mRNA transport in fungal top models - Wiley Online Library

Focus Article

mRNA transport in fungal top models Dierk Niessing,1,2 Ralf-Peter Jansen,3 Thomas Pohlmann4 and Michael Feldbrügge

4*

Eukaryotic cells rely on the precise determination of when and where proteins are synthesized. Spatiotemporal expression is supported by localization of mRNAs to specific subcellular sites and their subsequent local translation. This holds true for somatic cells as well as for oocytes and embryos. Most commonly, mRNA localization is achieved by active transport of the molecules along the actin or microtubule cytoskeleton. Key factors are molecular motors, adaptors, and RNA-binding proteins that recognize defined sequences or structures in cargo mRNAs. A deep understanding of this process has been gained from research on fungal model systems such as Saccharomyces cerevisiae and Ustilago maydis. Recent highlights of these studies are the following: (1) synergistic binding of two RNA-binding proteins is needed for high affinity recognition; (2) RNA sequences undergo profound structural rearrangements upon recognition; (3) mRNA transport is tightly linked to membrane trafficking; (4) mRNAs and ribosomes are transported on the cytoplasmic surface of endosomes; and (5) heteromeric protein complexes are, most likely, assembled co-translationally during endosomal transport. Thus, the study of simple fungal model organisms provides valuable insights into fundamental mechanisms of mRNA transport boosting the understanding of similar events in higher eukaryotes. © 2017 Wiley Periodicals, Inc. How to cite this article:

WIREs RNA 2018, 9:e1453. doi: 10.1002/wrna.1453

INTRODUCTION

L

ocalized translation is essential for a variety of cellular events such as asymmetric cell division, polarized growth, development, and neuronal processes.1–3 The underlying concept is active transport of mRNAs combined with their local translation. The general scheme involves molecular motors that transport mRNAs along actin microfilaments or microtubules. Transport along actin toward the plus-

*Correspondence to: [email protected] 1

Department of Cell Biology, Biomedical Center, Ludwig-Maximilians-University München, Planegg-Martinsried, Germany

2

Institute of Structural Biology, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany

3

Interfaculty Institute of Biochemistry, Eberhard Karls Universität Tübingen, Tübingen, Germany

4

Centre of Excellence on Plant Sciences, Institute for Microbiology, Heinrich-Heine University Düsseldorf, Düsseldorf, Germany Conflict of interest: The authors have declared no conflicts of interest for this article.

Volume 9, January/February 2018

ends of microfilaments has been observed in Saccharomyces cerevisiae and mammalian fibroblasts, and is mediated by type II or V myosins.4 Transport along microtubules has been studied in Drosophila melanogaster, Xenopus laevis, neuronal cells, and Ustilago maydis. Kinesins or dynein motors mediate movement of mRNA cargo toward plus- or minusends, respectively.1,5,6 Cargo mRNAs are recognized by RNA-binding proteins (RBPs) that interact with defined RNA localization elements (LEs, see Box 1). The majority of LEs in mRNAs are located in their 30 -untranslated region (30 -UTR)3 but LEs can also be found in the 50 -UTR, or even in the coding region, especially in unicellular eukaryotes like S. cerevisiae.7,8 LEs show hardly any sequence conservation across species. Although relatively little is known on how LEs function, the few well-studied examples suggest that it is their structure that is mainly important for their recognition.9,10 Specific recognition of LEs is mediated by dedicated RBPs that interact with structural elements or short sequence stretches. Some of these RBPs already bind

© 2017 Wiley Periodicals, Inc.

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BOX 1 LOCALIZATION ELEMENTS IN EUKARYOTES Cis-acting LEs determine which mRNAs are selected as cargo for transport. Among the LEs identified so far, only few consist of linear motifs that are recognized based on sequence specificity. For instance, the bipartite linear LE in the β-actin mRNA contains two sequence elements that are separated by a defined spacer. This element is recognized by the protein ZBP1.12,13 However, most known LEs consist of stem-loop structures10 with mismatches or structural deviations from canonical stem-loops. Such structural ‘abnormalities’ confer the specificity for their protein-interaction partners. For instance, the 44-nt LE in the fs(1)K10 mRNA from D. melanogaster adopts an unusual A0 form conformation via purine stacking, resulting in a widened major groove and specific recognition by the transport machinery.14 Other examples with structural information are the Oskar Entry Signal (OES) and the Spliced Oskar Localization Element (SOLE) from D. melanogaster.15–17 While the OES stem contains a number of bulges, the SOLE forms a continuous helical structure with noncanonical base pairs that result in a widened major groove. However, to date, the only example for which the recognition of a structured LE by its cognate RBPs is comprehensively understood is the E3 LE of the ASH1 mRNA from S. cerevisiae.18 This element folds into a stemloop with bulges and undergoes dramatic rearrangements upon binding by its transport machinery. For details on the specific LE recognition of the ASH1 mRNA, see main text.

