The RNA Binding Domains of the Nuclear poly(A)-binding Protein

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 ϳ11 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 basespecific 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.In the cell, mRNA molecules and their precursors are always bound by proteins. These proteins not only protect the RNA from nucleases and undesirable interactions of its highly charged surface but influence enzymes and other proteins that act upon the RNA at all stages of its maturation, function, and decay (1). Characteristically, a single RNA-binding protein very often contains more than one RNA binding domain. Different kinds of RNA binding domains have been described (2). Among them, the RNA recognition motif or RNP 1 -type RNA binding domain is probably the best understood (1, 3, 4). The RNP domain consists of ϳ90 amino acids forming a ␤␣␤␤␣␤ fold, in which a four-stranded ␤-sheet is backed by two ␣-helices. The two central antiparallel ␤-strands carry the highly conserved amino acids of the RNP1 and RNP2 motifs. Different members of the RNP protein family can bind structured or extended RNA molecules in a sequence-specific manner. As seen in several co-crystals, the RNA is bound on the surface of the ␤-sheet by hydrogen bonds and stacking interactions between bases and amino acid side chains (5-8).Another common RNA binding domain is the so-called RGG domain, characterized by multiple copies of the amino acid sequence arginine-glycine-glycine, interspersed with phenylalanine and tyrosine residues (9). The structure of the domain is not known, although a spiral of ␤-turns has been proposed based on spectroscopic data (10). A possibly related argininerich domain found at the C termini of several of the spliceosomal Sm core proteins was not ordered in a crystal structure in the absence of RNA (11). The RGG domain usually occurs in proteins in conjunction wit...

Regulation of the Nuclear Poly(A)-binding Protein by Arginine Methylation in Fission Yeast

“…5A, lanes 7 and 9). These results are consistent with studies indicating that the argininerich C-terminal domain of mammalian PABP2 contributes to RNA binding (15).…”

“…binding studies indicate that the C-terminal domain of mammalian PABP2 promotes self-association (15,51). Because our data (Fig.…”

“…It was previously reported that arginine methylated mammalian PABP2 binds synthetic poly(A) chains as efficiently as unmethylated PABP2 (15). Two different sources of PABP2 were used in these binding experiments: methylated PABP2 purified from calf thymus and unmethylated PABP2 purified from E. coli.…”

“…These results indicate that arginine methylation does not affect Pab2 ability to be imported into the nucleus. (15,50). Fission yeast Pab2 and a deletion mutant lacking the C-terminal arginine-rich domain were expressed in E. coli as GST fusions and purified on glutathione-Sepharose columns to investigate their ability to bind poly(A) RNA.…”

“…Mammalian PABP2 is characterized by a putative coiled-coil region, a single RNA recognition motif (RRM), and a C-terminal arginine-rich domain (6,13). The affinity of PAPB2 to synthetic poly(A) tails is in the nanomolar range and requires both the RRM and the C-terminal arginine-rich domain for optimal and specific interaction with poly(A) (15,16). Experiments using in vitro polyadenylation assays led to a model in which PABP2 stimulates processive poly(A) synthesis by direct and simultaneous interactions with the growing poly(A) tail and the poly(A) polymerase (17).…”