| 2006 |
CPSF-73 (CPSF3) is the pre-mRNA 3'-end-processing endonuclease. Crystal structure at 2.1 Å resolution revealed a metallo-β-lactamase domain and a novel β-CASP domain with a two-zinc-ion active site at their interface. Purified recombinant CPSF-73 possesses RNA endonuclease activity, and mutations disrupting zinc binding in the active site abolish this activity. |
X-ray crystallography (2.1 Å), in vitro RNA endonuclease assay with recombinant protein, active-site mutagenesis of zinc-coordinating residues |
Nature |
High |
17128255
|
| 2005 |
CPSF-73 is both the endonuclease and the 5'-3' exonuclease in histone pre-mRNA 3'-end processing. UV-crosslinking with site-specifically labeled RNA substrates (at the cleavage site) identified an ~85 kDa protein that crosslinked in a U7 snRNP-dependent manner; immunoprecipitation identified this as CPSF-73. |
UV-crosslinking with site-specific radiolabeled/phosphorothioate RNA substrates, immunoprecipitation |
Cell |
High |
16213211
|
| 2004 |
CPSF-73 contacts the pre-mRNA cleavage site in an AAUAAA-dependent manner, and mutation of key residues in the yeast CPSF-73 homolog (metallo-β-lactamase domain) causes lethality. The 3'-cleavage reaction is metal (Zn2+)-dependent, consistent with CPSF-73 being a metallo-β-lactamase-family endonuclease. |
UV-crosslinking with site-specific labeling in HeLa nuclear extract, yeast lethality assay with active-site mutants, metal-dependence assay (chelation/rescue) |
RNA (New York, N.Y.) |
High |
15037765
|
| 2009 |
CPSF73, CPSF100, and Symplekin form a stable core subcomplex that associates with histone-specific processing factors and is required for histone pre-mRNA 3'-end processing; chromatin immunoprecipitation showed that CPSF73 and Symplekin (but not CstF50) cotranscriptionally associate with histone genes. This core complex can function in two alternative cleavage machineries (poly(A) and histone mRNA). |
Co-immunoprecipitation, chromatin immunoprecipitation (ChIP), RNAi knockdown with functional pre-mRNA processing readout |
Molecular cell |
High |
19450530
|
| 2008 |
Conserved residues in the metallo-β-lactamase motifs of both CPSF73 and CPSF100 are required for the endonuclease activity that cleaves histone pre-mRNAs, indicating that CPSF73 and CPSF100 act together (analogous to homodimeric RNase Z/J family members) to form the active endonuclease. |
In vitro point mutagenesis of conserved MBL residues in mammalian CPSF73 and CPSF100, histone pre-mRNA cleavage assay |
EMBO reports |
High |
18688255
|
| 2008 |
CPSF-73 functions as a 5'-3' exonuclease that degrades the downstream cleavage product (DCP) of histone pre-mRNA in a U7 snRNP-dependent manner; degradation requires the U7 snRNP only for initiation and is processive. UV-crosslinking showed CPSF-73 directly contacts the DCP. RNAi showed that Xrn2 is not required for DCP degradation in vivo, implicating CPSF-73 as the in vivo 5'-3' exonuclease. |
UV-crosslinking with modified RNA substrates, in vitro degradation assay with U7 snRNP, RNAi knockdown of Xrn2 in HeLa cells |
Molecular and cellular biology |
High |
18955505
|
| 2012 |
The N-terminal regions of FLASH and Lsm11 form a platform that recruits a specific combination of polyadenylation factors—symplekin, CstF64, and all CPSF subunits including CPSF73—to the U7 snRNP. This interaction is abolished by point mutations in FLASH that are detrimental for processing. The same polyadenylation factors are associated with endogenous U7 snRNP and are recruited to histone pre-mRNA in a U7-dependent manner. |
Co-immunoprecipitation, pulldown assays, mass spectrometry, point mutagenesis of FLASH/Lsm11, chromatin association assays |
Molecular and cellular biology |
High |
23071092
|
| 2018 |
CPSF73 endonuclease activity is required for efficient transcriptional termination of protein-coding genes by RNA Pol II. Catalytically inactive CPSF73 cannot restore termination to cells lacking functional CPSF73, placing RNA cleavage by CPSF73 upstream of Xrn2-mediated cotranscriptional degradation and termination. CPSF73 loss causes more extensive readthrough transcription than Xrn2 elimination, indicating CPSF73 has a more foundational role. |
Conditional depletion via gene editing (auxin-inducible degron), catalytic mutant rescue experiments, mammalian native elongating transcript sequencing (mNET-seq) genome-wide |
Genes & development |
High |
29432121
|
| 2008 |