the signal co-transcriptionally3 and chaperone the mRNAs all the way to their final destination. These messenger ribonucleoprotein particles (mRNPs) are initially formed in the nucleus and subsequently mature by binding of additional RBPs before and/or after export of the complexes to the cytoplasm. Following export, the complexes are linked to motor proteins for active transport via adaptor proteins.3 In recent years, co-transport of mRNAs with membrane structures such as endoplasmic reticulum (ER) or endosomes has also been described.11 Because of the comparably low complexity of fungal transport machineries, the ease of genetic manipulation, and their biochemical accessibility, 2 of 13

fungal model systems are among the best choices to study mechanistic principles of mRNA localization. Although not all RBPs or LEs are evolutionarily conserved, the underlying mechanisms for active transport seem very similar. Hence, basic concepts have often been uncovered by studying fungal model systems such as S. cerevisiae and U. maydis. Here, we describe recent advances in this field, and first focus on how RBPs specifically recognize transcripts for their translationally silenced transport along actin cytoskeleton. We then describe what the structural requirements are for LE recognition and summarize our current knowledge about membrane attachment of RBPs in S. cerevisiae. In the second part, we portray the endosomal transport of translationally active mRNAs along microtubules in U. maydis.

ACTIN-DEPENDENT TRANSPORT OF mRNAs mRNA transport toward the plus-ends of actin filaments by type II or V myosins is found throughout eukaryotes. One of the best-studied examples is the type V myosin-dependent transport of ASH1 (Asymmetric Synthesis of HO) mRNA along actin tracks in S. cerevisiae. ASH1 encodes a transcriptional inhibitor of the site-specific endonuclease HO (HOmothallic switching), which is responsible for the genomic rearrangement at the MAT locus and thus switching of the mating type during asymmetric cell division.19 Mating-type switching in S. cerevisiae occurs after cell division in the mother cell and allows the formation of diploid zygotes from haploid progeny with initially identical mating type. Prior to cytokinesis, ASH1 mRNA is transported to the distal pole of the daughter cell where the mRNA is translated and Ash1p is produced. Thereby, the transcriptional repressor specifically enters the daughter cell nucleus, prevents HO expression, and consequently inhibits mating-type switching20,21 (Figure 1(a)). Thus, asymmetric gene expression is achieved by combining mRNA transport and local translation. Four LEs have been mapped in the ASH1 mRNA, of which three reside in its open reading frame and one is located in the 30 -UTR.7,8,22,23 Each of these LEs is recognized by the transport machinery via the RBP She2p24–26 shuttling between the cytoplasm and the nucleus,27 where it co-transcriptionally recognizes the LEs of the ASH1 mRNA28 (Figure 1 (a)). This initial binding in the nucleus is weak and of modest specificity.29 It is stabilized by the RBP Loc1p,30,31 albeit still with modest specificity. After transition through the nucleolus the complex is



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mRNA transport in fungal top models

