Review

mRNA transport meets membrane traffic Ralf-Peter Jansen1, Dierk Niessing2, Sebastian Baumann3, and Michael Feldbru¨gge3 1

Eberhard Karls Universita¨t Tu¨bingen, Interfaculty Institute of Biochemistry, Hoppe-Seyler-Strasse 4, 72076 Tu¨bingen, Germany Helmholtz Zentrum Mu¨nchen, German Research Centre for Environmental Health, Institute of Structural Biology, 85764 Neuherberg, Germany 3 Heinrich Heine University Du¨sseldorf, Institute for Microbiology, Cluster of Excellence on Plant Sciences, 40204 Du¨sseldorf, Germany 2

Active transport and local translation of mRNAs ensure the appropriate spatial organization of proteins within cells. Recent work has shown that this process is intricately connected to membrane trafficking. Here, we focus on new findings obtained in fungal model systems. Important highlights are that RNA-binding proteins recognize cargo mRNA synergistically and that mRNAs are co-transported with membranous compartments such as the endoplasmic reticulum (ER) and endosomes. We further discuss a novel concept of endosome-coupled translation that loads shuttling endosomes with septin cargo, a process important for correct septin filamentation. Interestingly, evidence is accumulating that RNA and membrane trafficking are also tightly interwoven in higher eukaryotes, suggesting that this phenomenon is a common theme and not an exception restricted to fungi. Active transport of mRNPs and membranous compartments Active cytoskeletal transport mediated by molecular motors is essential for cellular logistics in eukaryotes and transport defects often cause death or disease. An important class of molecular cargo is mRNA [1,2]. Actively transported mRNAs contain cis-acting regions, termed zip codes (see Glossary) or localization elements. These zip codes constitute binding sites for RNA-binding proteins of the transport complex, which themselves interact with adapter proteins and molecular motors such as kinesin, dynein, or myosin [2–4]. Furthermore, these complexes usually contain accessory factors such as helicases, translational repressors, RNA stability factors, or ribosomal proteins [5,6]. Therefore, mRNAs are never naked in the cell but form macromolecular complexes called mRNPs (messenger ribonucleoprotein particles). These mRNPs do not only contain the mRNA information for the encoded amino acid sequence but also determine the precise spatiotemporal regulation of translation and thereby guarantee the correct subcellular localization of the translated protein. Thus, ‘the message is the mRNP’ [7]. Corresponding author: Feldbru¨gge, M. ([email protected]). Keywords: actin; endoplasmic reticulum; endosomes; microtubule; mRNP; septin. 0168-9525/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tig.2014.07.002

408

Trends in Genetics, September 2014, Vol. 30, No. 9

Comprised in a second class of important molecular cargo are membranous compartments such as endosomes and small vesicles that transport lipids and proteins. The underlying transport events are known as membrane Glossary BAR domain (Bin–Amphiphysin–Rvs): this domain was first identified in BIN1, amphiphysins, and the yeast proteins Rvs167p and Rvs161p. It forms a bananashaped dimer that interacts with curved membranes and thereby functions in membrane shaping. Calmodulin: evolutionarily conserved regulatory protein that interacts with calcium via a characteristic binding motif called the EF hand. It functions in intracellular calcium signaling by interaction with other signaling proteins such as kinases. Cortical endoplasmic reticulum (cER): specific type of ER found in plants, yeasts, and some metazoan cell types. cER forms tubular networks juxtaposed to the plasma membrane. Early endosome: large vesicle-type membranous structure involved in endocytosis. Characteristic marker proteins for this endosomal compartment are the small G proteins Rab4 and Rab5. Early endosomes mature to late endosomes or are involved in rapid recycling towards the plasma membrane. ELAV-type: specific family of RNA-binding proteins initially identified in D. melanogaster. Named after the mutant phenotype Embryonic Lethal Abnormal Vision. ESCRT complex: a macromolecular machinery designated Endosomal Sorting Complexes Required for Transport. It consists of four subcomplexes called ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III. The machinery assembles in an ordered manner and functions in membrane remodeling. Golgi apparatus: a biological structure named after its discoverer, the Italian physician Camillo Golgi. The Golgi apparatus is a membranous compartment functioning in secretion of proteins. Transport vesicles from the ER deliver cargo to the Golgi complex and after maturation transport vesicles leave the Golgi for fusion with the plasma membrane. Late endosome: large vesicle-type membranous structure involved in endocytosis. A characteristic marker protein for this compartment is the small G protein Rab7. Late endosomes mature to multivesicular bodies/vacuolar compartments. Multivesicular body (MVB): specific endosomal compartment that is formed during maturation of late endosomes. MVBs are formed by pinching off vesicles inside the lumen of large endosomes. Similar to late endosomes they carry the small G protein Rab7 as a marker. Pleckstrin homology domain (PH): domain found in the protein Pleckstrin, a major target of protein kinase C. The 120 amino acid PH domain interacts with phosphatidylinositols and therefore functions in lipid binding and recognition. RNA-induced silencing complex (RISC): ribonucleoprotein complex that contains small RNAs and the Argonaute protein. It functions in the degradation or translational control of target mRNAs that are recognized by their complementarity to the bound small RNAs. Rough endoplasmic reticulum (rER): specific type of ER that is covered with translating ribosomes for co-translational import of proteins into its lumen and therefore functioning in active protein secretion. Signal recognition particle (SRP): ribonucleoprotein complex that recognizes specific signals in secreted and membrane proteins. Unconventional secretion: conventional secretion in eukaryotes is mediated by the interaction of the signal recognition particles with specific signal sequences at the N terminus of secreted proteins. To differentiate from this classic export pathway the term ‘unconventional secretion’ is used. Zip code: RNA sequence that functions as a recognition site for RBPs involved in subcellular mRNA localization.

