Arginine methylation of hnRNP A2 does not directly govern its subcellular localization

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 A2(R254A) 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.

Figure 1. HnRNP A/B proteins in oligonucleotide pulldowns.

Proteins pulled down from rat brain lysate using either unlabelled or dTelo4 oligonucleotide-labelled magnetic beads were resolved by SDS-PAGE and either stained with Coomassie Blue G250, or transferred to an Immobilon-Fl membrane for Western blotting. HnRNPs A1 and A2/B1 were identified by the application of anti-hnRNP A1 (Sigma, clone 4B10) followed by secondary anti-mouse IgG IRD800 or anti-rabbit A2/B1 followed by secondary anti-rabbit IgG Alexa-680 fluorophore.

Figure 2. Detection of diagnostic arginine and asymmetric dimethylarginine ions.

Zoomed view from region m/z 20–175 of MALDI-TOF/TOF analysis of (A) m/z 623.2, corresponding to unmodified peptide 251-GGGRGGY-257 from recombinant hnRNP A2 and (B) m/z 651.3, corresponding to peptide 251-GGGRGGY-257 with a dimethylarginine modification from HPLC-purified rat brain hnRNP A2. Structures representing diagnostic ions of arginine* and dimethylarginine** are labeled as previously described . See Supporting Information Fig. S4 for full spectrum.

Figure 3. Edman degradation detects methylated arginine residues.

Elution profiles from rat brain (A) and HeLa cell hnRNP A2 (B) showing degradation cycles 10–12 aligned with standards (stds) containing a mixture of unmodified (R), monomethylated (MMA), asymmetrically dimethylated (aDMA), and symmetrically dimethylated (sDMA) arginine residues. In each case, the residue released in cycle 11 is residue 254. The retention time of the residue released in this cycle indicates that residue 254 of hnRNP A2 is aDMA (i.e. N ^G,N ^G–dimethylarginine). In both cases, a proportion of unmodified arginine and MMA is observed that is small in the rat brain protein but significant for the protein isolate from HeLa cells. No sDMA is detected in either sample. The retention time of amino acids was determined by comparison to chemically pure standards (trace labelled “stds” at the bottom of each panel). The transposition of retention times observed for aDMA and tyrosine (Y) between panels A and B was caused by a small change in the HPLC buffer.

Figure 4. LC-MS/MS identification of monomethylated and unmodified Arg-254 residues in hnRNP A2 sourced from different cell types.

Comparative LC-MS/MS analysis of the peptide 244-FGGSPGYGGGRGGYGGGGPGYGNQGGGYGGGY-275, derived from hnRNP A2 protein isolated by pulldown assay (boxed band indicated in representative Coomassie-stained gel lanes, shown as insets). The modified peptides were obtained by thermolysin/AspN double digestion of endogenous protein purified from either (A) rat brain tissue homogenate, (B) HeLa, (C) B104 or (D) SH-SY5Y cultured cell lysates. The position of ion series indicative of either unmodified Arg, MMA or DMA at position Arg-254 is shown. The box identifies the band extracted for digestion and LC-MS/MS analysis. A cluster of peaks (rather than a single ion) is observed at each m/z, due to the natural abundance of heavy isotopes (predominantly ¹³C).

Figure 5. Nuclear localization of transiently expressed wild type and A2^R254A in interphase HeLa cells.

HeLa cells were transfected with GFP vector (first row) wild-type A2-GFP (second row) or A2^R254A-GFP. For the control vector, GFP is diffusely localized throughout out the nucleus and extranuclear region. Both A2-GFP and the A2^R254A-GFP point mutant, which lacks the sole methylated arginine residue, are exclusively observed in the nucleus. The signal from Hoechst dye (blue) is overlaid on both the GFP signal (centre panels) and images obtained by digital interference contrast (DIC) microscopy (right panels) to highlight the location of the nucleus. Scale bar = 10 µm.

Figure 6. Nuclear localization of endogenous methylated and unmethylated hnRNP A2 in interphase cells.

Endogenous A2/B1 in primary (A) rat brain hippocampal neurons and (B) HeLa, B104 and SH-SY5Y cells was stained with rabbit polyclonal A2/B1 antibody followed by anti-rabbit Alexafluor-488-conjugated secondary antibody and imaged by confocal microscopy. While the four cell systems imaged contain variable proportions of unmethylated, monomethylated and dimethylated hnRNP A2, all cells show an exclusively nuclear pattern of localization with no signal corresponding to hnRNP A2 detected outside the nucleus. Nuclear staining (Hoechst dye) and DIC images (B only) are overlaid. Scale bar = 10 µm for HeLa and B104, 5 µm for SH-SY5Y.

Figure 7. A comparison of arginine methylation between the A/B hnRNPs.

Represented schematically are magnified views of the RGG box domains of hnRNPs A1, A2 and A3, respectively (A–C). Each representation begins with the residues that lie immediately outside the second RRM and includes residues encoded by the alternatively spliced exons 8b and 9 for hnRNPs A1 and A2/B1 and the corresponding residues of hnRNP A3. The sequences are shown as “omega loops”, expanding on a structure first proposed by Steinert et al. . While these structures are hypothetical, biophysical data consistent with the GRD adopting such a fold have been reported for both hnRNP A2 and other RNA-binding, Gly-rich proteins . Large open circles represent hydrophobic residues; small black circles represent hydrophilic residues; small, hydrophobic residues (predominantly glycine) are represented by small open circles. Sites of modified arginine residues (red) for A1, A2 and A3 have been annotated from the findings discussed in this study: many of these sites are on or close to the apex of the glycine loop motif. Unmethylated arginine residues are coloured yellow. The position labelled “UP1” indicates the cleavage site that generates unwinding protein 1 from hnRNP A1. The corresponding residue is similarly demarcated for hnRNP A2 while for hnRNP A3, Arg-216 is aDMA-modified and thus unlikely to be cleaved by the trypsin-like protease. Residue numbering reflects the most abundant isoform hnRNP A2 (NCBI: NP_112533) and the full length isoforms hnRNP A1^B (NCBI: P09651) and hnRNP A3^A (NCBI: P51991).