FI GU RE 1 | Actin-dependent mRNA transport in Saccharomyces cerevisiae. (a) In a budding cell ASH1 mRNA is transported along actin tracks to the daughter cell for translation. Ash1p ends up in the daughter cell, where it is imported into the nucleus (n) for specific inhibition of HO expression. (b) Assembly of the transport mRNP is initiated in the nucleus. ASH1 mRNA is bound co-transcriptionally by She2p as well as Loc1p and Puf6p. This complex has modest specificity (open lock). Upon transition of the nucleolus (no), export and binding by She3p the mRNP is remodeled and locked in its transport competent form. (c) During transport, the ASH1 mRNA is translationally silenced by the action of the RBPs Khd1p, Puf6p, and Dhh1p. At its destination in the daughter cell Khd1p and Puf6p are released by phosphorylation. This results in the local translation of Ash1p, which is subsequently imported in the daughter nucleus (n). (d) Alternative actin-dependent transport mechanism involves the ER. Two RBPs associate transported mRNA cargo with the ER. Scp160 binds its mRNA cargo, which is translated at, and thereby tethered to, the ER. In contrast, She2p is able to bind specifically to the ER tubules by recognizing their membrane curvature, as well as by potentially interacting with an ER-associated orphan adaptor protein. exported to the cytoplasm.27 Loc1p is a strictly nuclear protein30,32,33 and is thus removed from the complex during or shortly after nuclear exit (Figure 1 (b)). This mRNP remodeling is achieved by the action of another RBP, She3p25,31,34 at or close to the nuclear pore. In biochemical assays, She3p is able to outcompete Loc1p from the ASH1 mRNA–She2p complex,31 indicating that a very similar event might occur in vivo. Notably, the addition of She3p results in a highly synergistic and specific complex in which She2p and She3p interact with each other and both proteins also contact the RNA.18,34 This synergism explains why She3p is able to outcompete Loc1p from the complex. As She3p is constitutively bound to the type V myosin motor Myo4p,24–26,35–40 the


resulting complex consists of ASH1 mRNA, She2p, She3p, and Myo4p (Figure 1(b)). Recent in vitroreconstitution and single-particle motility assays demonstrated that these three proteins together with the myosin chaperone She4p and myosin light chains are sufficient to support processive transport of ASH1 mRNA along actin filaments.41–43 In addition to this core complex, the transport particle is loaded with the translational repressors Dhh1p, Khd1p, and Puf6p44–48 that ensure translational silencing of the transcript during transport49 (Figure 1(c)). To release translational repression, Khd1p and Puf6p become phosphorylated by the casein kinases Yck1p and Yck2p.50,51 Owing to kinase anchoring at the plasma membrane, Yck1p–


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Khd1p interaction seems to be limited to the plasma membrane of the bud.51,52 Although not directly shown, such local interaction is similarly postulated for Puf6p and Yck2p. As mentioned above, this achieves local production of Ash1p, exerts the repression of HO endonuclease in the daughter cell, and results in a different cell fate than that of the mother cell. Although the reported translational repression of ASH1 mRNA is consistent with in vivo observations, there are unresolved issues with regard to Puf6p action. While some studies showed specific ASH1 mRNA binding by Puf6p,28,50 other studies reported that it belongs to an unusual class of Pumilio proteins with unspecific affinity for doublestranded RNA/DNA.31,34,53 Hence, despite intense research, the complete in vivo picture of translational repression, including the exact role of all components, awaits further clarification. Recently, X-ray crystallography was used to understand the molecular principles underlying the specific LE recognition in the ASH1 mRNA by She2p and She3p.18 While the LE alone was found to form a highly flexible stem loop with bulges, joining of She2p trapped the stem loop in a kinked conformation. Except for three base-specific contacts, the RNA is recognized mainly by its shape and by its ability to undergo conformational rearrangements. Unexpectedly, the increase in specificity upon joining of She3p is only based on additional conformational constraints and no additional base-specific contacts.18 Furthermore, the contributing part of She3p was found to be disordered. Interestingly, recent proteome-wide studies have identified a plethora of disordered proteins with RNA-binding capabilities but unknown functions.54 The RNA binding by She2p and She3p provides an intriguing example for how a disordered RBP can modulate an RNAbinding event of a globular protein toward higher specificity. The concept of transporting mRNAs encoding transcription factors for asymmetric gene expression is conserved in fungi and, for instance, also found during hyphal growth in the human pathogen Candida albicans.55 Here, a She3p-dependent transport system is responsible for the localization of ASH1 mRNA to the hyphal tip. Thereby, the encoded Ash1p transcription factor is specifically targeted to the most distal nucleus of the growing hyphae,56 where it probably triggers a defined gene expression program. Loss of CaAsh1 causes defects in hyphal growth and reduced virulence.57 However, this is independent of endonuclease HO regulation, because this ascomycete does not switch its mating type. 4 of 13

Another difference to S. cerevisiae is that no clear She2p homologue can be identified. Thus, either another protein such as the recently suggested SR-like RBP Slr158 needs to substitute the She2p function in this human pathogen or, alternatively, She3p has adapted to achieve highly specific RNA recognition on its own. One peculiarity is the observation that ASH1 mRNA from C. albicans is also actively transported when expressed in S. cerevisiae, suggesting that at least one LE in the C. albicans transcript is conserved and recognized by the She2p–She3p complex in budding yeast.59 Besides the ASH1 transcript, several other mRNAs encoding transcription factors were identified as potential She3p-associated cargo mRNAs in C. albicans. Thus, She3p might spatially control transcriptional programs by restricting the presence of several transcription factors to specific nuclei.56 Such a function might be widespread, because it was observed that the basic zipper-type transcription factor FlbB is specifically enriched in the most apical nucleus during hyphal growth in Aspergillus nidulans.60,61 In essence, transport of translationally silenced mRNAs combined with activated translation after deposition determines asymmetric gene expression in fungi.

