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SHOWCASE ON RESEARCH Post-Transcriptional Regulation of Gene Expression: the Many Hats of hnRNP Proteins Ross Smith and Jillian Dunphy School of Molecular and Microbial Sciences, University of Queensland, QLD 4072 The cellular expression of a protein is governed not only by the rate at which its RNA is transcribed but also by the subsequent fate of this RNA. Alternative splicing, nuclear export, cytoplasmic RNA trafficking, localisation, translation and degradation all play a significant role in determining the level and subcellular location of many proteins. Much of our research is focussed on RNA trafficking and splicing in mammalian cells. mRNA Trafficking The development of cellular and organismal asymmetry requires selective localisation of proteins, including morphogenic proteins, within cells. Many proteins possess motifs that determine where they will ply their trade. But proteins are not the only species to display sorting motifs; many mRNA molecules also have elements that direct them to specific subcellular locations, resulting in localisation of the proteins they encode. The nature of RNA localisation pathways has emerged primarily from studies of Xenopus and Drosophila oocytes and embryos (1, 2). In recent years, studies of mammalian oligodendrocytes, neurons, fibroblasts and epithelia have added considerably to the picture (3, 4). Cis-Acting Elements Selective trafficking of RNA involves the recognition of cis-acting elements, commonly in the 3' untranslated region (3' UTR), by cognate trans-acting proteins followed by cytoplasmic transport in what typically appear to be 0.5-1 µm diameter granules that incorporate motor proteins. Despite the numerous studies of RNA localisation in Drosophila and Xenopus, two of the best characterised RNA trafficking/localisation systems are those involved in transport of the mRNAs encoding βactin in chicken fibroblasts and myelin basic protein (MBP) in rat oligodendrocytes and neurons. Two of the first mammalian RNA trafficking elements defined were the 'zipcode' motif in the β-actin mRNA (5) and the 21-nucleotide cis-acting sequence now termed the heterogeneous nuclear ribonucleoprotein (hnRNP) A2 response element (A2RE), which was originally identified by deletion analysis in MBP mRNA (6). The A2RE was subsequently whittled down to an 11 nucleotide element (GCCAAGGAGCC) that retains the A2RE transport properties (7). These elements recruit their mRNAs to specific transport granules. Many RNA segments containing cis-acting elements involved in trafficking have been identified, but in most the precise location of the element is poorly defined. From the few published 3D structures of ssDNA or RNA in complexes with proteins, one would expect the Vol 36 No 3 December 2005 binding sites on trans-acting factors to accommodate 8-10 nucleotides. In the A2RE these nucleotides are contiguous, whereas in larger elements one would anticipate that the 3D structure brings together nucleotides separated in the linear sequence into the binding site. This hypothesis, and the ability of proteins to recognise even the A2RE11 cis-acting element with a range of point mutations (8), makes identification based solely on nucleotide sequence difficult. Recognition of cis-acting elements at this stage requires experimental data. Trans-Acting Factors A small number of RNA-binding motifs are found in trans-acting factors, including the common RNA recognition motif (RRM) and hnRNP K-homology (KH) domain. From a comprehensive set of experiments including pull-downs, cell microinjection, biosensor and gel mobility shift assays (7, 9, 10), we concluded that the RRM-containing hnRNP A2 is a trans-acting factor for cytoplasmic trafficking of RNAs possessing the A2RE11 motif. This element is necessary and sufficient for trafficking of MBP mRNA in oligodendrocytes and neurons, and the A2RE11/hnRNP A2 pathway is likely to operate in any cell with a subset of RNAs having A2RE-like motifs. The specificity of this interaction is striking: in the context of a kilobase RNA, mutation of a single A2RE11 nucleotide (A8G) is sufficient to eliminate binding to hnRNP A2 and thus RNA trafficking (Fig. 1). Oligodendrocyte treatments that reduce hnRNP A2 levels also disrupt the transport of A2REcontaining RNA. hnRNP A2 is ubiquitously expressed with high levels in brain, testis and skin. Although primarily nuclear, it is detected in the cytoplasm of many cells including neurons, oligodendrocytes and astrocytes. It is localised together with