mRNA localisation is an important mechanism for generating cellular asymmetry, as it allows cells to translate mRNAs in specific cytoplasmic regions. Two of the better characterised mRNA localisation systems in somatic cells are those responsible for localisation of beta-actin mRNA in fibroblasts and neurons, and myelin basic protein mRNA in oligodendrocytes. Beta-actin mRNA localisation is dependent upon a 54 nt localisation signal, the "zipcode", while MBP mRNA is dependent upon an 11 nt signal within its 3'UTR, the heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2) response element (A2RE). The microfilament-dependent localisation of beta-actin mRNA transport in fibroblasts is driven by the interaction between the trans-acting factor ZBP-1 (zipcode binding protein-1) and the zipcode, but the proteins responsible for the microtubule-dependent transport in neurons have not been identified. MBP mRNA transport in
oligodendrocytes, and possibly transport of A2RE-containing mRNAs in neurons, are dependent upon the interaction between the protein hnRNP A2 and the A2RE. We identified the brain zipcode-binding proteins to determine whether zipcode- and A2RE-dependent localisation systems use some of the same trans-acting factors for localisation, or whether they constitute separate and distinct localisation systems.
A group of approximately 10 brain proteins interacted specifically with the zipcode. ZBP-1 did not appear to be one of these proteins, but six other zipcode-binding proteins were identified by N-terminal protein sequencing. KH-type splicing regulatory protein, FUSE-binding protein, HuC, hnRNPs L, El, and E2 have an affinity for the rat zipcode sequence, but not for the A2RE. Conversely, proteins that bind to the A2RE (hnRNPs Al, A2, A3, and Bl) do not bind to the zipcode. Therefore, the targeting of A2RE- and zipcode- containing RNAs in brain
appears to employ different trans-acting factors. We also identified some of the proteins that bind to an mRNA stability element, the AU-rich element. KH-type splicing regulatory protein, and possibly FUSE-binding protein, bind to the AU-rich element The cellular and intracellular distributions of zipcode- and A2RE-binding proteins in rat brains were examined by immunostaining. KH-type splicing regulatory protein and FUSE-binding protein are concentrated in the nucleoplasm of most cells in 15- day old rat brains, but KH-type splicing regulatory protein is also concentrated in the nucleolus and is detected in granular structures in the cytoplasm. These structures resemble RNA transport complexes, suggesting that KH-type splicing regulatory protein has cytoplasmic functions that include a role in beta-actin mRNA localisation. The A2RE-binding proteins are predominantly nuclear in 21-day old rat brain cells, but are also detected at low levels in the cytoplasm.
Neurons and oligodendrocytes express hnRNP A2, hnRNP A3, and variable levels of hnRNP Al and hnRNP Bl, while astrocytes express hnRNP A2, hnRNP A3, hnRNP Bl, but do not express hnRNP Al. This shows that some A2RE- and zipcode-binding proteins are coexpressed in some neurons, oligodendrocytes, and astrocytes.
Although they are co-expressed by many cells, hnRNP A2 and KH-type splicing regulatory protein reside in different subcellular regions. These proteins are concentrated in different regions in the nucleoplasm and stain separate granular structures in the cytoplasm of cultured glial cells. This suggests that not only do these proteins bind different localisation signals, but that separate transport complexes are responsible for localisation of zipcode- and A2RE-containing mRNAs.