ACTIN-DEPENDENT CO-TRANSPORT OF mRNAs AND CORTICAL ER Interestingly, co-transport with cortical ER (cER) was uncovered as an alternative mode of mRNA localization62,63 (Figure 1(d)). cER is a specialized form of the ER that consists of tubular ER structures present in close contact to the plasma membrane. It functions, for example, in the delivery of lipids to the plasma membrane.64 During budding, this specialized ER is inherited via multiple steps: tubular ER structures emanate from the ER surrounding the nucleus (perinuclear ER) in the mother cell and migrate into the emerging bud by an actin- and Myo4p/She3p-dependent mechanism. In the bud, they are anchored at the bud tip and spread underneath the plasma membrane. The requirement of Myo4p and She3p in this process suggested a potential connection of ER tubule movement and mRNA transport. In fact, localization of several mRNAs to the bud depends on proper migration and anchoring of cER tubules. These mRNAs include two transcripts (WSC2, SRL1) encoding secreted proteins involved in cell wall maintenance and one (IST2) coding for a cER-specific membrane protein required for tethering cER to the plasma membrane.



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Furthermore, three mRNAs (SRO7, SEC4, and CDC42) encode proteins functioning in polarity establishment and polarized secretion. In addition one mRNA (EAR1) encodes an endosomal protein required for cargo sorting.63,65 As membrane and secreted proteins are generally translated at the ER, co-transport of mRNAs and cER tubules would facilitate local synthesis of these proteins at the cER close to the bud membrane and might support compartmentalization of cER as well as polarized secretion (Figure 1(d)). Consistently, several mRNAs are bound and localized by She2p, including IST2, TCB2, and TCB3,66,67 that encode proteins of a complex responsible for attachment of cER at the plasma membrane.68 Restricted synthesis of these proteins could support, for example, correct anchoring of cER. All of the above-mentioned mRNAs are bound by She2p, and several studies have demonstrated that it links these mRNAs to the ER. She2p can bind to membranes independently of translation, of Myo4p, and even independently of its target mRNAs.62,69 Loss of She2p results in a shift of target mRNAs from membranes into the cytosol.63 Thus, She2p serves as an example for a membrane-associated RBP. Although She3p is not required for She2p association with ER, it might still be involved in tight binding of cargo mRNAs as She3p strongly enhances binding to an LE from EAR1 mRNA, which is cotransported with ER.31,65 Binding of She2p to membranes appears to be specific for ER, because in vitro binding studies demonstrated no affinity to membranes derived from mitochondria.69 In addition, She2p preferentially binds to membranes with a high curvature as can be found in ER tubules.69 Membrane curvature is influenced by lipid content, especially by the ratio of phosphatidylethanolamine and phosphatidylcholine. Consistently, She2p binding in vitro and association of localized mRNAs with ER in vivo is sensitive to changes in the concentration of the lipid phosphatidylethanolamine and to mutations in enzymes involved in phospholipid conversion.70 As She2p does not contain known lipid-binding domains, its molecular mechanism of membrane association is still unclear. Association of co-transported mRNAs with ER tubules has also been observed during pheromone signaling. This targeted mRNA transport appears to be critical to orchestrate the morphological changes occurring during the pheromone response, a process that is independent of She2p.71 Instead, the key RBP involved is Scp160p belonging to the vigilin protein family. It is a 140-kDa protein with 14 RNA-binding domains of the K homology (KH) domain type.72 It


is interesting to note that, analogous to She2p, Scp160p interacts with Myo4p as well as ER and is essential for the delivery of pheromone-induced mRNAs (like FUS3, KAR3, and STE7) during polarized growth in mating cells71 (Figure 1(d)). However, in contrast to She2p, binding of Scp160p to ER depends on its associated RNAs. This suggests that translation of these mRNAs determines recruitment of Scp160p to ER, most likely via binding to a complex containing the ribosome, mRNA, nascent peptide chain, and signal recognition particles.

