The human U5 snRNP assembly factor, AAR2, regulates gene expression dependent on the PRPF8 protein
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ABSTRACT:
PROVIDER: PRJNA758575 | ENA |
REPOSITORIES: ENA
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Project description:Small nuclear ribonucleoprotein complexes (snRNPs) represent the main subunits of the spliceosome. While the assembly of snRNP core particles has been well characterized, the incorporation of snRNP-specific proteins and the mechanisms of snRNP recycling after splicing are less well characterized. Furthermore, little is known about how targeted down-regulation of snRNP assembly factors impacts gene expression. U5 snRNP assembly in yeast requires the Aar2 protein, but the role of the human AAR2 homolog has not yet been investigated in detail. Here, we report a crystal structure of human AAR2 in complex with the RNase H-like domain of the U5-specific PRPF8 protein and in vitro interaction studies of AAR2 with U5 proteins, revealing similarities and differences in the interaction patterns compared to yeast Aar2p. Based on the structure, we probed the importance of interacting regions and residues, and designed AAR2 variants that failed to stably bind PRPF8 in vitro. SiRNA-mediated aar2 knock-down affected the expression levels of many genes. These effects were reversed by expressing siRNA-resistant PRPF8-binding AAR2 variants, but not by a phospho-mimetic AAR2 variant defective in PRPF8 binding. Our results reveal an unexpected role of an snRNP assembly factor in adjusting gene expression levels.
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Project description:U5 snRNP is a complex particle essential for RNA splicing. U5 snRNPs undergo an 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 a next round of splicing. Here, we show that a previously uncharacterized protein TSSC4 is a new 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.
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Project description:Pre mRNA splicing is orchestrated by the spliceosome; a dynamic and highly regulated ribonucleoprotein complex composed of five small nuclear ribonucleoprotein particles (snRNPs). Despite extensive studies, the biogenesis of snRNPs remains incompletely understood. Here, we identify neurochondrin (NCDN) as a critical regulator of pre-U5 snRNP biogenesis in the cytoplasm. NCDN directly binds to AAR2 and facilitates its release from the PRPF8−AAR2−EFTUD2 assembly intermediate, a key step required for U5 snRNP maturation. Loss of NCDN causes the accumulation of this intermediate, resulting in a decreased level of mature U5 snRNP. Spliceosome dysregulation often leads to alternative splicing abnormalities implicated in cancer. Indeed, NCDN deficiency suppresses tumor cell proliferation and induces apoptosis, while high NCDN expression promotes tumor cell growth and correlates with poor survival in glioblastoma patients. Transcriptome analysis reveals that loss of NCDN causes widespread alternative splicing defects and changes in gene expression. Aberrant splicing of tumor-associated genes may contribute to tumor progression in glioblastoma. Collectively, these results establish NCDN as an essential chaperone factor for U5 snRNP assembly and spliceosome function, and highlight NCDN as a potential therapeutic target in the treatment of glioma with elevated NCDN expression.
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Project description:The human ecdysoneless protein (ECD) plays a pivotal role in cellular and physiological processes, including the regulation of the cell cycle, cell survival, and embryogenesis. More recently, ECD has been implicated in RNA biogenesis through its regulatory effects on mRNA nuclear export and RNA splicing. In the current study, we identified the mechanism of ECD’s function in RNA splicing. We demonstrate a novel role of ECD as an RNA-binding protein and, through mutational analyses, we show that it utilizes its N-terminal region to interact with RNAs. By employing unbiased approaches such as eCLIP-seq and RNA-seq analyses in hTERT-immortalized mammary epithelial cells (76NTERT), we demonstrate that ECD directly binds to RNA and predominantly utilizes its RNA-binding abilities to regulate RNA splicing. This regulation occurs either through direct binding of ECD to RNA sequences located near splicing sites, one of the top ECD binding motifs identified being purine-rich exonic splicing enhancers or by influencing the biogenesis of the U5 small nuclear ribonucleoprotein particle (U5 snRNP) complex via its direct interaction with U5 snRNA. Notably, this interaction not only maintains the expression of U5 snRNA but also is essential for maintenance of protein components of the U5 snRNP complex, such as PRPF8 and EFTUD2. Consistently, ECD KO in 76NTERT cells lead to widespread aberrant splicing of numerous RNAs leading to alteration in expression of several genes. Importantly, ECD KO or the loss of its RNA-binding function resulted in the downregulation of U5 snRNP components, which functionally culminates into cellular proliferation block. Significantly, full-length ECD but not its RNA binding site mutant failed to rescue cell proliferation blocks upon ECD KO. Collectively, we provide compelling evidence that ECD regulates RNA splicing by directly associating with U5 snRNP and regulating U5snRNP complex to maintain cell proliferation and survival.
