Abstract
The hnRNP A/B paralogs A1, A2/B1 and A3 are key components of the nuclear 40S hnRNP core particles. Despite a high degree of sequence similarity, increasing evidence suggests they perform additional, functionally distinct roles in RNA metabolism. Here we identify and study the functional consequences of differential post-translational modification of hnRNPs A1, A2 and A3. We show that while arginine residues in the RGG box domain of hnRNP A1 and A3 are almost exhaustively, asymmetrically dimethylated, hnRNP A2 is dimethylated at only a single residue (Arg-254) and this modification is conserved across cell types. It has been suggested that arginine methylation regulates the nucleocytoplasmic distribution of hnRNP A/B proteins. However, we show that transfected cells expressing an A2R254A point mutant exhibit no difference in subcellular localization. Similarly, immunostaining and mass spectrometry of endogenous hnRNP A2 in transformed cells reveals a naturally-occurring pool of unmethylated protein but an exclusively nuclear pattern of localization. Our results suggest an alternative role for post-translational arginine methylation of hnRNPs and offer further evidence that the hnRNP A/B paralogs are not functionally redundant.
Highlights
In eukaryotic cell nuclei, nascent pre-mRNA transcripts are packaged into ribonucleoprotein (RNP) complexes by a group of highly conserved, abundant proteins, the heterogeneous nuclear ribonucleoproteins A/B
It had been postulated that post-translational arginine methylation regulates the nucleocytoplasmic distribution of heterogeneous nuclear ribonucleoproteins (hnRNPs) A2 [19] since deletion of most of the RGG box region (Fig. 7B), results in a four-fold increase in the cytoplasmic-tonuclear ratio of the shortened protein [19] while treatment with adenosine dialdehyde (AdOx), an indirect inhibitor of arginine methyltransferase activity, reportedly results in an altered ratio consistent with redistribution of hnRNP A2 from the nucleus to the cytoplasm [19]
The simplicity of the hnRNP A2 system, where only a single arginine is modified, allowed us to investigate the influence of arginine methylation on subcellular localization using a transiently-expressed point mutant conjugated to GFP (A2R254A)
Summary
Nascent pre-mRNA transcripts (hnRNA) are packaged into ribonucleoprotein (RNP) complexes by a group of highly conserved, abundant proteins, the heterogeneous nuclear ribonucleoproteins (hnRNPs) A/B. These complexes, visualized on electron micrographs of non-nucleolar transcription units, appear as repeating globular RNP structures approximately 250 Ain diameter [1]. The hnRNP A/B proteins were isolated from cell nuclei in the form of RNA-protein particles sedimenting at around 40S [2] and later were found to package around 500–700 nucleotides of newly transcribed RNA [3,4] The RNP particle arrangement on nascent hnRNA is nonrandom and sequence-dependent [1,3,5] and serves to condense and stabilize the transcripts and minimize tangling and knotting: this is especially relevant for long tracts of unspliced pre-mRNA [3,6,7]. Despite some progress made in determining their positioning and assembly properties during transcript packaging [4,7,8] the mechanism(s) by which this group of proteins is selected for, or excluded from nascent transcripts within the nuclear milieu, has not yet been established
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