MICROTUBULE-DEPENDENT TRANSPORT OF mRNAs Long-distance transport along microtubules is essential to support efficient cell expansion at the growth tips of fungal hyphae. Important carriers are early endosomes that serve as multipurpose platforms and transport a variety of cargoes, such as mRNAs, ribosomes, protein complexes, as well as whole organelles, including peroxisomes.73–78 One of the best-studied examples for endosomal transport during hyphal growth is the plant pathogen U. maydis. In this fungus, the switch from yeast-like to hyphal growth is a prerequisite for infection of its host, which is corn.79 This process depends on mating of two compatible haploid yeast cells resulting in the formation of dikaryotic hyphae with two separated nuclei after cell fusion.80,81 Hyphae expand at the apical poles while the nuclei are positioned in the center. During this phase of infection the cell cycle is arrested. The cell cycle is reactivated after plant penetration resulting in cell proliferation within the host. At the basal pole, septae are inserted at regular time intervals, leading to the formation of characteristic empty sections devoid of cytoplasm82 (Figure 2(a)). Destruction of microtubules specifically results in aberrant hyphal growth.83 Instead of the normal unipolar expansion, hyphae grow out bipolarly and the insertion of basal septa is delayed.76,84,85 Thus, microtubules are important in maintaining the correct axis of polarity. The cytoskeletal elements are organized into two to four antiparallel microtubular bundles with plus-ends of incorporated microtubules facing to the poles. Microtubules serve as tracks for the active transport of endosomes positive for the small G protein Rab5a, a specific marker protein for early endosomes.77 Plus-end-directed transport is mediated by the kinesin-3-type motor Kin3,86,87 which is attached to endosomes via its pleckstrin Minus-end-directed homology domain.84,88,89


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(a) Apical pole

Basal pole

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Nucleus

Empty section

Remnants of initial cell

Septum Septin filaments

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(c) 11 12

Pab1 AAAA

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? Did2

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Peroxisome

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Kin3

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F I G U R E 2 | Microtubule-dependent mRNA transport in Ustilago maydis. (a) Micrograph (top) and model (bottom) of hypha that expands from an initial yeast-like cell by polarized growth at the apical pole. Septa are inserted in regular intervals resulting in empty sections at the basal pole. Note that the lab strain used (AB33) was genetically modified so that hyphal growth can be elicited efficiently and independent from mating. In contrast to dikaryotic wild-type hyphae, the lab strain contains only a single nucleus positioned in the cell center. Organelles such as ribosomes, peroxisomes, as well as mRNPs are transported by early endosomes along antiparallel microtubule arrays. Scale bar: 10 μm. (b) Schematic model of the transport complex of endosomes with an mRNP. Early endosomes are transported by Kin3 and Dynein. According to the results obtained in Aspergillus nidulans, the small GTPase Rab5a is proposed to directly bind to components of the FHF complex that interact with the dynein supercomplex. The RBPs Rrm4 and Pab1 bind directly to endosomal protein Upa1. This interaction is mediated by the MLLE domains of Rrm4 and Pab1. Further components associated with this transport complex are peroxisomes and ribosomes. The presence of the latter suggests active translation of transported mRNAs. (c) Coordinated transport and translation of septin mRNA on shuttling endosomes promotes the assembly of heteromeric septin complexes, and subsequently, the formation of higher order structures like septin filaments at the hyphal tip. transport is executed by the cytoplasmic dynein complex consisting of a split dynein Dyn1/Dyn2.87 Interestingly, loss of Kin3 or Dyn2 from U. maydis results in the formation of bipolar hyphae, indicating that bidirectional movement of early endosomes is a key function for microtubule-dependent transport during polar growth.84,85 Recently, it was shown in 6 of 13

A. nidulans and vertebrate neurons that Rab5 directly interacts with components of the FHF complex, which in turn binds the dynein supercomplex for recruitment of the minus-end-directed transport machinery90–92 (Figure 2(b)). Studying the function of the RBP Rrm4 in U. maydis disclosed the first link between mRNA