CSR1 (a tumor suppressor) binds the C-terminus of CPSF3 (amino acids 440–543) via yeast two-hybrid and further direct binding assays. This interaction causes CPSF3 translocation from the nucleus to the cytoplasm, inhibiting polyadenylation activity both in vitro and in vivo. A CSR1 mutant unable to bind CPSF3 neither re-localizes it nor inhibits polyadenylation. CPSF3 knockdown by siRNA mimics CSR1-induced cell death. |
Yeast two-hybrid, co-immunoprecipitation, subcellular fractionation/localization, in vitro and in vivo polyadenylation assay, siRNA knockdown |
Oncogene |
Medium |
18806823
|
| 2006 |
The C-terminal domain of yeast Brr5/Ysh1 (CPSF3 ortholog) lying outside β-lactamase homology is essential for cell viability and mRNA 3'-end processing. A homologous protein Syc1, by mimicking the essential Brr5 C-terminus, acts as a negative regulator of mRNA 3'-end formation. |
Yeast genetics (deletion analysis), in vitro 3'-end processing assay with yeast extracts, genetic rescue experiments |
RNA (New York, N.Y.) |
Medium |
16431986
|
| 2019 |
CPSF3 is the direct molecular target of JTE-607 (a prodrug converted by ester hydrolysis to its active form), which binds CPSF3 as identified by compound-immobilized affinity chromatography. JTE-607 treatment causes accumulation of pre-mRNAs, and CPSF3 knockdown similarly causes pre-mRNA accumulation and suppresses inflammatory cytokine expression. |
Compound-immobilized affinity chromatography, CPSF3 knockdown, pre-mRNA accumulation assay |
Biochemical and biophysical research communications |
Medium |
31399191
|
| 2019 |
CPSF3 is the target of JTE-607 in AML and Ewing's sarcoma. Chemical genetics/phenotypic screening identified CPSF3. Inhibition of CPSF3 prevents release of newly synthesized pre-mRNAs and causes transcriptional readthrough and formation of DNA-RNA R-loop structures. |
Phenotypic screening, chemical genetics, gene editing resistance mutations, pre-mRNA accumulation assays, R-loop detection |
Nature chemical biology |
High |
31819276
|
| 2019 |
Crystal structures of Cryptosporidium CPSF3 reveal that the oxaborole AN3661 blocks mRNA processing activity by binding at the metal-dependent catalytic center of CPSF3, with the oxaborole group directly occupying the active site. |
X-ray crystallography of recombinant Cryptosporidium CPSF3 with inhibitor bound, growth inhibition assay, in vivo efficacy in mouse models |
Science translational medicine |
High |
31694928
|
| 2020 |
Reconstituted recombinant U7 snRNP (all 13 components) demonstrates that CPSF73 is both an endonuclease and a 5'-3' exonuclease: it cleaves histone pre-mRNAs endonucleolytically and degrades the downstream cleavage product 5'-to-3'. Both activities require base-pairing of U7 snRNA, presence of the symplekin N-terminal domain (NTD), and are abolished by active-site mutations in CPSF73. Binding of symplekin NTD to SSU72 phosphatase also abolishes activity. |
In vitro reconstitution of recombinant U7 snRNP from 13 components, endonuclease and exonuclease assays, active-site mutagenesis of CPSF73, substrate modification studies |
RNA (New York, N.Y.) |
High |
32554553
|
| 2020 |
In yeast, the Ysh1 (CPSF3 ortholog) endonuclease is regulated by ubiquitin-mediated proteasomal degradation. Ubiquitination of Ysh1 is mediated by the Ubc4 ubiquitin-conjugating enzyme and the RING ubiquitin ligase Mpe1. Ipa1 mutation causes striking depletion of Ysh1 protein (but not mRNA), rescued by proteasome inactivation or YSH1 overexpression. |
Yeast genetics (ipa1-1 mutant), proteasome inhibition, ubiquitination assays, YSH1 overexpression rescue, mRNA level analysis |
RNA biology |
Medium |
32009536
|
| 2019 |
Yeast Ipa1 interacts with the Ysh1 (CPSF3 ortholog) endonuclease and promotes proper transcription termination and poly(A) site selection globally. Ipa1 mutation leads to decreased Ysh1 levels and poor recruitment of the cleavage/polyadenylation complex to transcribed genes. Ysh1 overexpression in the Ipa1 mutant rescues the termination defect. |
Chromatin immunoprecipitation, yeast genetic suppression (Ysh1 overexpression rescue), Pol II elongation kinetics assay |
Cell reports |
Medium |
30759400
|
| 2021 |
UBE3D, a HECT-like E3 ligase, stabilizes CPSF73 protein by preventing its ubiquitin-mediated proteasomal degradation. Depletion of UBE3D leads to CPSF73 downregulation, a pre-mRNA cleavage defect, and dysregulated gene expression. |
UBE3D depletion, ubiquitination assay, pre-mRNA cleavage assay, protein stability assay |
iScience |
Medium |
35992060