Review

Trends in Genetics September 2014, Vol. 30, No. 9

trafficking [8]. For outbound traffic, secreted proteins or those of the endomembrane system are directly translated at the rough endoplasmic reticulum (rER) where the translation products enter the secretory pathway. Subsequently, the respective proteins are transported by vesicles via the Golgi apparatus to the plasma membrane for secretion or for plasma membrane insertion [9]. During inbound traffic, distinct areas of the plasma membrane pinch off in the form of vesicles that fuse with specific endosomal compartments, called early endosomes. These can mature into late endosomes and fuse with the vacuole or lysosome for protein degradation. Alternatively, they can be redirected to the plasma membrane for receptor recycling [8]. To fulfill their function, compartments involved in membrane trafficking such as vesicles, endosomes, or ER substructures are actively distributed along the cytoskeleton. In this review, we summarize how these two seemingly independent classes of intracellular transport are tightly linked. We focus on recent results obtained in fungal model systems describing mechanistic insights on the co-transport of mRNPs and ER as well as endosomes. Assembly of mRNP cargo requires multiple RNAbinding proteins Localizing mRNPs have been observed throughout all eukaryotic kingdoms. One of the best-studied model systems to investigate mRNP transport is Saccharomyces cerevisiae. Core factors of the mRNA trafficking machinery (A)

are the myosin motor Myo4p, the adaptor protein She3p, and the RNA-binding protein She2p (Figure 1) [10]. During cell division and polar growth, this so-called SHE machinery transports approximately 30 types of transcripts along actin tracks from the mother cell to the tip of the growing daughter cell, the bud (Box 1) [10,11]. Cargo mRNAs thereby accumulate at the growth pole of daughter cells, resulting in a specific subcellular localization in the bud. Currently, the best-studied example is the asymmetric localization of ASH1-containing mRNPs, which facilitates daughter cell specific expression of the transcriptional repressor Ash1p. The resulting selective distribution of Ash1p leads to inhibition of mating type switching specifically in the daughter cell nucleus [10]. The ASH1 mRNA is already co-transcriptionally recognized by the RNA-binding protein She2p via its four zip code elements, forming an initial nuclear pre-complex [12]. Surprisingly, She2p binds to ASH1 mRNA with low affinity and specificity in vitro [13,14], which is incompatible with processive transport of mRNA in the cell. This seeming paradox was recently resolved by the observation that joining of the nuclear ribosome biogenesis factor Loc1p dramatically stabilizes the complex and improves the specificity of She2p for ASH1 mRNA [15]. The Loc1pstabilized complex accumulates in the nucleolus [15], where it is most likely loaded with the translational regulators Puf6p and Khd1p (Figure 1) [10]. During nuclear export, the ASH1 mRNP is remodeled and Loc1p is (C)

ER

She2p Khd1p

Adaptor AAAA

She3p

Puf6p AAAA

Myo4p

Acn

Khd1p

She2p (D)

(B)

AAAA

RNA Pol II

DNA

Acn She3p

ASH1

Myo4p AAAA AAAA

AAAA

Loc1p Puf6p

TRENDS in Genetics

Figure 1. Messenger ribonucleoprotein particle (mRNP) transport in Saccharomyces cerevisiae. On the left two different stages of budding cells are depicted. Broken rectangles indicate subcellular regions enlarged on the right. (A) Early phase: co-transport of mRNA (blue line with 50 cap structure as circle and 30 poly[A] tail) with cortical endoplasmic reticulum (cER; gold) along actin (black line). Note that the membrane adaptor for cER transport by Myo4p/She3p is unknown. (B) Late phase: cERindependent transport of ASH1 mRNA. (C) Detailed view of ER/mRNA co-transport. (D) Detailed view of nuclear events during She2p-dependent mRNP transport. She2p (green) is loaded co-transcriptionally on the mRNA elements (red hairpin). The nucleolar protein Loc1p (yellow) is replaced during cytoplasmic remodeling by She3p (red).

