Activating Compound | Comment | Organism | Structure |
---|---|---|---|
additional information | PARN interacts with the 7-methylguanosine cap, which enhances its deadenylation activity | Mus musculus |
Cloned (Comment) | Organism |
---|---|
The DNA fragment encoding the cap-binding domain of PARN (residues 430-516) is amplified from a cDNA clone by PCR and is subcloned into pCR2.1. The cap-binding domain is expressed with a His6-tail, a protease cleavage site, a (Gly-Gly-Ser)2-Gly sequence at the N-terminus and a Ser-Gly-Pro-Ser-Ser-Gly sequence at the C-terminus. Deletion mutants of PARN that contain the cap-binding domain and its N- and/or C-terminal flanking regions, encoding residues 420-506, 420-516, 420-536, 430-506, 430-516 and 430-536, are amplified by PCR and subcloned into pET15b. Selected point mutations are introduced into the cap-binding domain (residues 430-516) using a site-directed mutagenesis kit. The wild type and mutant forms of the cap-binding domain of PARN are overexpressed in Escherichia coli. | Mus musculus |
Protein Variants | Comment | Organism |
---|---|---|
D471A | point mutation is introduced into the cap-binding domain usin site-directed mutagenesis kit, pull-down assay shows no significant impact on the cap-binding activity of PARN | Mus musculus |
K447A | point mutation is introduced into the cap-binding domain using site-directed mutagenesis kit, pull-down assay shows no significant impact on the cap-binding activity of PARN | Mus musculus |
K450A | point mutation is introduced into the cap-binding domain using site-directed mutagenesis kit, pull-down assay shows no significant impact on the cap-binding activity of PARN | Mus musculus |
W468L | point mutation is introduced into the cap-binding domain using site-directed mutagenesis kit, mutation significantly decreases the interaction between PARN RNA-recognition motif and the cap analog, the aromatic ring of W468 is directly responsible for the cap recognition and is a functionally critical residue for the cap-binding activity of PARN RNA-recognition motif | Mus musculus |
Localization | Comment | Organism | GeneOntology No. | Textmining |
---|---|---|---|---|
cytoplasm | - |
Mus musculus | 5737 | - |
nucleus | - |
Mus musculus | 5634 | - |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
additional information | PARN is a divalent metal ion-dependent 3'-exonuclease | Mus musculus |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | Mus musculus | PARN is a divalent metal ion-dependent 3'-exonuclease that specifically catalyzes the 3'-5'-degradation of the single-stranded poly(A) tail of mRNA with a free 3'-hydroxyl group | ? | - |
? | |
additional information | Mus musculus | PARN is one of the major mammalian 3'-specific exoribonucleases involved in the degradation of the mRNA poly(A)-tail and it is also involved in the regulation of translation in early embryonic development, it is the key enzyme involved in the deadenylation of mRNA in a cap-dependent manner | ? | - |
? | |
additional information | Mus musculus | the interaction between PARN and the 7-methylguanosine cap of mRNA plays a key role in stimulating the rate of deadenylation | ? | - |
? | |
poly(A)-mRNA + H2O | Mus musculus | - |
5'-AMP | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Mus musculus | Q8VDG3 | - |
- |
Purification (Comment) | Organism |
---|---|
The wild type and mutant forms of the cap-binding domain of PARN are overexpressed in Escherichia coli BL21(DE3) cells, harvested by centrifugation and suspended in 20 mM Tris-HCl buffer containing 1 M NaCl, 30 mM imidazole, 1 mM 1,4-DL-dithiothreitol, 0.1 mg/ml lysozyme, DNaseI and protease inhibitor cocktail, and lyse with a sonicator. Cell debris and inclusion bodies are removed by centrifugation. The supernatant is loaded on a Ni2+-NTA-agarose column and the proteins are eluted with 20 mM Tris-HCl, 1 M NaCl and 200 mM imidazole. | Mus musculus |
Unlabeled and [15N], [13C]-labeled PARN cap-binding domains used for NMR experiments are synthesized by the cell-free protein expression system. After reaction, proteins are isolated by Ni2+-affinity chromatography before removing of the His6-tag. Subsequent cation-exchange chromatography yields the purified cap-binding domain. | Mus musculus |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
additional information | - |
10-mer poly(A) oligonucöleotide can not bind to the cap-binding site on PARN RNA-recognition motif | Mus musculus |
additional information | - |
Kd value of 45 microM for the interaction between PARN RNA-recognition motif and 7-methylguanosine | Mus musculus |
additional information | - |
PARN RRM has stronger affinity for the 7-methylguanosine than for the GpppG and GpppA cap analogs | Mus musculus |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | PARN is a divalent metal ion-dependent 3'-exonuclease that specifically catalyzes the 3'-5'-degradation of the single-stranded poly(A) tail of mRNA with a free 3'-hydroxyl group | Mus musculus | ? | - |
? | |
additional information | PARN is one of the major mammalian 3'-specific exoribonucleases involved in the degradation of the mRNA poly(A)-tail and it is also involved in the regulation of translation in early embryonic development, it is the key enzyme involved in the deadenylation of mRNA in a cap-dependent manner | Mus musculus | ? | - |
? | |
additional information | the interaction between PARN and the 7-methylguanosine cap of mRNA plays a key role in stimulating the rate of deadenylation | Mus musculus | ? | - |
? | |
poly(A)-mRNA + H2O | - |
Mus musculus | 5'-AMP | - |
? |
Synonyms | Comment | Organism |
---|---|---|
PARN | - |
Mus musculus |
poly(A)-specific ribonuclease | - |
Mus musculus |