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and endosomal transport. Rrm4 contains three Nterminal RRMs (RNA recognition motifs) with an architecture characteristic for ELAV-type proteins (embryonic lethal abnormal vision).93 In this class of RBPs, two tandem RRMs are followed by a linker and a third RRM. Loss of Rrm4 results in the formation of bipolar hyphae, similar to the aberrant phenotype observed upon loss of microtubule function. Rrm4 co-localizes extensively with early endosomes.84 However, unlike Rab5a, which is also found in static membranous compartments, Rrm4 localizes exclusively to motile endosomes. In fact, it thus serves as an excellent marker for this endosomal compartment.94 A combination of in vivo UV crosslinking and RNA live imaging revealed Rrm4 target mRNAs like ubi1, rho3, and septin cdc3.73 Loss of Cdc3 leads to defects during establishment of unipolar hyphal growth, suggesting a link between septin function and microtubule-dependent processes specifically during this phase of hyphal growth.74 Importantly, when Rrm4 is missing and hence cdc3 mRNA transport is abolished, the subcellular localization of the translation product Cdc3 is altered. First, Cdc3 protein localization to early endosomes no longer occurs and second, a gradient of septin filaments is no longer formed at the growing tip.74 Septin mRNAs and protein localize to identical endosomes, and without mRNA transport no septin protein is present on the cytoplasmic surface of endosomes. Because ribosomes are also present on shuttling endosomes in an Rrm4-dependent setting, transport of translationally active mRNAs is the most succinct explanation6,74 (Figure 2(b)). Consistently, Rrm4 is also needed for the distribution of ribosomes in hyphae.76 Albeit specifically present on endosomes, Rrm4 does not harbor any obvious motif for direct lipid binding. Instead, the protein contains two regions at the C-terminus that are predicted to fold into MLLE domains known from the C-terminus of the human cytoplasmic poly(A)-binding protein PABPC1.95,96 The domain contains a conserved KITGMLLE amino acid signature and serves as a peptide-binding pocket for PAM2 sequences (PABP-associated motif 2). In the case of PABPC, these specific protein/protein interactions mediate PABPC binding to the translational release factor eRF3.97 Interestingly, mutations in critical amino acids of the C-terminal MLLE domain of Rrm4 abolish movement,98 suggesting that protein interaction partner(s) are responsible for Rrm4 attachment. Analyzing candidates revealed a novel PAM2-containing protein Upa1 (Ustilago PAM2-containing protein 1).99 This protein not only contains a PAM2 motif for interaction with the


poly(A)-binding protein Pab1 from U. maydis, but in addition two functionally important PAM2-like sequences for interaction with the MLLE domains of Rrm4.99 Its C-terminus resembles the endosomal protein Pib1 from S. cerervisiae, which contains a FYVE domain for binding of phosphatidylinositol 3-phosphate lipids in membranes of early endosomes.100–102 Indeed, Upa1 serves as a linker protein that binds both to shuttling early endosomes via its FYVE domain and to Rrm4 by the PAM2-like sequences (Figure 2(b)). Loss of Upa1 causes a bipolar phenotype. Furthermore, the processive movement of Rrm4 is affected, whereas the movement of other endosomal proteins such as Rab5a is unchanged.99 Interestingly, it was found in A. nidulans that the protein PxdA functions as an adaptor protein to link peroxisomes to early endosomes.103 Thus, it appears to be a common theme that specific adaptor proteins are needed to direct individual cargoes like mRNPs or peroxisomes to early endosomes for long-distance transport.78 A recent study answered the question how the Rrm4-dependent special functions of early endosomes are linked to their classic function in endocytic trafficking,104 namely the internalization of plasma membrane proteins for degradation via late endosomes and vacuole/lysosomes.105 Loss of the crucial ESCRT regulator Did2 in U. maydis resulted in defective vacuolar targeting of proteins as well as in altered unipolar growth, resembling mutants affected in microtubule function.104 The latter mutant phenotype was due to disturbed endosomal maturation. Thereby, the membrane composition of early endosomes was altered so that molecular motors could not be attached efficiently and the lipid-associated linker Upa1 did not bind correctly. This caused defects in endosomal mRNA transport. Thus, the ESCRT regulator Did2 coordinates the two seemingly separate functions of early endosomes, longdistance transport and endocytic trafficking.104 In line with these findings, in higher eukaryotes Rab5positive endosomes are involved in axonal growth and the ESCRT-II complex participates in neuronal mRNA transport.106,107 Studies in U. maydis have revealed that transcripts encoding all four septins are important cargo for the mRNA transport machinery. Each septin as well as its cognate mRNA is present on shuttling endosomes suggesting that all subunits are translated locally on the vesicles.75 Interestingly, the presence of the individual septin proteins on endosomes depends on each other, indicating that heteromeric complexes could directly assemble on endosomes.75 These heteromers are then transported toward the growing tip