409

Review Box 1. Cell division and polarized growth in S. cerevisiae Saccharomyces cerevisiae cells divide asymmetrically by budding. During the cell cycle, the bud emerges from the mother cell in late G1 at a specific site that is defined by polarization cues [70]. The bud initially exhibits a focused, apical growth pattern by targeted delivery of membranes and proteins. Later, during G2, the bud expands evenly over its entire surface until it has reached a size similar to the mother. Subsequently, a septum is inserted for cell division. A key regulator is the evolutionary conserved small GTPase Cdc42p that accumulates at the site of bud emergence [70]. Local translation of its mRNA might contribute to its subcellular localization but is not essential for it [24]. Cdc42p activates a series of cell polarity effectors, including those that induce the formation of bundled actin filaments, called actin cables. These grow from the site of polarization deeply into the mother cell. Actin cables, facing with their plus ends towards the bud tip, represent the major transport roads to deliver proteins, lipids, RNA, vesicles, and even organelles to the growing bud. Two plus end-directed class V myosins, Myo2p and Myo4p, are the molecular motors that mediate cargo movement [71]. The essential myosin Myo2p is required for polarized delivery of secretory vesicles to the bud tip. In addition, the delivery of these vesicles and their regulated fusion with the plasma membrane involves small GTPases of the Rab protein family such as Sec4p, SNARE complexes and their regulators, as well as a large protein complex called exocyst. Most of these components are found at the bud tip. Myo2p also moves several organelles such as the vacuole and peroxisomes into the growing bud for their proper inheritance [72], ensuring the presence of organelles that cannot be made de novo. The nonessential myosin Myo4p delivers mRNAs and is involved in inheritance of a specific ER compartment, the cER (Box 2). cER inheritance is not essential due to the later symmetric segregation of perinuclear ER in mitosis ensuring the presence of ER in both mother cell and bud. During mating, a pheromone-induced response activates a different polarization process called shmooing [73]. Interestingly, this polarization process shares components with budding, including small G proteins, formins, secretory machinery components, the actin cytoskeleton, and myosin motors.

replaced by the RNA-binding protein She3p [15]. The result is a highly specific and stable complex, consisting of She2p, She3p, and ASH1 mRNA (Figure 1) [14]. Similar to what was observed for the nuclear pre-complex, the synergistic binding of these two RNA-binding proteins is essential for achieving specific mRNA localization, as both She2p and She3p alone bind nonspecifically to RNA [14]. She3p is also constitutively bound to the type V myosin Myo4p [16–18], resulting in a defined, motor-containing transport complex. Within minutes this mRNP moves along actin filaments to the tip of the daughter cell, where it becomes anchored. Translation of the ASH1 mRNA is finally initiated by the phosphorylation and subsequent release of Puf6p and Khd1p at the bud tip [10]. The comparably low complexity of this transport system provides an appealing opportunity to uncover detailed mechanistic insights. Partially or fully reconstituted ASH1 mRNPs in vitro consisting of She2p, She3p, Myo4p, and motor-bound calmodulins function as minimal mRNPs. To assess their mechanistic properties, these reconstituted mRNPs were used to perform single particle motility assays [16,19–21], which demonstrated that the minimal mRNPs are indeed motile and have a defined size with fixed protein stoichiometries [16,19]. Furthermore, this approach unambiguously proved that mRNAs are dispensable for the activation of processive movement of 410

