Binding of an RNA Trafficking Response Element to Heterogeneous Nuclear Ribonucleoproteins A1 and A2

Abstract

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 binds a 21-nucleotide myelin basic protein mRNA response element, the A2RE, and A2RE-like sequences in other localized mRNAs, and is a trans-acting factor in oligodendrocyte cytoplasmic RNA trafficking. Recombinant human hnRNPs A1 and A2 were used in a biosensor to explore interactions with A2RE and the cognate oligodeoxyribonucleotide. Both proteins have a single site that bound oligonucleotides with markedly different sequences but did not bind in the presence of heparin. Both also possess a second, specific site that bound only A2RE and was unaffected by heparin. hnRNP A2 bound A2RE in the latter site with a K d near 50 nM, whereas the K d for hnRNP A1 was above 10 M. UV cross-linking assays led to a similar conclusion. Mutant A2RE sequences, that in earlier qualitative studies appeared not to bind hnRNP A2 or support RNA trafficking in oligodendrocytes, had dissociation constants above 5 M for this protein. The two concatenated RNA recognition motifs (RRMs), but not the individual RRMs, mimicked the binding behavior of hnRNP A2. These data highlight the specificity of the interaction of A2RE with these hnRNPs and suggest that the sequence-specific A2RE-binding site on hnRNP A2 is formed by both RRMs acting in cis.The family of more than 20 heterogeneous nuclear ribonucleoproteins (hnRNPs) 1 appears to play diverse roles in the post-transcriptional processing of hnRNA and subsequent packaging, transport, and translation of mRNA (1-4). hnRNPs A1, A2, B1, B2, C1, and C2 are the major components of 40S "core particles," studied primarily in HeLa cells, which are thought to package nascent ssRNA in the nucleus in a histonelike fashion (5-10). The hnRNP A/B proteins have a modular structure with two N-terminal RNA recognition motifs (RRMs) followed by a glycine-rich region ( Fig. 1) (11). The three-dimensional structures of the tandem RRMs of hnRNP A1, which are assumed to be the principal mediators of the RNA-protein interactions, have been determined (12, 13). Although the three-dimensional structures of other hnRNPs of the A/B family have not been determined, their RRMs share sufficient sequence identity with those of hnRNP A1 to suggest that they share the same tertiary fold: a four-stranded antiparallel ␤-sheet flanked by two helices. This module adopts a comparable fold in the RRMs of more distantly related proteins, such as U1A (14 -17).Although a number of the hnRNPs have a general role in intranuclear packaging of RNA, some also manifest sequencespecific binding (1, 4, 18 -22). In addition to being a major component of core particles (23), hnRNP A2 binds a telomeric sequence (24, 25, 43) and a small RNA segment known to be necessary and sufficient for transport of the message encoding myelin basic protein in the cytoplasm of oligodendrocytes, the A2RE (26 -28). The binding of hnRNP A2 to A2RE has been shown, by mutational and antisense approaches, to be essential for cytoplasmic RNA trafficking in oligodendrocytes (29).Although hnRNP A2 has been shown to ...

Heterogeneous Nuclear Ribonucleoprotein A3, a Novel RNA Trafficking Response Element-binding Protein

“…The biosensor responses with purified recombinant hnRNP A3 (Fig. 6) closely paralleled those recorded for hnRNP A2 (43), indicating that hnRNP A3, like hnRNP A2, possesses two RNA-binding sites; one of them is sequencespecific, binding to A2RE, and the other binds with little discrimination between sequences. Although the RNA recognition motifs of hnRNP A3 are closer in sequence to hnRNP A1 than to hnRNP A2, hnRNP A3 mimics hnRNP A2 more closely than hnRNP A1 in its binding to A2RE, although with dissociation constants for A2RE binding that are severalfold higher than for hnRNP A2.…”

“…Biosensor measurements in which A2RE or nonspecific oligoribonucleotide (NS1) was added to purified, expressed Histagged hnRNP A3 immobilized on the cuvette showed that the oligonucleotide binding to this protein closely parallels the binding to hnRNP A2 (43). A saturating concentration of NS1 (30 M) or A2RE (4 M) was added to the biosensor, resulting in a response with A2RE double that with NS1.…”

Multiplexed Dendritic Targeting of α Calcium Calmodulin-dependent Protein Kinase II, Neurogranin, and Activity-regulated Cytoskeleton-associated Protein RNAs by the A2 Pathway

“…Because hnRNP A2 contains two RNA recognition motifs and because previous studies identified both specific and nonspecific binding sites in hnRNP A2 (Shan et al, 2000), SPR data were fitted to a heterogeneous two-site ligand model to determine separate on rates (k a ) off rates (k d ) and affinity constants (K D ) for each binding site. It is possible that the SPR data could be fitted equally well with a different model but the two-site model is consistent with the known structure of hnRNP A2 and with previous reports of specific and nonspecific binding sites in hnRNP A2 (Shan et al, 2000). Sensorgrams for A2RE sequences from rat MBP, mouse ␣CaMKII, rat ␣CaMKII, rat NG, rat ARC, and rat PKM RNAs and for antisense MBP A2RE sequence as a nonspecific binding control are shown in Figure 4, A-G.…”

“…Analyte in HEPESbuffered saline buffer (10 mM HEPES, 3 mM EDTA, 0.15 M NaCl, and 0.05% surfactant p20, pH 7.5) was injected at different concentrations (12.5, 25, 50, 100, and 200 nM) at a flow rate of 30 l/min for 180 s, followed by HBS at 30 l/min for 180 s. After each injection the surface of the chip was regenerated using 2.5 M NaCl at 100 l/min for 20 s. Sensorgrams were globally fitted with Biacore T100 evaluation software. Because hnRNP A2 contains two independent RNA binding sites (Shan et al, 2000) SPR data were fitted with a heterogeneous two-site ligand binding model to determine k on , k off , and K D values for two distinct binding sites in hnRNP A2. Control experiments with hnRNP E1 as ligand showed Ͻ100 RU binding at 200 nM for all analytes.…”

Nucleic Acid-Based Strategies for the Treatment of Coxsackievirus-Induced Myocarditis

“…Furthermore, ASONs can also disrupt RNA trafficking by occupying protein-RNA interaction sequences necessary for correct intracellular localization. For example, hnRNP A2 response element (A2RE) is identified as a key sequence required for the trafficking of myelin basic protein (Shan et al, 2000). Due to major problems including instability, non-specific delivery and unwanted side effects of the ASONs, the structure of this molecule has been modified extensively at different components (i.e., the bases, sugar or phosphate backbone) and has entered its third generation (Fig.…”