A2RE-containing mRNA in the cytoplasmic transport granules. In cultured oligodendrocytes, the levels of hnRNP A2 in the cytoplasm correlate with the high MBP mRNA levels seen in the most active period of process formation (11). After this peak period, hnRNP A2 reverts to allnuclear. This may reflect changes in isoform levels with the more cytoplasmic isoforms (lacking exon 9) being expressed during process formation (see below). Thus changes in alternative splicing may accompany cellular development. Our conclusion that hnRNP A2 is a trans-acting factor for RNA trafficking has been strengthened by numerous subsequent studies. Confocal microscopy colocalisation analysis of assembling transport granules (12) showed that hnRNP A2 is found only in AUSTRALIAN BIOCHEMIST Page 5 Post-Transcriptional Regulation: hnRNP Proteins SHOWCASE ON RESEARCH Fig. 1. (A) RNA transport in live, cultured hippocampal neurons microinjected with fluorescently labelled A2RE-containing RNA or A2RE11-containing RNA (green) and Texas Red-labelled dextran (red: to show cell morphology) and observed by confocal microscopy. The RNA assembles into granules and moves along the processes (arrows in top left panel). A single nucleotide change (A8G) in the A2RE element of the ~200 nucleotide RNA stops its assembly into granules, resulting in a diffuse RNA distribution in the soma and proximal neurites (arrowhead in bottom left panel) with only an occasional granule visible in the processes. Scale bar, 10 µm. (B) The proportions of cells positive for transport. This figure is from ref 10. Copyright 2003 by the Society for Neuroscience. granules containing MBP RNA, and not in granules containing RNA that lacks the A2RE sequence. A2RE binding to hnRNP A2 induces the latter to cooperatively self-associate and associate with granules. RNAs with normally different trafficking patterns (MBP to cell periphery; Connexin 32 to ER; GFP to perikaryon) are segregated into different granules, but if the A2RE is incorporated into connexin or GFP RNAs they are incorporated into the same granules as co-injected MBP RNA. The A2RE-mediated sorting pathway is thus epistatic to the intrinsic sorting pathways for GFP and connexin mRNAs. Another trafficking pathway in mammalian cells, in which an identified trans-acting factor binds a well defined cognate cis-acting element, is the zipcode binding protein (ZBP)/zipcode pathway (13, 14). ZBP1, a KH-domain protein, binds a 54 nucleotide cis element (the 'zipcode') that contains short tandem ZBP-binding repeats in fibroblast β-actin RNA. A larger homologue, ZBP2, which is enriched in the brain, also binds the actin zipcode and has a role in localisation of β-actin mRNA. Other families of cis-acting elements/trans-acting factors are represented by the highly helical testis-brain RNA-binding protein (TBRBP) which binds the Y element (AGAAGCCCTATGCT), and the cytoplasmic polyadenylation element binding protein (CPEB), an RRM/zinc finger protein which binds a UUUUAU sequence. Like hnRNP A2, many of the other trans-acting proteins involved in RNA trafficking and localisation Page 6 are present at high concentrations in the nucleus, where they have other functions, and many are hnRNP-like or are hnRNP homologues. RNA Trafficking Models From studies of oligodendrocytes and neurons the following picture has emerged (Fig. 2). In the nucleus, RNAs possessing trafficking elements combine with their cognate trans-acting factors prior to export to the cytoplasm. In the cytoplasm, the protein-RNA complex associates with other components of the transport granules (including ribosomes, tRNA synthetases, elongation factors, and other molecules needed for translation), which then move towards the cell periphery. The early studies of β-actin mRNA transport showed that movement of its granules is dependent on microfilaments in fibroblasts (14) whereas the transport of both A2RE-containing RNA (7) and β-actin RNA (15) in neurons is microtubule-dependent. RNA granule movement on microtubules is governed by the molecular motors kinesin and dynein, both of which are present in individual granules. Translation appears to be arrested during trafficking and a granule protein that suppresses translation has been identified (E. Barbarese, personal communication). However, the mechanisms by which the RNA-protein complexes dissociate from the granules, and the mRNAs become anchored at their destination and translated, are largely unknown. AUSTRALIAN BIOCHEMIST Vol 36 No 3 December 2005 SHOWCASE ON RESEARCH Dendritic mRNA Transport Post-Transcriptional