Trends in Genetics September 2014, Vol. 30, No. 9

the protein complex along actin filaments [16]. Thus, in contrast to what has been reported for dynein-dependent transport in Drosophila melanogaster [22] mRNA cargo does not influence processive transport in S. cerevisiae. In summary, results obtained in this simple model show that functional, motile mRNPs can be assembled in vitro and that this example can be used to understand the underlying molecular mechanisms of mRNA transport in vivo. Furthermore, the interaction between multiple RNAbinding proteins to ensure specific RNA recognition is an important emerging concept in mRNA localization. This observation might also help to explain why so few specific in vitro interactions have been reported between individual RNA-binding proteins and their target RNAs in higher eukaryotes. Actin-dependent co-transport of mRNPs and ER in S. cerevisiae As discussed above, the minimal transport machinery has been well defined in vitro and was shown to function without additional components such as accessory factors. However, several observations indicated that in vivo mRNP transport is more complex. Apparently, the SHE machinery can use several transport pathways and there are additional mRNA transport routes independent of the SHE machinery (see below). Several lines of evidence point towards a potential molecular link to membrane trafficking. (i) ASH1 mRNA was detected in ER-derived fractions [23,24]. (ii) Mutations in the regulatory protein Arf1p, which is required for Golgi–ER vesicle trafficking, disturbed mRNA localization [25]. (iii) The core components for mRNP transport, Myo4p and She3p, but not She2p, are needed for inheritance of cortical ER (cER) (Box 2) [26,27]. This active cER transport to the bud is important to supply the growing bud with ER during the initial phase of cell division (Box 1). RNA live imaging has proven that mRNAs are indeed co-transported with the ER towards the bud and that this movement depends on the SHE machinery [28]. This observation was recently corroborated by showing that She2p is able to directly bind to membranes and by demonstrating that besides RNA binding it also possesses lipid-binding properties. She2p preferentially binds to membrane structures with high curvature, which is consistent with the observed colocalization of mRNPs and tubular ER [29]. In addition, She2p binds more strongly to ER-derived membranes than to those derived from mitochondria, indicating the presence of unknown specificity determinants on the ER [29]. It is of note that only a subset of bud-localizing mRNAs is transported in an ER-dependent manner [24,30]. Many of these mRNAs encode membrane and secreted proteins as well as proteins required for establishment of polarized secretion such as the highly conserved small GTPase Cdc42p (Box 1) [31]. These examples provide a possible explanation for the intimate link between mRNP and ER co-transport. Membrane and secreted proteins require translation at the ER. Thus, mRNA/ER co-transport might facilitate local translation of these proteins at the bud ER or even during movement of cER and mRNPs. This could permit a specific protein composition at the cER or lead to

Review Box 2. Targeting mRNAs to the ER The ER is a large, membranous organelle with a diversity of structural domains, ranging from regions that are flat and cisternal to others that are highly curved and tubular. Its elaborate structure is determined by both integral membrane proteins and interactions with the cytoskeleton [74]. Specific domains are adapted to form contacts with other organelles and with the plasma membrane. In Saccharomyces cerevisiae, the cER forms a tubular network in close proximity to the plasma membrane and functions as a local supplier of lipids [75]. Although ultrastructural analysis suggests cER to be smooth ER lacking ribosomes, new results contrarily indicate that protein synthesis occurs at this ER domain [76]. In general, proteins destined for the ER lumen or its membrane are translated by ER-bound ribosomes, which are co-translationally directed to the ER surface by the signal recognition particle (SRP). However, several observations suggest alternative modes of targeting mRNAs to the ER [11]. In humans, ER-associated polysomes not only bind mRNAs coding for proteins of the endomembrane system but also contain a significant fraction of cytoplasmic mRNAs [77]. After translation initiation, ribosomes with cytoplasmic mRNAs detach from the ER to complete translation in the cytoplasm. Hence, translation at the ER is abundant and not restricted to proteins that enter the organelle or its membrane. In addition, SRP-independent targeting signals in mRNAs and cognate receptors exist at the ER. One example is the mammalian ER membrane protein p180. It contains a single transmembrane domain and a large lysine-rich domain that directly binds to mRNAs and sequesters them in the ER membrane [37]. Analysis of the distribution of several mRNAs encoding membrane proteins in S. cerevisiae revealed that mRNA targeting to the ER is independent of their 30 untranslated region (UTR) and translation but requires the presence of the coding region [33]. Interestingly, the degree of RNA colocalization with the ER coincides with the uracil content of their coding sequence, which is consistent with a previous observation that mRNAs of membrane proteins are rich in uracil [78]. Thus, high uracil content might preselect mRNAs coding for membrane proteins for ER association and could be recognized by ER-associated RNA-binding proteins.

the establishment of special ER subdomains. For instance, Ist2p, a cER specific membrane protein that tethers cER to the plasma membrane [32], is encoded by a bud-localized mRNA [30]. These examples clearly indicate that mRNP transport is more complex than previously anticipated even in simple eukaryotic models. With regard to membrane-independent and cER-dependent transport in vivo, we propose a timeresolved model differentiating early and late events during budding (Figure 1A,B). It is based on the fact that ASH1 mRNA is expressed exclusively in late mitosis, when cER transport is no longer prominent. By contrast, bud localized mRNAs that encode membrane proteins are expressed and transported early during the cell cycle, contemporaneous with cER tubule movement. Therefore, localizing mRNAs encoding membrane proteins might travel first using the cER co-transport pathway (Figure 1), whereas transcripts expressed at later stages are most likely transported via a cER membrane-independent pathway (Figure 1B). The observation that ASH1 mRNA has been detected in microsomal (i.e., ER-derived) fractions [23,24] is probably due to ER association during anchoring after transport to the bud tip. Although transcripts encoding polarity factors also hitchhike into the daughter cell by association with cER (Box 2) [24], some mRNAs encoding secreted proteins might move to the bud associated with perinuclear ER [33]. However, this process would be independent of the