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for delivery and assembly into higher ordered filamentous structures75 (Figure 2(a) and (c)). Septin filaments might also be needed to organize other cytoskeletal elements like actin bundles or microtubules to initiate unipolar hyphal growth. This hypothesis is supported by studies with other organisms, where septins organize the actin cytoskeleton or alter microtubule stability by posttranslationally modifying the structures.110,117 Consistently, septin assembly is promoted in the vicinity of membranes

BOX 2 SEPTINS Septins are highly conserved cytoskeletal elements present from fungi to animals but absent in plants.108,109 They exhibit important functions in a variety of processes such as cytokinesis, cell polarity, motility, as well as during bacterial infections.110–112 In plant pathogenic fungi, septins organize the formation of specialized infection entities, called appressoria, and are therefore important for host penetration.113–115 Important for septin function is the ability to form higher-ordered structures such as rings or filaments. These serve as membrane barriers to organize membranous subdomains or they function as scaffolds for the recruitment of signaling or cytoskeletal components like kinases and actin, respectively.109,116 Furthermore, septin filaments influence the spatial posttranslational modifications of microtubules and thereby regulate their dynamics.117 In S. cerevisiae, four core septins form palindromic heterooctameric building blocks, that is, Cdc11:Cdc12:Cdc3:Cdc10::Cdc10:Cdc3:Cdc12: Cdc11, following a defined order of assembly.118 These nonpolar elements anneal headto-tail to form rod-like structures that associate into elaborate higher-ordered structures such as extended filaments. Interestingly, assembly is promoted by lipid interactions and septins can sense membrane curvature, suggesting a close link between septin assembly and membrane function.119,120 In U. maydis, septins are important for the cell shape of yeast cells121 and unipolar growth of infectious hyphae.74,75 Unipolar growth depends on endosomal transport of septin mRNAs and endosomal assembly of the translation products (see main text).

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and septin complexes can sense membrane curvature (see Box 2).110,120,122

CONCLUSION As outlined in this Focus Article, fungi constitute powerful model systems to uncover the fundamental basics of actin- and microtubule-dependent mRNA transport. A unifying view of the transport machinery supports the presence of cis-acting LEs in cargo mRNAs that are recognized by key RBPs. Accessory factors are needed to connect cargo mRNPs with molecular motors for active transport along the cytoskeleton. Recent highlights uncovered in fungi are the synergistic recognition of LEs to achieve specificity in binding as well as profound changes of the RNA structure upon recognition.18 These findings are not only essential to understand mechanistic details during remodeling of transport-competent mRNPs, but the identified mechanistic rules may serve as paradigms for mRNP assembly and function in other organisms. Moreover, a connection to membrane trafficking is exemplified by the co-transport of mRNAs with ER or endosomes for targeting of membrane proteins or septin complexes, respectively.11,62,75 The intimate link of RNA and membrane biology might be widespread, because an increasing number of examples is emerging, like secretory factor Sec2 from C. albicans binding its own mRNA123 or ESCRT components interacting with mRNAs orchestrating localized translation.107,124 Studying endosomal mRNA transport during polar growth led to the hypothesis that the transport of translationally active septin mRNAs is needed for efficient assembly of heteromeric septin complexes on the cytoplasmic surface of endosomes.74,75 Intriguingly, similar processes might take place during neuronal mRNA transport. Rab5positive early endosomes participate in axon growth106 and the endosomal component ESCRT-II binds mRNAs.107 Furthermore, bidirectional movement of translating polysomes with velocities indicative for a motor-based transport was recently reported in neurons.125 Besides the well-established transport of translationally silent mRNAs, the transport of translationally active mRNAs might be more common than previously anticipated. Future research on fungal model organisms will uncover not only further details of mRNA transport but also its detailed mechanistic implications in protein distribution. Central remaining questions are how protein translation and transport are synchronized for efficient delivery of the translation products,



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and how specific local delivery is achieved. Finally, basic research on mRNA transport led to the discovery of a novel mechanism of unconventional secretion, which is currently being exploited for

export of heterologous proteins for industrial application.126,127 In essence, studying eukaryotic microorganisms as role models deciphers fundamental processes of intracellular trafficking.

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