RESUMO
Cleavage and polyadenylation specificity factor 30 (CPSF30) is a zinc finger protein that regulates pre-mRNA processing. CPSF30 contains five CCCH domains and one CCHC domain and recognizes two conserved 3' pre-mRNA sequences: an AU hexamer and a U-rich motif. AU hexamer motifs are common in pre-mRNAs and are typically defined as AAUAAA. Variations within the AAUAAA hexamer occur in certain pre-mRNAs and can affect polyadenylation efficiency or be linked to diseases. The effects of disease-related variations on CPSF30/pre-mRNA binding were determined using a construct of CPSF30 that contains just the five CCCH domains (CPSF30-5F). Bioinformatics was utilized to identify the variability within the AU hexamer sequence in pre-mRNAs. The effects of this sequence variability on CPSF30-5F/RNA binding affinities were measured. Bases at positions 1, 2, 4, and 5 within the AU hexamer were found to be important for RNA binding. Bioinformatics revealed that the three bases flanking the AU hexamer at the 5' and 3' ends are twice as likely to be adenine or uracil as guanine and cytosine. The presence of A and U residues in these flanking regions was determined to promote higher-affinity CPSF30-5F/RNA binding than G and C residues. The addition of the zinc knuckle domain to CPSF30-5F (CPSF30-FL) restored binding to AU hexamer variants. This restoration of binding is connected to the presence of a U-rich sequence within the pre-mRNA to which the zinc knuckle binds. A mechanism of differential RNA binding by CPSF30, modulated by accessibility of the two RNA binding sites, is proposed.
Assuntos
Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Poli U/metabolismo , Poliadenilação , Precursores de RNA/metabolismo , RNA Mensageiro/metabolismo , Animais , Sítios de Ligação , Bovinos , Fator de Especificidade de Clivagem e Poliadenilação/química , Fator de Especificidade de Clivagem e Poliadenilação/genética , Ligação Proteica , Precursores de RNA/genética , RNA Mensageiro/química , RNA Mensageiro/genética , Dedos de ZincoRESUMO
Cleavage and polyadenylation specificity factor 30 (CPSF30) is a key protein involved in pre-mRNA processing. CPSF30 contains five Cys3His domains (annotated as "zinc-finger" domains). Using inductively coupled plasma mass spectrometry, X-ray absorption spectroscopy, and UV-visible spectroscopy, we report that CPSF30 is isolated with iron, in addition to zinc. Iron is present in CPSF30 as a 2Fe-2S cluster and uses one of the Cys3His domains; 2Fe-2S clusters with a Cys3His ligand set are rare and notably have also been identified in MitoNEET, a protein that was also annotated as a zinc finger. These findings support a role for iron in some zinc-finger proteins. Using electrophoretic mobility shift assays and fluorescence anisotropy, we report that CPSF30 selectively recognizes the AU-rich hexamer (AAUAAA) sequence present in pre-mRNA, providing the first molecular-based evidence to our knowledge for CPSF30/RNA binding. Removal of zinc, or both zinc and iron, abrogates binding, whereas removal of just iron significantly lessens binding. From these data we propose a model for RNA recognition that involves a metal-dependent cooperative binding mechanism.
Assuntos
Fator de Especificidade de Clivagem e Poliadenilação/química , Ferro/química , Sinais de Poliadenilação na Ponta 3' do RNA/genética , RNA Mensageiro/química , Enxofre/química , Fatores de Poliadenilação e Clivagem de mRNA/química , Sítios de Ligação , Fator de Especificidade de Clivagem e Poliadenilação/genética , Simulação por Computador , Humanos , Modelos Químicos , Poliadenilação/genética , Ligação Proteica , Fatores de Poliadenilação e Clivagem de mRNA/genéticaRESUMO
Neural Zinc Finger Factor-1 (NZF-1) and Myelin Transcription Factor 1 (MyT1) are two homologous nonclassical zinc finger (ZF) proteins that are involved in the development of the central nervous system (CNS). Both NZF-1 and MyT1 contain multiple ZF domains, each of which contains an absolutely conserved Cys2His2Cys motif. All three cysteines and the second histidine have been shown to coordinate Zn(II); however, the role of the first histidine remains unresolved. Using a functional form of NZF-1 that contains two ZF domains (NZF-1-F2F3), mutant proteins in which each histidine was sequentially mutated to a phenylalanine were prepared to determine the role(s) of the histidine residues in DNA recognition. When the first histidine is mutated, the protein binds Zn(II) in an analogous manner to the native protein. Surprisingly, this mutant does not bind to target DNA (ß-RAR), suggesting that the noncoordinating histidine is critical for sequence selective DNA recognition. The first histidine will coordinate Zn(II) when the second histidine is mutated; however, the overall fold of the protein is perturbed leading to abrogation of DNA binding. NZF-1-F2F3 selectively binds to a specific DNA target sequence (from ß-RAR) with high affinity (nM); while its homologue MyT1 (MyT1-F2F3), which is 92% identical to NZF-1-F2F3, binds to this same DNA sequence nonspecifically. A single, nonconserved amino acid residue in NZF-1-F2F3 is shown to be responsible for this high affinity DNA binding to ß-RAR. When this residue (arginine) is engineered into the MyT1-F2F3 sequence, the affinity for ß-RAR DNA increases.