Trends in Genetics September 2014, Vol. 30, No. 9

SHE machinery and stresses once again the complexity of the mRNP transport system. A SHE machinery-independent pathway has also been proposed to act during mating (Box 1), when mRNAs (e.g., encoding polarity factors) can also be transported in association with the ER [34]. A possible candidate that may function in connecting mRNPs to the ER is the RNA-binding protein Scp160p. It colocalizes with the ER and has been suggested to specifically interact with the Ga subunit of an activated G protein (Box 1). Because it binds to ER and Myo4p, Scp160p might replace She2p and She3p in the membrane-dependent delivery of mRNAs to sites of polarization during mating. This observation provides the first example of regulated RNA transport during chemotropism [34]. Finally, ABP140 mRNA is transported in the opposite direction towards the distal pole of the mother cell. This localization is independent of the SHE machinery but depends on the translation of the N-terminal actin binding peptide of Abp140p and on actin bundles growing towards the distal pole [35]. Co-trafficking of endomembranes and mRNAs as well as membrane-independent transport occurring in the same cell has also been observed in higher organisms (e.g., Xenopus oocytes). Here, mRNA trafficking to the vegetal pole at early stages of oocyte development occurs together with a membrane-containing structure called the mitochondrial cloud that contains mitochondria and a dense network of ER. At later stages, active transport of mRNPs along microtubules becomes more prominent. The latter is apparently membrane-independent [36]. A potential RNA-binding protein involved in mRNA/ER co-transport in animals is, for example, the ER-associated RNA transport protein Vg1RBP/Vera from Xenopus [36]. Alternatively, general RNA-binding proteins in the ER membrane such as the recently identified p180 [37] might anchor mRNAs to the ER also during transport. p180 is an integral ER membrane protein containing an RNA-binding domain (Box 2). In summary, the co-transport of ER and mRNPs demonstrates the intensive connection between mRNPs and membranes. She2p provides the first example of a key RNA-binding protein involved in transport that directly binds membranes and thereby links mRNP localization to membrane trafficking. Microtubule-dependent transport of endosomes in Ustilago maydis Microtubule-dependent transport is the main alternative route for active intracellular trafficking. In recent years, the fungus Ustilago maydis (Box 3) has matured to serve as an attractive model system to study this mode of transport [38]. This transport becomes particularly important when cells switch from yeast-like to hyphal growth during the early phase of plant infection. In filamentous fungi, microtubule-dependent transport is essential for efficient polar extension at the hyphal growth pole [39] and associated defects disturb hyphal growth. In U. maydis, microtubule defects lead to formation of shorter hyphae that often initiate a second growth pole resulting in characteristic aberrant bipolar cells (see Figure IC in Box 3) [40–42]. Endosomes constitute the main 411

Review

Trends in Genetics September 2014, Vol. 30, No. 9

Box 3. U. maydis, a eukaryotic model microorganism on the rise Ustilago maydis is a basidiomycete that infects corn and causes smut disease. The term refers to the formation of large tumor-like structures, developing black spores that lead to a dirty appearance [79,80]. Traditionally, U. maydis has been studied intensively as a plant pathogen with a focus on effector biology [79]. However, the fungus also serves as a powerful model for diverse cellular processes such as DNA repair, the unfolded protein response, peroxisomal protein targeting, and RNA biology [49,81–83]. U. maydis can be easily handled in the laboratory and is amenable to genetic manipulation by homologous recombination. Sophisticated genetic tools have been established including various protein tags, regulated promoters, resistance marker recycling, and vectors for Golden Gate cloning [80,84]. There is also a high quality annotation of the genome sequence, transferring this organism into the postgenomic era [79,80].

(A)

∗

Infection by U. maydis requires a morphological switch from yeast-like cells to polarly growing hyphae (Figure I). This switch is regulated by the key transcription factor bW/bE, whose activity is regulated at the level of heterodimerization. Because monomers originate from two different mating partners, the formation of infectious hyphae is tightly coupled to mating of two compatible cells [79,80]. This detailed knowledge has allowed the design of laboratory strains in which expression of an active bW/bE heterodimer is under control of a nitrogen source-dependent promoter. Thus, filamentous growth can be elicited highly synchronously and reproducibly in liquid culture. This is prerequisite for in vivo studies of intracellular trafficking at defined stages of hyphal growth allowing sophisticated protein, RNA, and membrane live cell imaging.

