Splicing and the Cytoplasmic Localisation of Mrna Dispatch

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RNA Processing: Splicing and the Dispatch Cytoplasmic Localisation of mRNA

Isabel M. Palacios Mago is a Component of the Exon–Exon Junction Complex There is now substantial evidence that post-transcrip- An unexpected link has been discovered between tional events in the nucleus and the cytoplasm can be pre-mRNA splicing in the nucleus and mRNA linked [10]. Pre-messenger RNA (mRNA) splicing can localisation in the cytoplasm. The new findings affect the structure and composition of the ribonucle- suggest that recruitment of the Mago Nashi and oprotein particles (mRNPs), and thereby influence Y14 proteins upon splicing of oskar mRNA is an downstream processing events. For example, the essential step in the localisation of the RNA to the presence of an intron can enhance the efficiency of posterior pole of the Drosophila oocyte. both the nuclear export and translation of mRNA. It has also been shown that mRNAs containing an exon–exon junction more than 50 nucleotides down- Intracellular localisation of mRNA is a common way of stream of an in-frame termination codon are targeted targeting proteins to the regions of the cell where they for nonsense-mediated mRNA decay. This implies that are required. Some of the best characterised exam- mRNAs may carry a ‘mark’ that indicates where an ples of localised mRNAs are found in the Drosophila intron was located in their precursors. In human cell oocyte, where their localisation is essential to define extracts, the spliceosome has been shown to deposit the anterior-posterior and the dorsal-ventral body a multiprotein complex at a conserved position 20–24 axes [1]. For example, the microtubule-dependent nucleotides upstream of an exon–exon junction [11]. localisation of oskar mRNA to the posterior pole of the The first five components of this exon–exon junction oocyte specifies the formation of the pole plasm, a complex to be identified were SRm160, DEK, RNPS1, specialised cytoplasm that contains the determinants Aly/REF and Y14 [11–14]. required for the development of the abdomen and the Y14 is an RNA-binding protein that binds preferen- germline lineage. tially to spliced mRNAs immediately upstream of the The Drosophila egg chamber is composed of 16 exon–exon junctions, and remains bound to the mRNA germ cells, including 15 nurse cells and one oocyte, after nuclear export. Y14 thus has the expected char- surrounded by a layer of somatic follicle cells. The acteristics of an ‘intron marker’ [13,15]. Furthermore, oocyte grows as the nurse cells transfer their cyto- Y14 interacts with the nonsense-mediated mRNA plasmic contents into it. A transcript that localises at decay factor hUpf3, a further component of the the posterior pole of the oocyte, such as oskar mRNA, exon–exon junction complex [14,16], and RNPS1 has is therefore transcribed in the nurse cell nuclei, carried recently been shown to recruit other factors that into the oocyte and finally transported from the ante- mediate nonsense-mediated mRNA decay [17]. Finally, rior to the posterior pole of the oocyte. In mago nashi the exon–exon junction complex facilitates recruit- (mago) mutant oocytes, oskar mRNA remains at the ment of the heterodimeric nuclear export receptor anterior of the oocyte and never reaches the posterior TAP/p15 to spliced mRNAs, probably by interact- pole (Figure 1) [2–5]. ing with Aly/REF [14,18]. The splicing-dependent Thus, Mago protein has a specific function in oskar exon–exon junction complex thus has a function in mRNA localization and co-localizes with the transcript loading the mRNA with the right sort of proteins for at the posterior of the oocyte. However, Mago is an nuclear export, as well as for nonsense-mediated essential, highly conserved and predominantly nuclear mRNA decay. protein that also plays a role in the polarisation of the The human homologue of Mago (Magoh) has oocyte. These observations suggest functions beyond recently been shown to interact with Y14, forming a oskar mRNA localisation, and the discovery that Mago stable heterodimeric complex [19,20]. This suggested is a component of the exon–exon junction complex that Mago may be a functional component of the may begin to answer the remaining questions about exon–exon junction complex, and recent studies [8,9] Mago function. indicate that this is indeed so. Drosophila Mago Recent studies [6–9] — one published recently (DmMago) has been found to bind avidly and specifi- in Current Biology [6] — indicate that the recruitment cally to Drosophila Y14 (also called Tsunagi), forming of Mago and interacting proteins upon splicing of a stable complex that localises in the nucleus, with oskar mRNA is an essential step in the localization nucleolar exclusion. DmMago and its human homo- of the mRNA to the posterior pole of the Drosophila logue are both components of the exon–exon junction oocyte. complex, and like Y14, accompany spliced mRNAs to the cytoplasm. Mago, a protein essential for the pos- Wellcome/CRC Institute, Department of Genetics, University terior localisation of oskar mRNA, is thus a component of Cambridge, Tennis Court Road, CB2 1QR Cambridge, UK. of the exon–exon junction complex that binds directly E-mail: [email protected] to Y14 [8,9]. Current Biology R51

