. Here, we have characterized a 52-kDa protein associated with the human U5 snRNP, designated U5-52K. Protein sequencing revealed that U5-52K is identical to the CD2BP2, which interacts with the cytoplasmic portion of the human T-cell surface protein CD2. Consistent with it associating with an snRNP, immunofluorescence studies demonstrated that the 52K protein is predominantly located in the nucleoplasm of HeLa cells, where it overlaps, at least in part, with splicing-factor compartments (or "speckles"). We further demonstrate that the 52K protein is a constituent of the 20S U5 snRNP, but is not found in U4/U6.U5 tri-snRNPs. Thus, it is the only 20S U5-specific protein that is not integrated into the tri-snRNP and resembles, in this respect, the U4/U6 di-snRNP assembly factor Prp24p/p110. Yeast two-hybrid screening and pulldown assays revealed that the 52K protein interacts with the U5-specific 102K and 15K proteins, suggesting that these interactions are responsible for its integration into the U5 particle. The N-terminal two-thirds of 52K interact with the 102K protein, whereas its C-terminal GYF-domain binds the 15K protein. As the latter lacks a proline-rich tract, our data indicate that a GYF-domain can also engage in specific protein-protein interactions in a polyproline-independent manner. Interestingly, the U5-102K protein has been shown previously to play an essential role in tri-snRNP formation, binding the U4/U6-61K protein. The interaction of 52K with a tri-snRNP bridging protein, coupled with its absence from the tri-snRNP, suggests it might function in tri-snRNP assembly.

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Project description:The U4/U6·U5 tri-small nuclear ribonucleoprotein particle (tri-snRNP) is an essential pre-mRNA splicing factor, which is assembled in a stepwise manner before each round of splicing. It was previously shown that the tri-snRNP is formed in Cajal bodies (CBs), but little is known about the dynamics of this process. Here we created a mathematical model of tri-snRNP assembly in CBs and used it to fit kinetics of individual snRNPs monitored by fluorescence recovery after photobleaching. A global fitting of all kinetic data determined key reaction constants of tri-snRNP assembly. Our model predicts that the rates of di-snRNP and tri-snRNP assemblies are similar and that ?230 tri-snRNPs are assembled in one CB per minute. Our analysis further indicates that tri-snRNP assembly is approximately 10-fold faster in CBs than in the surrounding nucleoplasm, which is fully consistent with the importance of CBs for snRNP formation in rapidly developing biological systems. Finally, the model predicted binding between SART3 and a CB component. We tested this prediction by Förster resonance energy transfer and revealed an interaction between SART3 and coilin in CBs.

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Project description: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. We show here that Sad1 plays a role in maintaining a functional form of the tri-snRNP by promoting the association of U5 snRNP with U4/U6 di-snRNP. In the absence of Sad1, the U4/U6.U5 tri-snRNP dissociates into U5 and U4/U6 upon ATP hydrolysis and cannot bind to the spliceosome. The separated U4/U6 and U5 can reassociate upon incubation more favorably in the absence of ATP and in the presence of Sad1. Brr2 is responsible for mediating ATP-dependent dissociation of the tri-snRNP. Our results demonstrate a role of Sad1 in maintaining the integrity of the tri-snRNP by counteracting Brr2-mediated dissociation of tri-snRNP and provide insights into homeostasis of the tri-snRNP.

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Project description: Not available

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Project description:The de novo assembly and post-splicing reassembly of the U4/U6.U5 tri-snRNP remain to be investigated. We report here that ZIP, a protein containing a CCCH-type zinc finger and a G-patch domain, as characterized by us previously, regulates pre-mRNA splicing independent of RNA binding. We found that ZIP physically associates with the U4/U6.U5 tri-small nuclear ribonucleoprotein (tri-snRNP). Remarkably, the ZIP-containing tri-snRNP, which has a sedimentation coefficient of ∼35S, is a tri-snRNP that has not been described previously. We also found that the 35S tri-snRNP contains hPrp24, indicative of a state in which the U4/U6 di-snRNP is integrating with the U5 snRNP. We found that the 35S tri-snRNP is enriched in the Cajal body, indicating that it is an assembly intermediate during 25S tri-snRNP maturation. We showed that the 35S tri-snRNP also contains hPrp43, in which ATPase/RNA helicase activities are stimulated by ZIP. Our study identified, for the first time, a tri-snRNP intermediate, shedding new light on the de novo assembly and recycling of the U4/U6.U5 tri-snRNP.

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Human pre-B spliceosome and U4/U6.U5 tri-snRNP

Project description: Not available

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Project description:U4/U6.U5 tri-snRNP represents a substantial part of the spliceosome before activation. A cryo-electron microscopy structure of Saccharomyces cerevisiae U4/U6.U5 tri-snRNP at 3.7?Å resolution led to an essentially complete atomic model comprising 30 proteins plus U4/U6 and U5 small nuclear RNAs (snRNAs). The structure reveals striking interweaving interactions of the protein and RNA components, including extended polypeptides penetrating into subunit interfaces. The invariant ACAGAGA sequence of U6 snRNA, which base-pairs with the 5'-splice site during catalytic activation, forms a hairpin stabilized by Dib1 and Prp8 while the adjacent nucleotides interact with the exon binding loop 1 of U5 snRNA. Snu114 harbours GTP, but its putative catalytic histidine is held away from the ?-phosphate by hydrogen bonding to a tyrosine in the amino-terminal domain of Prp8. Mutation of this histidine to alanine has no detectable effect on yeast growth. The structure provides important new insights into the spliceosome activation process leading to the formation of the catalytic centre.

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Project description:Splicing is catalyzed by the spliceosome, a complex of five major small nuclear ribonucleoprotein particles (snRNPs). The pre-mRNA splicing factor PRPF8 is a crucial component of the U5 snRNP, and together with EFTUD2 and SNRNP200, it forms a central module of the spliceosome. Using quantitative proteomics, we identified assembly intermediates containing PRPF8, EFTUD2, and SNRNP200 in association with the HSP90/R2TP complex, its ZNHIT2 cofactor, and additional proteins. HSP90 and R2TP bind unassembled U5 proteins in the cytoplasm, stabilize them, and promote the formation of the U5 snRNP. We further found that PRPF8 mutants causing Retinitis pigmentosa assemble less efficiently with the U5 snRNP and bind more strongly to R2TP, with one mutant retained in the cytoplasm in an R2TP-dependent manner. We propose that the HSP90/R2TP chaperone system promotes the assembly of a key module of U5 snRNP while assuring the quality control of PRPF8. The proteomics data further reveal new interactions between R2TP and the tuberous sclerosis complex (TSC), pointing to a potential link between growth signals and the assembly of key cellular machines.

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