(E)

∗

Growth pole

∗

wt (B)

(C)

wt

rrm4Δ

(D)

Growth pole

Growth pole

Tub1G

TRENDS in Genetics

Figure I. Dimorphism of the plant pathogen Ustilago maydis. (A) Monokaryotic laboratory strain (arrowhead, nucleus) 6 h after induction of hyphal growth. Hypha extends at the apical pole and inserts retraction septa at the basal pole leading to the formation of sections devoid of cytoplasm (asterisks). (B) Yeast form of the same strain before induction. (C) Monokaryotic hypha of a strain carrying a deletion in rrm4. A typical growth behavior is the formation of bipolar growing hyphae. (D) Hypha expressing a-tubulin fused to the green fluorescent protein visualizing flexible microtubule bundles. Cell boundaries are given as broken lines (size bars, 10 mm). (E) Maize cob of an infected plant with characteristic disease symptoms (photo by K. Schipper).

entities for microtubule-dependent transport. They move bidirectionally along bundles of microtubules that are arranged in an antipolar array extending along the whole hypha (see Figure ID in Box 3) [43,44]. Plus end-directed transport is mediated by the kinesin-3 type motor Kin3 [45], which interacts with the endosomal membrane by a pleckstrin homology domain [41]. Minus end-directed transport is mediated by split dynein Dyn1/2, which is particularly important for retrograde transport from the hyphal poles (Figure 2A) [45]. Because dynein can dissociate from endosomes during plus end-directed transport, the conventional kinesin Kin1 is crucial for recycling dynein to the plus ends for the next round of minus enddirected transport [43,44]. Shuttling endosomes share characteristic features of early endosomes such as the marker proteins Rab4 and Rab5a and might function in endocytosis or recycling or act as signaling endosomes [44,46]. But neither a cargo for endocytosis nor a receptor for endosomal signaling have been identified to date [44]. Despite this, several diverse biological functions have been linked to these endosomes [43]. During mating, Rab5a-positive endosomes have been shown to be important for the accumulation of the pheromone receptor at the growth pole of conjugation tubes [43,44]. During hyphal growth they mainly function in 412

the transport of mRNAs, ribosomes, and septins (see below) [40,41,46,47], stressing current views that endosomes function as versatile multipurpose platforms [48]. Moreover, mRNA shuttling on dynamic endosomes constitute yet another example of an intimate connection between membrane and mRNA trafficking. Endosome-coupled translation for efficient septin filamentation in U. maydis The molecular link between mRNA transport and endosomes was uncovered while studying the role of the ELAVtype RNA-binding protein Rrm4 in U. maydis. Rrm4 was found to be important for hyphal growth and its shuttling along microtubules suggested a function in long-distance mRNA transport [49]. In fact, the endosomal motors Kin3 and Dyn1/2 also mediated Rrm4 movement, and colocalization studies showed co-transport of Rrm4-containing mRNPs and endosomes (Figure 2A,B) [41]. These observations uncovered endosomal co-transport of mRNPs as a novel trafficking mechanism [41]. The phenomenon was further studied using the poly(A)binding protein Pab1 as a molecular marker. Pab1 colocalizes with Rrm4-positive endosomes and this localization is dependent on the RNA binding activity of Rrm4. Without Rrm4-mediated mRNA transport, Pab1 forms a

Review (A)

Trends in Genetics September 2014, Vol. 30, No. 9

wt

⊕ ⊕ ⊕

(B)

wt

rrm4Δ

(C)

A

AA

AAA

AAA

AAA

AAA

c c c

AAA

c c c

c

AAA

c

⊕ c

AAA

c

⊕

AAA

c

c

c c

A AA

c c

AAA

AAA AA

A

Key:

(D)

Endosome

Ribosome

Rrm4

Adaptor

Pab1

Ribosome

Rrm4

AAAA cdc3

Pab1

AAA mRNA

Adaptor

Cdc3/12

Rab4

c c

Dyn1/2 dynein

Yup1

c Rab5a

Cdc3/12 (sepns)

c Kin3

Microtubule bundles

⊕

c c

Rab4/ Rab5a

Sepn filament with gradient Kin3 (kinesin-3)