Figure 1. Drosophila oogenesis. A Anterior Posterior (A) The Drosophila ovariole is composed Germarium Stage Stage Stage 9 Stage 13 of the germarium and a series of egg 2-4 5-7 chambers. A single cystoblast, situated at the anterior tip of the germarium, under- Oocyte goes four incomplete divisions to form a Nurse cyst of 16 germline cells interconnected cells by specialised structures called ring canals. Only one cell within the cyst adopts the oocyte fate, and localises to oskar mRNA the posterior of the egg chamber, while B Follicle cells the other 15 cells undergo DNA endo- reduplication and develop as nurse cells. (B) The oocyte grows as it receives cytoplasmic contents (orange) from the tran- scriptionally active nurse cells, which Oocyte ultimately undergo apoptosis at the end Nurse cells of oogenesis. A layer of somatic follicle Mago/Y14 cells surrounds the germ line cells. At stage 8/9 of oogenesis, oskar mRNA C (i) (ii) (blue) is transported from the nurse cells through the ring canals into the oocyte and, within the oocyte, from the anterior to the posterior pole. At this stage, Mago and Y14 proteins (red) are localised in the nuclei and at the posterior pole. (C) Local- isation of oskar mRNA at stage 9 of ooge- Current Biology nesis in (i) wild-type and (ii) mago nashi mutant egg chambers. In wild-type egg chambers, oskar mRNA localises to the posterior pole of the oocyte. This localisation is completely abolished in mago nashi mutant oocytes, and oskar mRNA is only detected at the anterior pole. In both panels, oskar mRNA is detected by in situ hybridisation.

Interestingly, Magoh also binds to the nuclear posterior pole of the oocyte. Last but not least, in y14 export factor TAP, though not to other constituents of mutants, oskar mRNA localisation to the posterior the complex [8]. This observation, together with the pole of the oocyte is abolished, without affecting the interaction of Y14 with Aly/REF and TAP, suggested a nuclear export and translocation of the mRNA from possible role for the Magoh–Y14 complex in mRNA the nurse cells into the oocyte [6,7]. nuclear export. This seems not to be the case, Mago and Y14, two proteins required for oskar mRNA however, as depletion of DmMago–Y14 by double- localization to the posterior pole in the Drosophila stranded RNA-mediated interference (RNAi) in oocyte, are thus components of the exon–exon junction Drosophila cultured cells was found to have no effect complex (Figure 2). These findings suggest that recruit- on nuclear export of bulk mRNAs, although involve- ment of the Mago–Y14 complex upon splicing of oskar ment in the export of specific mRNAs cannot be ruled mRNA is an essential step in the assembly of the poste- out. RNAi against DmMago and Y14 was found to rior localization machinery. Several questions remain to cause a similar inhibition of cell growth to that be answered. For example, is splicing of oskar mRNA observed on depletion of Upf1, suggesting that the essential for its localisation to the posterior pole of the effect may be due to the deficiency of nonsense- oocyte, or could the Y14–Mago complex be deposited mediated mRNA decay [9]. on the transcript by some other means? Is the machinery of nonsense-mediated mRNA decay required for Y14 is Required for Localisation of oskar mRNA mRNA localisation, and are the other components of the to the Posterior Pole of the Oocyte oskar mRNA localization machinery reciprocally The identification of the heterodimeric Y14–Mago required for nonsense-mediated mRNA decay? Finally, complex in Drosophila also raises the possibility that do Mago and Y14 function in the post-splicing metabo- the exon–exon junction complex plays a role in the lism of other RNAs? posterior localisation of oskar mRNA in the oocyte. Two groups have recently shown that this is indeed References the case for Y14 [6,7]. First, the heterodimer 1. Palacios, I.M., and St. Johnston, D. (2001) Getting the message across: the intracellular localization of mRNAs in DmMago–Y14 was found to localise in the nuclei of all higher eukaryotes. Annu. Rev. Cell Dev. Biol. 17, 569–614. the cells in the egg chamber. Second, Y14 was seen 2. Boswell, R.E., Prout, M.E., and Steichen, J.C. (1991) Muta- to accumulate at the posterior pole with oskar mRNA, tions in a newly identified Drosophila melanogaster gene, and in mutant oocytes in which oskar mRNA is mislo- mago nashi, disrupt germ cell formation and result in the calised, Y14 co-localises with the mRNA. These formation of mirror-image symmetrical double abdomen embryos [published erratum appears in Development 1991 results suggest that Y14 is a component of the locali- Dec;113(4):preceding Table of Contents]. Development sation machinery that transports oskar mRNA to the 113, 373–384. Dispatch R52

