Abstract
The nuclear poly(A)-binding protein (PABPN1) is involved in the synthesis of the mRNA poly(A) tails in most eukaryotes. We report that the protein contains two RNA binding domains, a ribonucleoprotein-type RNA binding domain (RNP domain) located approximately in the middle of the protein sequence and an arginine-rich C-terminal domain. The C-terminal domain also promotes self-association of PABPN1 and moderately cooperative binding to RNA. Whereas the isolated RNP domain binds specifically to poly(A), the isolated C-terminal domain binds non-specifically to RNA and other polyanions. Despite this nonspecific RNA binding by the C-terminal domain, selection experiments show that adenosine residues throughout the entire minimal binding site of approximately 11 nucleotides are recognized specifically. UV-induced cross-links with oligo(A) carrying photoactivatable nucleotides at different positions all map to the RNP domain, suggesting that most or all of the base-specific contacts are made by the RNP domain, whereas the C-terminal domain may contribute nonspecific contacts, conceivably to the same nucleotides. Asymmetric dimethylation of 13 arginine residues in the C-terminal domain has no detectable influence on the interaction of the protein with RNA. The N-terminal domain of PABPN1 is not required for RNA binding but is essential for the stimulation of poly(A) polymerase.
Highlights
In the cell, mRNA molecules and their precursors are always bound by proteins
We report that the protein contains two RNA binding domains, a ribonucleoprotein-type RNA binding domain (RNP domain) located approximately in the middle of the protein sequence and an arginine-rich C-terminal domain
A significantly improved expression was achieved by use of a partially synthetic cDNA in which the GC content of the first 300 nucleotides was reduced to 47%, compared with 80% in the wild-type sequence, without a change in the encoded amino acid sequence
Summary
DNA—Sequences of DNA oligonucleotides used in the following procedures are available upon request. Gel-purified C-spiked A12 and homogeneous A12 used as a control were 5Ј-labeled, and about 2.5 nM labeled oligonucleotides (as 5Ј-ends) were incubated at ambient temperature for 15 min in 100 l of RNA binding buffer containing different concentrations of calf thymus PABPN1. Binding reactions were done in 100 l of RNA binding buffer (see above) that contained 2 nM (as 5Ј-ends) of either of the two modified oligonucleotides in the presence of either 50 nM His-tagged wild-type PABPN1 or 500 nM of the deletion variants, respectively. After 30 min of irradiation at 312 nm, the aliquots from each binding reaction were recombined, and 20 l of each irradiated RNA/ protein mix were digested with 200 ng of protease Lys-C (sequencing grade; Roche Diagnostic) at ambient temperature. The gel was dried, and radioactive proteins were identified by phosphorimaging
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