Literature summary for 3.1.13.4 extracted from

Nagata, T.; Suzuki, S.; Endo, R.; Shirouzu, M.; Terada, T.; Inoue, M.; Kigawa, T.; Kobayashi, N.; Guentert, P.; Tanaka, A.; Hayashizaki, Y.; Muto, Y.; Yokoyama, S.
The RRM domain of poly(A)-specific ribonuclease has a noncanonical binding site for mRNA cap analog recognition (2008), Nucleic Acids Res., 36, 4754-4767.

Activating Compound

Activating Compound	Comment	Organism	Structure
additional information	PARN interacts with the 7-methylguanosine cap, which enhances its deadenylation activity	Mus musculus

Cloned(Commentary)

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

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

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

Localization	Comment	Organism	GeneOntology No.	Textmining
cytoplasm	-	Mus musculus	5737	-
nucleus	-	Mus musculus	5634	-

Metals/Ions

Metals/Ions	Comment	Organism	Structure
additional information	PARN is a divalent metal ion-dependent 3'-exonuclease	Mus musculus

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
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

Organism	UniProt	Comment	Textmining
Mus musculus	Q8VDG3	-	-

Purification (Commentary)

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

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 [micromol/min/mg]

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 and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
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

Synonyms	Comment	Organism
PARN	-	Mus musculus
poly(A)-specific ribonuclease	-	Mus musculus