The cis-acting response element, A2RE, which is sufficient for cytoplasmic mRNA trafficking in oligodendrocytes, binds a small group of rat brain proteins. Predominant among these is heterogeneous nuclear ribonucleoprotein (hnRNP) A2, a trans-acting factor for cytoplasmic trafficking of RNAs bearing A2RE-like sequences. We have now identified the other A2RE-binding proteins as hnRNP A1/A1 B , hnRNP B1, and four isoforms of hnRNP A3. The rat and human hnRNP A3 cDNAs have been sequenced, revealing the existence of alternatively spliced mRNAs. In Western blotting, 38-, 39-, 41-, and 41.5-kDa components were all recognized by antibodies against a peptide in the glycine-rich region of hnRNP A3, but only the 41-and 41.5-kDa bands bound antibodies to a 15-residue N-terminal peptide encoded by an alternatively spliced part of exon 1. The identities of these four proteins were verified by Edman sequencing and mass spectral analysis of tryptic fragments generated from electrophoretically separated bands. Sequence-specific binding of bacterially expressed hnRNP A3 to A2RE has been demonstrated by biosensor and UV cross-linking electrophoretic mobility shift assays. Mutational analysis and confocal microscopy data support the hypothesis that the hnRNP A3 isoforms have a role in cytoplasmic trafficking of RNA.Establishment of asymmetry in cells requires selective localization of proteins. This may be accomplished by directed protein transport, a well established pathway for plasma membrane and secreted proteins, or by trafficking and subsequent localization of mRNA. Localization of RNA has been intensively studied in Drosophila and Xenopus oocytes (for reviews see Refs. 1-6) and more recently in mammalian somatic cells (7-12).In 1982 Subsequent experiments demonstrated that MBP mRNA is translated close to myelin and the protein rapidly incorporated into the nascent membrane (14 -16) and lead to a model in which MBP mRNA is recruited into RNA transport granules in the perikaryon and then transported, by indirect attachment to the microtubule-bound motor protein kinesin, to the myelin compartment at the cell periphery (10, 17-21). The granules are localized in the myelin compartment, and the RNA cargo is translated, with the MBP being incorporated into the myelin membrane. Deletion studies led to the conclusion that a small element in the 3Ј-untranslated region of the MBP mRNA, the RNA transport sequence (RTS), is sufficient and necessary for this cytoplasmic RNA transport in oligodendrocytes (17). Cytoplasmic trafficking of RNA encoding ␤-actin is also dependent on inclusion in transport granules that are attached to the cytoskeleton. In fibroblasts ␤-actin mRNA transport is microfilament-dependent (9, 22), whereas microtubules are implicated in transport of this mRNA in neurons (23,24).trans-Acting factors have been isolated in pull-down experiments with RTS-labeled magnetic particles. The predominant RTS-binding protein from a number of rat tissues is heterogeneous nuclear ribonucleoprotein (hnRNP) A2 (25), a constituent of...