The development of cellular and organismal asymmetry requires selective localization of morphogenic proteins within cells. In oocytes, localization of such proteins often results from targeting of their messages. In comparison with oocytes, RNA targeting in mammalian somatic cells has attracted limited attention but localization in a number of organisms and in cells including glia, neurons, fibroblasts and epithelia, has been reported. Two pathways for mRNA trafficking in somatic cells have been described in detail. In oligodendrocytes, RNAs containing the cw-acting heterogeneous nuclear ribonucleoprotein (hnRNP) A2 response element (A2RE) are recruited to transport granules through association with the trans-acting factor hnRNP A2. Granule movement from the soma into the cell processes is kinesin- and microtubule-dependent and results in RNA localization at the cell periphery. A parallel pathway exists for the microfilament-dependent trafficking of β-actin message in fibroblasts by zipcode binding protein.
To understand the molecular mechanism of mRNA transport in somatic cells, it is important and necessary to investigate the A2RE-hnRNPs interactions. In Chapter 3, expressed human hnRNPs Al, A2 and A3 (Chapter 5) were used in a resonant mirror biosensor to explore interactions with A2RE. All these proteins have a single site that bound oligonucleotides with markedly different sequences and did not bind RNA in the presence of heparin. hnRNPs A2 and A3, but not hnRNP A1, also possess second, specific sites which bound only A2RE and were unaffected by heparin. Gel electrophoresis mobility shift assays led to a similar conclusion but revealed differences in nonspecific binding to rat and recombinant proteins. Mutant A2RE sequences that in earlier, qualitative studies did not bind hnRNP A2 or support RNA trafficking in oligodendrocytes, had dissociation constants above 5 μM. The concatenated RNA recognition motifs (RRMs), but not the individual RRMs, mimicked the binding behaviour of hnRNP A2. These data highlight the specificity of the interaction of A2RE with a subset of the hnRNP A/B protein family and suggest that the binding site on hnRNP A2 is formed by both RRMs acting in cis.
Many neuronal mRNAs are localized in the dendrites where their translation may lead to protein localization that is responsive to synaptic activity and result in the long term potentiation needed for memory formation. To explore the mRNAs transport mechanism in neuron, microinjection and laser scanning confocal microscopy techniques were applied in Chapter 4 and it was demonstrated that hippocampal neurons in primary culture shared with oligodendrocytes a common mechanism for localization of some mRNAs. As in oligodendrocytes, microinjected RNAs bearing the A2RE sequence are packaged into granules, which are transported from the cell soma into the processes, suggesting that a parallel pathway for transport exist with hnRNP A2 as a trans-acting factor. Supporting evidence comes from antisense oligonucleotide treatment of neurons, which demonstrated a correlation between the level of cytoplasmic hnRNP A2 and RNA transport, and from the observation that a point mutation in the A2RE, which eliminates binding of the A2RE to hnRNP A2 also eliminates mRNA transport in hippocampal neurons. The observation of cytoplasmic granules containing both microinjected RNA and hnRNP A2 implies that transcripts assemble into multi-molecular complexes that may function as transport units to the processes of hippocampal neurons. Colocalization of hnRNP A2, RNA granules and kinesin suggests that this transport is dependent on intact microtubules.
Further experiments in Chapter 5 revealed that hnRNP A3, like hnRNP A2 binds to A2RE-RNA in cultured hippocampal neurons and assembles into hnRNP A3/A2RE granules which are transported from cell soma to neurites. The subcellular distribution of hnRNP A2 and A3 in hippocampal neurons indicated that hnRNP A2 did not localize with A3 in RNA granules, suggesting two A2RE trafficking pathways might exist in hippocampal neurons. These findings not only shed light on the molecular mechanisms of mRNA transport in hippocampal neurons, but also indicate that a single transport pathway may be used by different cell types.