Literature summary for 3.1.13.4 extracted from

Monecke, T.; Schell, S.; Dickmanns, A.; Ficner, R.
Crystal structure of the RRM domain of poly(A)-specific ribonuclease reveals a novel m(7)G-cap-binding mode (2008), J. Mol. Biol., 382, 827-834.

Cloned(Commentary)

Cloned (Comment)	Organism
The PARN fragment comprising amino acids 445-540 is amplified from a human cDNA library and cloned into the expression vector, the glutathione S-transferase-PARN-RNA-recognition motif fusion protein is expressed in Escherichia coli BL21(DE3). PARN mutants are generated from the wild-type clone (pGEX-6P-1 PARN445-540) using a Site-Directed Mutagenesis Kit. SeMet-containing PARN-RNA-recognition motif is expressed in Escherichia coli.	Homo sapiens

Cloned (Comment)

Organism

The PARN fragment comprising amino acids 445-540 is amplified from a human cDNA library and cloned into the expression vector, the glutathione S-transferase-PARN-RNA-recognition motif fusion protein is expressed in Escherichia coli BL21(DE3). PARN mutants are generated from the wild-type clone (pGEX-6P-1 PARN445-540) using a Site-Directed Mutagenesis Kit. SeMet-containing PARN-RNA-recognition motif is expressed in Escherichia coli.

Homo sapiens

Crystallization (Commentary)

Crystallization (Comment)	Organism
crystal structure of the PARN-RNA-recognition motif domain (residues 445-540) with a bound 7-methylguanosine triphosphate nucleotide shows a remarkable conformational flexibility of the RNA-recognition motif domain, crystal structure is refined at a resolution of 2.1 A, protein folds into a three-stranded antiparallel beta-sheet that is flanked by one alpha-helix connecting beta-strands beta1 and beta2	Homo sapiens

Crystallization (Comment)

Organism

crystal structure of the PARN-RNA-recognition motif domain (residues 445-540) with a bound 7-methylguanosine triphosphate nucleotide shows a remarkable conformational flexibility of the RNA-recognition motif domain, crystal structure is refined at a resolution of 2.1 A, protein folds into a three-stranded antiparallel beta-sheet that is flanked by one alpha-helix connecting beta-strands beta1 and beta2

Homo sapiens

Protein Variants

Protein Variants	Comment	Organism
D478A	generated by site-directed mutagenesis, Kd value for 7-methylguanosine triphosphate is 20.0 microM	Homo sapiens
K454A	generated by site-directed mutagenesis, Kd value for 7-methylguanosine triphosphate is 20.03 microM	Homo sapiens
T458A	generated by site-directed mutagenesis, Kd value for 7-methylguanosine triphosphate is 30.58 microM	Homo sapiens

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
additional information	Homo sapiens	PARN interacts not only with the 3'-end of the mRNA but also with its 5'-end as PARN contains an RNA-recognition motif domain that specifically binds both the poly(A) tail and the 7-methylguanosine cap. The interaction of PARN with the 5'-cap of mRNAs stimulates the deadenylation activity and enhances the processivity of this reaction	?	-	?

Organism

Organism	UniProt	Comment	Textmining
Homo sapiens	O95453	-	-

Purification (Commentary)

Purification (Comment)	Organism
Cell lysis is performed in a buffer containing 400 mM NaCl, 50 mM Tris/HCl, pH 8.0, 2 mM ethylenediaminetetraacetic acid and 2 mM dithiothreitol. The supernatant of the lysate is loaded onto a glutathione-Sepharose column and is eluted with lysis buffer containing 30 mM reduced glutathione. Glutathione S-transferase-PARN-RNA-recognition motif is incubated with protease and a final gel filtration is performed using a buffer containing 300 mM NaCl, 20 mM Tris/HCl, pH 8.0, and 2 mM DTT. PARN-RNA-recognition motif is concentrated to 8.8 mg/ml using a vivaspin concentrator, and 7-methylguanosine triphosphate is added in 6fold molar excess. SeMet-containing PARN-RNA-recognition motif purification is analogous, with the exception that the DTT concentration is elevated to 5 mM.	Homo sapiens

Purification (Comment)

Organism

Cell lysis is performed in a buffer containing 400 mM NaCl, 50 mM Tris/HCl, pH 8.0, 2 mM ethylenediaminetetraacetic acid and 2 mM dithiothreitol. The supernatant of the lysate is loaded onto a glutathione-Sepharose column and is eluted with lysis buffer containing 30 mM reduced glutathione. Glutathione S-transferase-PARN-RNA-recognition motif is incubated with protease and a final gel filtration is performed using a buffer containing 300 mM NaCl, 20 mM Tris/HCl, pH 8.0, and 2 mM DTT. PARN-RNA-recognition motif is concentrated to 8.8 mg/ml using a vivaspin concentrator, and 7-methylguanosine triphosphate is added in 6fold molar excess. SeMet-containing PARN-RNA-recognition motif purification is analogous, with the exception that the DTT concentration is elevated to 5 mM.

Homo sapiens

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
additional information	-	the Kd value of the wild type PARN-RNA-recognition motif enzyme for 7-methylguanosine triphosphate is 6.94 microM	Homo sapiens

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
additional information	PARN interacts not only with the 3'-end of the mRNA but also with its 5'-end as PARN contains an RNA-recognition motif domain that specifically binds both the poly(A) tail and the 7-methylguanosine cap. The interaction of PARN with the 5'-cap of mRNAs stimulates the deadenylation activity and enhances the processivity of this reaction	Homo sapiens	?	-	?

Subunits

Subunits	Comment	Organism
homodimer	gel filtration experiments show that the PARN-RNA-recognition motif domain predominantly exists as a monomer, but about 5% elute at a volume corresponding to a homodimer	Homo sapiens
monomer	gel filtration experiments show that the PARN-RNA-recognition motif domain predominantly exists as a monomer, but about 5% elute at a volume corresponding to a homodimer	Homo sapiens

Synonyms

Synonyms	Comment	Organism
PARN	-	Homo sapiens
poly(A)-specific ribonuclease	-	Homo sapiens