Aberrant sepn ring

⊕ Microtubule bundle

Yup1 TRENDS in Genetics

Figure 2. Model depicting endosomal mRNA transport in Ustilago maydis. (A) Schematic drawing of a hypha with endosomes shuttling bidirectionally along microtubule bundles. Messenger ribonucleoprotein particles (mRNPs) and associated ribosomes are transported by endosomes. (B) (Detail from A). Growth pole of hyphal tip. Rrm4-positive endosomes transport septins towards the hyphal tip. Septins are assembled in filaments forming a gradient emanating from the growth pole. (C) Growth pole of strain lacking Rrm4. Without Rrm4 septins are no longer present on endosomes and without endosomal septin transport the gradient in septin filaments is no longer formed. Instead, aberrant septin rings are observed. (D) (Detail from B). An endosome positive for markers of early endosomes such as Rab4 and Rab5a transports septins. Rrm4 is attached to these specific endosomes most likely by an unknown adaptor. Endosome-coupled translation serves as a mechanism to load newly synthesized septin cargo.

cytoplasmic gradient from the region around the nucleus towards the growth pole, which is not observed in wild type hyphae [47]. Thus, one function of endosomal mRNP transport might be the even distribution of mRNAs throughout the hypha. This could also include ribosomes because their presence on endosomes is Rrm4-dependent [46]. The observed defects in hyphal growth of rrm4D mutants might be explained by a pleiotropic defect, because ribosomes become limited particularly in regions distant from the nucleus, and hence numerous mRNAs

may be translated inefficiently. However, an earlier proteomic approach comparing wild type hyphae to those of rrm4 deletion strains demonstrated quantitative changes only for few proteins. This indicates that defective endosomal ribosome transport does not alter the amount of the vast majority of proteins [50]. Clear differences were only observed in membrane-associated fractions. The endochitinase Cts1 was among the few proteins with altered abundance, and further studies revealed that Rrm4-mediated transport is crucial for unconventional 413

Review Cts1 secretion [50,51]. This uncovered an unexpected molecular link between mRNP transport and unconventional secretion specifically during polar growth. Rrm4 binds numerous mRNAs in vivo encoding, for example, the small G protein Rho3 and the septin Cdc3 [47]. Genetic evidence provided a link between Cdc3 and Rrm4, because cdc3 and rrm4 deletion strains form similar aberrant hyphae in the early phase of hyphal growth [40]. Septins function in membrane compartmentalization and regulate the stability and activity of the actin and microtubule cytoskeleton [52]. The building blocks of septins are octamers that assemble in the cytoplasm and oligomerize in an annealing-like process on membranes into higherorder structures (Box 4) [52–54]. RNA live imaging confirmed that cdc3 mRNA was transported on Rrm4-positive endosomes. Of note, the translation product Cdc3 colocalized with its mRNA in the same endosomal compartment [40]. Together with the finding that the presence of cdc3 mRNA is a prerequisite for Cdc3 protein localization on endosomes, this is suggestive of local translation of septin mRNA on endosomes (Figure 2B). Consistent with this idea, ribosomes localize to moving endosomes but only in the presence of Rrm4 (Figure 2B,C) [40,46]. Remarkably, the translation of mRNAs during transport has also been reported for ABP140 mRNA in S. cerevisiae (see above) [35]. Why might septin mRNAs be translated on endosomes and why would the translation product shuttle? Besides its endosomal localization, Cdc3 forms intracellular filaments with a gradient emanating from the hyphal tip. Hence,

Trends in Genetics September 2014, Vol. 30, No. 9

Rrm4-dependent endosomal septin transport towards the tip could be important for setting up this gradient (Figure 2B,D). FRAP experiments indeed supported the hypothesis that endosomal transport rather than local translation at the tip is crucial for efficient septin filament formation. The observation that ribosomes are underrepresented at the hyphal growth pole [46] and the fact that target mRNAs do not accumulate at this subcellular site also argues against the classical view of local translation at the hyphal tip [49]. However, presently the possibility of both endosome-mediated cdc3 RNA anterograde transport to the hyphal tip and the retrograde trafficking of tip-translated Cdc3 protein cannot be ruled out (see model in [40]). Interestingly, a second septin, Cdc12, also localizes to endosomes and its presence is required for endosomal targeting of Cdc3 (Figure 2D). Thus, several lines of evidence suggest that endosome-coupled translation of septin mRNA facilitates oligomerization and efficient septin filamentation in U. maydis [40]. In line with this, neuronal septins can be purified from presynaptic vesicles and exhibit vesicle-like staining patterns, suggesting evolutionary conservation of this phenomenon (see below) [52]. Endosome-coupled translation of septin mRNA is the first example suggesting that fungal endosomes possess functions beyond the recycling of already synthesized material [44,55]. Indeed, endosomes might play an essential role in the delivery of newly synthesized proteins to support microtubule-dependent hyphal growth. This finding also provides an attractive explanation as to why microtubules are essential for efficient polar growth.