6. Hachet, O., and Ephrussi, A. (2001) Drosophila Y14 shut- Spliceosome pre-mRNA tles to the posterior of the oocyte and is required for oskar Splicing mRNA transport. Curr. Biol. 11, 1666–1674. Mago 7. Mohr, S.E., Dillon, S.T., and Boswell, R.E. (2001) The RNA- Aly/REF Y14 Intron binding protein Tsunagi interacts with Mago to establish polarity and localize oskar mRNA during Drosophila ooge- RNPS1 SRm160 TAP/p15 nesis. Genes Dev. 15, 2886–2899. 8. Kataoka, N., Diem, M.D., Kim, V.N., Yong, J., and Dreyfuss, DEK Upf3 Nucleus G. (2001) Magoh, a human homolog of Drosophila mago nashi protein, is a component of the splicing-dependent Export exon–exon junction complex. EMBO J. 20, 6424–6433. NPC 9. Le Hir, H., Gatfield, D., Braun, I.C., Forler, D., and Izaur- Cytoplasm ralde, E. (2001) The protein Mago provides a link between Barentsz splicing and mRNA localization. EMBO Rep. 2, in press. Upf2 10. Shyu, A.B., and Wilkinson, M.F. (2000) The double lives of shuttling mRNA binding proteins. Cell 102, 135–138. ? 11. Le Hir, H., Izaurralde, E., Maquat, L.E., and Moore, M.J. Barentsz Mago (2000) The spliceosome deposits multiple proteins 20–24 Y14 nucleotides upstream of mRNA exon–exon junctions. PTC-containing EMBO J. 19, 6860–6869. Upf3 Upf2 mRNA 12. Le Hir, H., Moore, M.J., and Maquat, L.E. (2000) Pre-mRNA NMD splicing alters mRNP composition: evidence for stable Nurse cell association of proteins at exon–exon junctions. Genes RC Dev. 14, 1098–1108. Oocyte Anterior 13. Kim, V.N., Yong, J., Kataoka, N., Abel, L., Diem, M.D., and Dreyfuss, G. (2001) The Y14 protein communicates to the Mago Barentsz cytoplasm the position of exon–exon junctions. EMBO J. Y14 Staufen 20, 2062–2068. oskar mRNA ? ? 14. Le Hir, H., Gatfield, D., Izaurralde, E., and Moore, M.J. ? Khc Localisation Upf3 Upf2 (2001) The exon–exon junction complex provides a binding platform for factors involved in mRNA export and non- Posterior sense-mediated mRNA decay. EMBO J. 20, 4987–4997. 15. Kataoka, N., Yong, J., Kim, V.N., Velazquez, F., Perkinson, R.A., Wang, F., and Dreyfuss, G. (2000) Pre-mRNA splicing Figure 2. A model for the assembly and function of the imprints mRNA in the nucleus with a novel RNA-binding exon–exon junction complex. protein that persists in the cytoplasm. Mol. Cell 6, In a splicing-dependent manner, the six components of the 673–682. exon–exon junction complex — including Mago, Y14, RNPS1, 16. Kim, V.N., Kataoka, N., and Dreyfuss, G. (2001) Role of the DEK, SRm160 and Aly/REF — are deposited onto the mRNA nonsense-mediated decay factor hUpf3 in the splicing- upstream of the exon–exon junction. In the nucleoplasm, this dependent exon–exon junction complex. Science 293, complex leads to the recruitment of Upf3 and the heterodimeric 1832–1836. export receptor TAP/p15. After translocation through the 17. Lykke-Andersen, J., Shu, M.D., and Steitz, J.A. (2001) nuclear pore complexes (NPC), several components of the Communication of the position of exon–exon junctions to exported mRNP dissociate and are recycled back to the the mRNA surveillance machinery by the protein RNPS1. nucleus. In contrast, Y14, Mago and Upf3 appear to remain Science 293, 1836–1839. bound to the mRNA in the absence of translation. Upf2, a 18. Conti, E., and Izaurralde, E. (2001) Nucleocytoplasmic nonsense-mediated mRNA decay factor, and Barentsz, a transport enters the atomic age. Curr. Opin. Cell Biol. 13, protein required for oskar mRNA localisation, are recruited after 310–319. translocation. Those mRNAs that contain a premature termina- 19. Zhao, X.F., Nowak, N.J., Shows, T.B., and Aplan, P.D. tion codon (PTC-containing mRNA) are degraded by the non- (2000) MAGOH interacts with a novel RNA-binding protein. sense-mediated mRNA decay mechanism. oskar mRNA is Genomics 63, 145–148. transported from the anterior to the posterior pole of the oocyte 20. Mingot, J.M., Kostka, S., Kraft, R., Hartmann, E., and upon binding of specific components of the localisation Gorlich, D. (2001) Importin 13: a novel mediator of nuclear machinery, such as Staufen and kinesin heavy chain (Khc) [1]. import and export. EMBO J. 20, 3685–3694. The recycling of SRm160 and the role of Upf2 and Upf3 on oskar mRNA localisation remain uncertain (indicated by question marks).

3. Micklem, D.R., Dasgupta, R., Elliott, H., Gergely, F., David- son, C., Brand, A., Gonzalez-Reyes, A., and St. Johnston, D. (1997) The mago nashi gene is required for the polarisation of the oocyte and the formation of perpendicular axes in Drosophila. Curr. Biol. 7, 468–478. 4. Newmark, P.A., and Boswell, R.E. (1994) The mago nashi locus encodes an essential product required for germ plasm assembly in Drosophila. Development 120, 1303–1313. 5. Newmark, P.A., Mohr, S.E., Gong, L., and Boswell, R.E. (1997) Mago nashi mediates the posterior follicle cell-to- oocyte signal to organize axis formation in Drosophila. Development 124, 3197–3207.