Mutational Analysis of a Heterogeneous Nuclear Ribonucleoprotein A2 Response Element for RNA Trafficking

Abstract

Cytoplasmic transport and localization of mRNA has been reported for a range of oocytes and somatic cells. The heterogeneous nuclear ribonucleoprotein (hnRNP) A2 response element (A2RE) is a 21-nucleotide segment of the myelin basic protein mRNA that is necessary and sufficient for cytoplasmic transport of this message in oligodendrocytes. The predominant A2RE-binding protein in rat brain has previously been identified as hnRNP A2. Here we report that an 11-nucleotide subsegment of the A2RE (A2RE11) was as effective as the fulllength A2RE in binding hnRNP A2 and mediating transport of heterologous RNA in oligodendrocytes. Point mutations of the A2RE11 that eliminated binding to hnRNP A2 also markedly reduced the ability of these oligoribonucleotides to support RNA transport. Oligodendrocytes treated with antisense oligonucleotides directed against the translation start site of hnRNP A2 had reduced levels of this protein and disrupted transport of microinjected myelin basic protein RNA. Several A2RE-like sequences from localized neuronal RNAs also bound hnRNP A2 and promoted RNA transport in oligodendrocytes. These data demonstrate the specificity of A2RE recognition by hnRNP A2, provide direct evidence for the involvement of hnRNP A2 in cytoplasmic RNA transport, and suggest that this protein may interact with a wide variety of localized messages that possess A2RE-like sequences.Vectorial transport and localized translation of mRNA in the cytoplasm affords a mechanism for establishing an asymmetric distribution of cytosolic proteins in cells. This is particularly important in oogenesis and embryonic development (1-8). In somatic cells, some mRNAs are also transported to discrete locations within the cell. These include myelin basic protein (MBP) 1 mRNA, which is localized in the myelinating periphery of oligodendrocytes (9 -11), microtubule-associated protein 2A (MAP2A) and tau, which are localized in neurites (12-14), and ␤-actin mRNA, which is localized in the leading edge of fibroblasts (15-17). An active, multistep, cytoplasmic transport pathway has been delineated for MBP mRNA in cultured oligodendrocytes. MBP transcripts microinjected into the soma assemble into granules that move out along the myelin-forming processes (18,19). Transport of these granules is dependent on the presence of kinesin and intact microtubules (20). Similar RNA-rich granules have been observed in other cells (21)(22)(23)(24).A cis-acting sequence sufficient and necessary for MBP mRNA transport has been identified within a 21-nucleotide stretch of the 3Ј-untranslated region. Incorporation of this segment, the hnRNP A2 response element (A2RE, formerly termed the RNA transport sequence (25)), into heterologous RNAs mediates transport into the processes and also enhances translation.2 A2RE-like sequences are present in other localized mRNAs including mouse MAP2A, mouse protamine 2, and rat glial fibrillary acidic protein (25), suggesting that the A2RE represents a general signal for RNA transport.Earlier we used biotin-labeled oligorib...

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

“…The A2RE-like sequence in mouse ␣CaMKII RNA differs from the second A2RE sequence in MBP RNA at one position (G6U). Substitution of C for G at this position (G6C) in MBP A2RE abrogated hnRNP A2 binding and dendritic targeting by the A2 pathway (Munro et al, 1999). However, our results indicate that the A2RE sequence in mouse ␣CaMKII RNA with U at this position binds to hnRNP A2 (Figure 4) and that it is necessary and sufficient for dendritic targeting by the A2 pathway (Figure 2).…”

“…A2RE-like sequences have been noted in dendritically localized neuronal RNAs protein kinase M (PKM ) (Muslimov et al, 2004) and BC1 (Muslimov et al, 2006), but it is not known whether these RNAs are targeted by the A2 pathway. Because polymorphisms at several positions in the A2RE sequence are compatible with hnRNP A2 binding and dendritic targeting (Munro et al, 1999) sequence homology alone is not sufficient to identify functional A2RE sequences. In this study, functional A2RE sequences in ␣ calcium calmodulin-dependent protein kinase II (␣CaMKII), neurogranin (NG), and activity-regulated cytoskeleton-associated protein (ARC) RNAs are identified by three criteria: significant sequence similarity to one of the A2RE sequences in MBP RNA, high-affinity binding to hnRNP A2, and necessary and sufficient for dendritic targeting in microinjected neurons.…”

“…Targeting by the A2 pathway requires a characteristic 11-nucleotide cis-acting RNA sequence, the hnRNP A2 response element (A2RE) , that is recognized by a cognate trans-acting factor, heterogeneous nuclear ribonucleoprotein (hnRNP) A2 (Hoek et al, 1998). MBP RNA contains two partially overlapping A2RE sequences (GCCAAGGAGCC and GCCAGAGAGCC) (Munro et al, 1999), and A2RE sequences have also been identified in the gag and vpr genes of human immunodeficiency virus (HIV) (Mouland et al, 2001). A2RE-like sequences have been noted in dendritically localized neuronal RNAs protein kinase M (PKM ) (Muslimov et al, 2004) and BC1 (Muslimov et al, 2006), but it is not known whether these RNAs are targeted by the A2 pathway.…”

The Microtubule-associated Protein Tumor Overexpressed Gene Binds to the RNA Trafficking Protein Heterogeneous Nuclear Ribonucleoprotein A2

“…HnRNP A2 binds to a specific cis-acting element, hnRNP A2 response element (A2RE), found in several dendritically localized mRNAs Munro et al, 1999). This interaction is necessary for transport of A2RE-mRNAs to the periphery of neural cells (Munro et al, 1999;Shan et al, 2003).…”

“…HnRNP A2 binds to a specific cis-acting element, hnRNP A2 response element (A2RE), found in several dendritically localized mRNAs Munro et al, 1999). This interaction is necessary for transport of A2RE-mRNAs to the periphery of neural cells (Munro et al, 1999;Shan et al, 2003).A2RE-mRNAs, hnRNP A2, and other components form complexes that look by light microscopy like granules that are transported to the cell periphery by a microtubule-and kinesin-based mechanism. In cultured oligodendrocytes and neurons, granules containing A2RE-mRNAs and hnRNP A2 are associated with the cytoskeleton (CSK) .…”