|
| 2023 |
X-ray crystallography demonstrated that benzoxaborole compounds bind to the active site of CPSF3 in a manner distinct from other known CPSF3 inhibitors. Forward genetics resistance mapping identified CPSF3 mutations that reduce benzoxaborole binding and confer resistance. Benzoxaboroles inhibit CPSF73 endonuclease activity in vitro and curb transcriptional termination in cells. |
X-ray crystallography of CPSF3-benzoxaborole complex, forward genetics resistance screen, in vitro endonuclease activity assay, transcription termination assay in cells |
Cell chemical biology |
High |
37967558
|
| 2023 |
The C-terminal domain 3 (CTD3) of CPSF73 is required for binding to Symplekin, as demonstrated by biochemical assays; the CPSF73-CPSF100 C-terminal heterodimer structure was determined by NMR for the Encephalitozoon cuniculi minimal construct, revealing extensive inter-protein contacts and TBP-like CTD2 folds. |
NMR solution structure of minimal CPSF73-CPSF100 C-terminal heterodimer, biochemical binding assays (CTD3–Symplekin interaction) |
Open biology |
Medium |
37989222
|
| 2023 |
The active site of CPSF73 contains a mixture of metal ions (Fe, Zn, Mn) rather than exclusively zinc ions. The abundance of the metal ions varies with the expression host, yet the enzyme retains endonuclease activity with less than 20% zinc occupancy, indicating that iron and/or manganese can support catalysis. |
Inductively coupled plasma mass spectrometry (ICP-MS), X-ray diffraction metal anomalous scattering, in vitro pre-mRNA cleavage assay |
The Journal of biological chemistry |
High |
36822327
|
| 2024 |
UBE3D is a binding partner and stabilizer of CPSF73 in the cytoplasm; structural studies showed that the conserved C-terminal cysteine of UBE3D likely coordinates the active-site metal ions of CPSF73, functioning as a cytoplasmic chaperone required for CPSF73's nuclear pre-mRNA processing function. Loss of UBE3D leads to CPSF73 downregulation. |
Structural studies (cryo-EM of INTS9-INTS11-BRAT1 complex providing mechanistic analogy), co-immunoprecipitation, depletion experiments with pre-mRNA processing readout |
Molecular cell |
Medium |
39032490
|
| 2024 |
RBBP6 E3 ligase mediates K63-linked ubiquitination of CPSF3, which stabilizes CPSF3 and regulates alternative polyadenylation events. RBBP6 depletion induces shortening of 3'UTRs of MYC competing-endogenous RNAs, releasing miR-590-3p to decrease MYC expression in glioblastoma stem cells. |
CRISPR/Cas9 knockout screens, Co-immunoprecipitation, ubiquitination assay (K63-linkage), 3'UTR sequencing, alternative polyadenylation profiling, CPSF3 inhibitor (JTE-607) functional assay |
Cell discovery |
Medium |
38503731
|
| 2025 |
UBE3D interacts with CPSF3 and can de-ubiquitinate CPSF3; deficiency of UBE3D leads to reduced CPSF3 protein levels in both mouse and human cells. Overexpression of dominant negative CPSF3 mutants partially reduces mRNA levels of Homeobox genes, linking CPSF3-mediated pre-mRNA 3'-end processing to Homeobox gene expression during early embryogenesis. |
Co-immunoprecipitation, CRISPR/Cas9 knockout mice, in situ hybridization, immunofluorescence, RNA-seq, dominant-negative CPSF3 overexpression |
Cell death discovery |
Medium |
40075082
|
| 2026 |
A conserved helix in the Lsm11 N-terminal extension directly contacts the metallo-β-lactamase domain of CPSF73 and is required to hold CPSF73 in the correct position for the cleavage reaction in U7 snRNP. Cryo-EM also revealed that CPSF73 can achieve an open (active) conformation independent of RNA binding in its active site. Additionally, the C-terminal end of CstF77 contacts CPSF100 at the CPSF73-CPSF100 interface, with a small effect on cleavage activity. |
Cryo-EM structure determination of reconstituted U7 snRNP complexes, mutagenesis/deletion of Lsm11 helix, in vitro pre-mRNA cleavage assays |
Nucleic acids research |
High |
41495886
|
| 2026 |
Heat shock causes loss of CPSF73 endonucleolytic cleavage activity at thousands of genes despite CPSF73 remaining present. Overexpression of RBBP6, an activator of CPSF73, during heat shock rescues the loss of cleavage and dampens readthrough transcription. Heat shock also increases Tyr1 and Ser2 phosphorylation of the Pol II CTD at 3'-ends, which is attenuated at readthrough genes. |
mNET-seq or equivalent nascent RNA sequencing, overexpression rescue of RBBP6, cleavage assay, ChIP for Pol II CTD phosphorylation marks |
Cell reports |
Medium |
41758649
|