Regulation: hnRNP Proteins In oligodendrocytes, localisation of MBP mRNA appears to serve the function of delivering MBP directly to nascent myelin where it is needed for formation of compact myelin. There is no evidence that RNAs encoding proteins other than MBP and myelin-associated oligodendrocytic basic protein (MOBP) are localised in these cells. By contrast, a substantial subset of mRNAs is localised to the dendrites in neurons, where many are translated (16). They include mRNAs encoding microtubuleassociated protein 2 (MAP2), activity-regulated cytoskeleton-associated protein (ARC), and the glycine and glutamate receptors. Localised RNAs are often found associated with ribosomes and other components of the translation apparatus near dendritic spines. Some of these localised RNAs, such as that encoding the alpha-chain of Ca++/calmodulindependent kinase II (CaMKIIα) may participate in activity-regulated protein synthesis (17). Disruption of the 3' UTR of CaMKIIα mRNA results in its mislocalisation (restricted to soma), a marked reduction in post-synaptic densities, a reduction in long-term potentiation (LTP) and impairments in spatial memory, associative fear conditioning and object recognition (18, 19). Because several dendritically localised RNAs contain A2RE-like sequences (6) and the level of hnRNP A2 in the brain is higher than in most other cell types, interaction of this motif with hnRNP A2 may afford a general pathway for targeting these RNA molecules in the brain. In addition to hnRNP A2, the proteins TB-RBP, ZBP1, ZBP2 and MAP2-RNA trans-acting protein (MARTA) all have established interactions with at least one neuronal RNA element. However, it is likely that a protein such as hnRNP A2 binds to more than one cisacting element in the same or different mRNA molecules, and that more than one protein binds elements within a single mRNA. For example, both the cytoplasmic CPE and localisation elements in CaMKIIα RNA (see above) bind proteins that influence trafficking of this RNA in neurons, LTP and memory. hnRNPs as Regulators of Alternative Splicing Alternative splicing generates high protein diversity from a limited genetic repertoire with different isoforms potentially having distinct biological activities or locations. Proteins belonging to the hnRNP A family are multi-tasking; in addition to their roles in RNA trafficking they regulate alternative splicing of RNA. hnRNP A1, A2 and A3 isoforms have been detected in spliceosomes at various stages of splicing (20) and appear to regulate splice site selection by antagonising the activities of SR (splicing factor) proteins (21). High hnRNP levels favour exon skipping by the spliceosome, thus affecting mRNA stability, localisation and translatability. hnRNP A1, A2, and A3 are both regulators of and subject to alternative splicing. The two most abundant of the four isoforms of hnRNP A2 appear to be solely nuclear, but the minor forms are found at similar levels in the nucleus and cytoplasm. We have evidence that the minor but not the major isoforms participate in cytoplasmic RNA trafficking (J. Hatfield, M. Landsberg et al., unpublished data). The minor forms are translated primarily in early development in rats, when the need for RNA trafficking may peak (11). By contrast, the levels of the major forms, which perform housekeeping tasks in the nucleus, are unchanged from birth to maturity (J. Hatfield et al., unpublished data). Fig. 2. Model depicting trafficking of A2RE-containing RNAs. hnRNP A2 is imported into nuclei and binds to mRNA through the A2RE cis-acting element. The A2RE mRNA/hnRNP A2 complex is then exported to the cytoplasm. Multiple copies of the RNA-protein complex are recruited to individual transport granules that also contain inactive translation machinery. The transport granules attach to microtubules and are transported to the site of translation (in this case to dendritic spines). At its destination the mRNA is anchored and translated. Each of the steps in this process, including RNA degradation, is a potential post-transcriptional control point for gene expression. Vol 36 No 3 December 2005 AUSTRALIAN BIOCHEMIST Page 7 Post-Transcriptional Regulation: hnRNP Proteins SHOWCASE ON RESEARCH Overexpression of splice variants hnRNP A2 and B1 and an increase in hnRNP B1 relative to A2 are observed in lung carcinogenesis and recapitulate the changes during foetal lung development. This reinforces the view that hnRNP A2/B1 is an oncodevelopmental protein and suggests a role for the alternative splicing of this protein in