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Project description:U1 snRNP plays an essential role in initiating spliceosome assembly, yet the mechanism underlying its synergy with other splicing regulators for efficient spliceosome assembly remains elusive. Here we identify ZFP207 as a key regulator of U1 snRNP function that substantially promotes spliceosome assembly. Acute depletion of ZFP207 results in a series of molecular phenotypes indicative of U1 snRNP dysregulation. Mechanistically, the N-terminal zinc finger domains of ZFP207 directly bind to the stem-loop 3 (SL3) of U1 snRNA, while its C-terminal intrinsically disordered regions (IDRs) undergo phase separation to form biomolecular condensates with U1 snRNP. These condensates create a crowded molecular environment that increases the local concentration of splicing snRNPs and regulators, thereby accelerating the speed of spliceosome assembly by facilitating interactions between U1 snRNP and other snRNPs. Collectively, our study demonstrates the critical role of phase separation in ensuring effective U1 snRNP function and promoting efficient spliceosome assembly.
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Project description:U1 snRNP plays an essential role in initiating spliceosome assembly, yet the mechanism underlying its synergy with other splicing regulators for efficient spliceosome assembly remains elusive. Here we identify ZFP207 as a key regulator of U1 snRNP function that substantially promotes spliceosome assembly. Acute depletion of ZFP207 largely recapitulates the molecular phenotypes observed with the depletion of SNRNP70, a core component of U1 snRNP. Mechanistically, the N-terminal zinc finger domains of ZFP207 directly bind to U1 snRNA, while its C-terminus undergoes phase separation via intrinsically disordered regions (IDRs) to forms biomolecular condensates with U1 snRNP. These condensates create a crowded molecular environment that increases the local concentration of splicing snRNPs and regulators, thereby accelerating the speed of spliceosome assembly by facilitating interactions between U1 snRNP and other snRNPs. Collectively, our study demonstrates the critical role of phase separation in ensuring proper U1 snRNP function and efficient spliceosome assembly.
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Project description:U1 snRNP plays an essential role in initiating spliceosome assembly, yet the mechanism underlying its synergy with other splicing regulators for efficient spliceosome assembly remains elusive. Here we identify ZFP207 as a key regulator of U1 snRNP function that substantially promotes spliceosome assembly. Acute depletion of ZFP207 largely recapitulates the molecular phenotypes observed with the depletion of SNRNP70, a core component of U1 snRNP. Mechanistically, the N-terminal zinc finger domains of ZFP207 directly bind to U1 snRNA, while its C-terminus undergoes phase separation via intrinsically disordered regions (IDRs) to forms biomolecular condensates with U1 snRNP. These condensates create a crowded molecular environment that increases the local concentration of splicing snRNPs and regulators, thereby accelerating the speed of spliceosome assembly by facilitating interactions between U1 snRNP and other snRNPs. Collectively, our study demonstrates the critical role of phase separation in ensuring proper U1 snRNP function and efficient spliceosome assembly.
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Project description:U1 snRNP plays an essential role in initiating spliceosome assembly, yet the mechanism underlying its synergy with other splicing regulators for efficient spliceosome assembly remains elusive. Here we identify ZFP207 as a key regulator of U1 snRNP function that substantially promotes spliceosome assembly. Acute depletion of ZFP207 recapitulates the molecular phenotypes observed with the depletion of SNRNP70, a core component of U1 snRNP. Mechanistically, the N-terminal zinc finger domains of ZFP207 directly bind to U1 snRNA, while its C-terminus undergoes phase separation via intrinsically disordered regions (IDRs). The coordination between the N-terminus and C-terminus of ZFP207 drives the formation of biomolecular condensate with U1 snRNP, which creates a molecular environment to promote spliceosome assembly by facilitating the interactions between U1 snRNP and other splicing regulators. Collectively, our study demonstrates the critical role of ZFP207-mediated phase separation in ensuring proper U1 snRNP function and spliceosome assembly.
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