the regulation of tumourigenesis (22). Conclusions and Perspectives The multi-tasking hnRNP A/B family of proteins play important roles in cytoplasmic mRNA trafficking. Two prime needs for advancement of our understanding of RNA trafficking are the precise identification of further cis-acting elements and their trans-acting factors, and elucidation of the 3D structures of their complexes. Such information will define better the principles of recognition of RNA elements by proteins. We have focussed here on hnRNP A2, but this protein has two paralogues, hnRNP A1 and A3, and we have evidence that the latter is also involved in RNA transport. It will be interesting to define more fully the connections between these three proteins; although they have partially overlapping functions they also show some clear differences. For example, their levels change differently during the cell cycle, they have different effects on cell growth (23), and they are subject to different patterns of Arg dimethylation in their RGG boxes. It seems that cells do not simply produce the three paralogues as a molecular insurance policy. Additionally, these hnRNP paralogues have nuclear functions that include the regulation of pre-mRNA splicing and all have multiple splice variants that may differ in localisation and function. The possibility that the four isoforms of hnRNP A2 fulfil different functions warrants further investigation, both to improve our understanding of this protein and to clarify the general role played by alternative splicing, which is emerging as a critical step in the regulation of gene expression. hnRNP A2 should prove useful in investigating the roles of different isoforms; it is relatively abundant and there is evidence for some of its isoforms being confined to separate subcellular compartments. The dual roles of hnRNP A proteins in mRNA splicing and trafficking highlight the co-ordination of RNA processing and localisation. The importance of appropriate RNA processing and trafficking to the quantity, quality and localisation of proteins is readily apparent for both A2RE-containing RNAs and hnRNP A proteins themselves. Page 8 References 1. Lipshitz, H.D., and Smibert, C.A. (2000) Curr. Opin. Gen. Dev. 10, 476-488 2. St Johnston, D., and Nüsslein-Volhard, C. (1992) Cell 68, 201-219 3. Carson, J.H., Kwon, S., and Barbarese, E. (1998) Curr. Opin. Neurobiol. 8, 607-612 4. Bassell, G.J., Oleynikov, Y., and Singer, R.H. (1999) FASEB J. 13, 447-454 5. Singer, R.H. (1993) Curr. Biol. 3, 719-721 6. Ainger, K., Avossa, D., Diana, A.S., Barry, C., Barbarese, E., and Carson, J.H. (1997) J. Cell Biol. 138, 1077-1087 7. Munro, T.P., Magee, R.J., Kidd, G.J., Carson, J.H., Barbarese, L., Smith, L.M., and Smith, R. (1999) J. Biol. Chem. 274, 34389-34395 8. Moran-Jones, K., McLure, L., Kennedy, D., Reddel, R.R., Sara, S,. and Smith, R. (2005) Nucleic Acids Res. 33, 486-496 9. Shan, J., Moran-Jones, K., Munro, T.P., Kidd, G.J., Winzor, D.J., Hoek, K.S., and Smith, R. (2000) J. Biol. Chem. 275, 38286-38295 10. Shan, J., Munro, T.P., Barbarese, E., Carson, J.H., and Smith, R. (2003) J. Neurosci. 23, 8859-8866 11. Maggipinto, M., Rabiner, C., Kidd, G.J., Hawkins, A.J., Smith, R., and Barbarese, E. (2004) J. Neurosci. Res. 75, 614-623 12. Carson, J.H., Cui, H., and Barbarese, E. (2001) Curr. Opin. Neurobiol. 11, 558-563 13. Bassell, G.J., Zhang, H., Byrd, A.L., Femino, A.M., Singer, R.H., Taneja, K.L., Lifshitz, L.M., Herman, I.M., and Kosik, K.S. (1998) J. Neurosci. 18, 251-265 14. Farina, K.L., Huttelmaier, S., Musunuru, K., Darnell, R.B., and Singer, R.H. (2003) J. Cell Biol. 160, 77-87 15. Tiruchinapalli, D.M., Oleynikov, Y., Kelic, S., Shenoy, S.M., Hartley, A., Stanton, P.K., Singer, R.H., and Bassell, G.J. (2003) J. Neurosci. 23, 32513261 16. Kiebler, M.A., and DesGroseillers, L. (2000) Neuron 25, 19-28 17. Steward, O., and Schuman, E.M. (2003) Neuron 40, 347-359 18. Frey, U., and Morris, R.G.M. (1997) Nature 385, 533536 19. Miller, S., Yasuda, M., Coats, J.K., Jones, Y., Martone, M.E., and Mayford, M. (2002) Neuron 36, 507-519 20. Rappsilber, J., Ryder, U., Lamond, A.I., and Mann, M. (2002) Genome Res. 12, 1231-1245 21. Mayeda, A., Munroe, S.H., Cáceres, J.F., and Krainer, A.R. (1994) EMBO J. 13, 5483-5495 22. Zerbe, L.K., Pino, I., Pio, R., Cosper, P.F., DwyerNield, L.D., Meyer, A.M., Port, J.D., Montuenga, L.M., and Malkinson, A.M. (2004) Mol. Carcinog. 41, 187-96 23. He, Y., Brown, M.A., Rothnagel, J.A., Saunders, N.A,. and Smith, R. (2005) J. Cell Sci. 118, 3173-3183 AUSTRALIAN BIOCHEMIST Vol 36 No 3 December 2005