Box 4. Septins constitute the fourth cytoskeletal element Septins are highly conserved proteins found in fungi and animal cells but absent in plants. They function in a variety of processes such as cytokinesis, membrane dynamics, and exocytosis. Moreover, septins have been implicated in tumor formation and neurological diseases [52,53] and have also been described to be important for fungal infection of plants [85]. Septins form higherorder structures such as rings and filaments. The former are found at the cleavage furrow during cell division in Saccharomyces cerevisiae and at the base of cilia in epithelial cells or dendritic spines of neurons [52]. Septin rings function as molecular barriers for the compartmentalization of membranes [53,86]. Septin filaments are present in mammalian cells and filamentous fungi [87]. They colocalize with the actin and microtubule cytoskeleton and can modify these cytoskeletal components by regulating their stability and activity [52]. Characteristic for all septin subunits is a central GTP-binding domain that is flanked by a polybasic region implicated in membrane binding [52]. Four septins form palindromic heterooctamers (e.g., Cdc11-Cdc12-Cdc3-Cdc10-Cdc10Cdc3-Cdc12-Cdc11 in S. cerevisiae), which interact head to tail to form long rods. Two rods assemble like railroad tracks, which build higher-order structures (Figure I). Octamers are found in the cytoplasm but higher-order structures are assembled by annealing of these building blocks on membranes [54]. However, it is currently unclear how defined octamers are assembled in metazoans containing numerous septin genes. For example, in humans 13 septin genes are present and numerous alternative splicing products are known [52,53]. Gene deletion studies in S. cerevisiae revealed yet another level of complexity for septin assembly as some septins are able to replace each other, indicative of some functional redundancy [88].

414

Octamer

(A) Monomers 11 12

3

10

NC

G

NC

G

NC

G

G

Amino termini

Paired filament

Carboxy termini Amino termini (B)

Cdc3G

Filaments Rings

TRENDS in Genetics

Figure I. Septins form defined building blocks that assemble into rings and filaments. (A) Four septins (Cdc11:Cdc12:Cdc3:Cdc10) form palindromic octamers that assemble into paired filaments like railroad tracks. These are again part of higher-ordered structures. (B) Septin rings and filaments in budding cell of Ustilago maydis. Yeast cell expressing Cdc3 fused to the green fluorescent protein. Cell boundaries are given as broken lines.

Review Box 5. Outstanding questions  What distinguishes ER-dependent from ER-independent mRNA localization?  How are motor complexes tethered to the ER?  Which other organisms and processes rely on endosome-coupled translation?  How are mRNPs coupled to endosomes?  Which other proteins are synthesized on endosomes?

Outstanding questions for further research in the field are presented in Box 5. mRNP and membrane co-transport in higher eukaryotes Extensive bidirectional transport of mRNPs has also been observed in numerous cell types of higher eukaryotes. Although several hypotheses have been put forward to rationalize this seemingly futile transport [6], the role of this shuttling still remains elusive. In Drosophila oocytes, oskar mRNA-containing RNPs are transported along microtubules with a seemingly random orientation, but nevertheless end up at the posterior pole. It was proposed that a biased random walk along a weakly polarized cytoskeleton ultimately results in accumulation of mRNAs at the pole [56]. By contrast, mammalian neurons show similar extensive shuttling of mRNPs but without significant accumulations at specific subcellular sites. This transport process has been compared to a sushi belt continuously serving demanding synapses along the dendrite with mRNAs for local translation [6]. Alternatively, back and forth navigation of mRNPs offers the possibility of bypassing potential roadblocks on microtubules [4]. The abovedescribed results in polarized U. maydis hyphae provide an additional hypothesis: mRNPs may shuttle on bidirectionally moving endosomes in order to distribute mRNAs and ribosomes as well as to deliver translation products [38,40,46]. Is endosomal mRNA trafficking found in U. maydis conserved in higher eukaryotes? Several observations indicate that this may be the case. The idea of mRNP transport on endosomes was proposed more than 10 years ago when Cohen observed that loss-of-function mutants of Rab11, a recycling endosomal marker, showed defects in oskar mRNA localization [57]. However, the rab11 mutants also exhibited cytoskeletal defects and therefore endosomal mRNP transport could not be unambiguously demonstrated. Later studies reported that mutants in components of ESCRT-II (endosomal sorting complex required for transport II) abolish the final localization step of bicoid mRNA. Despite the RNA zip code binding activity of one subunit, VPS36, the role of ESCRT-II in RNA localization seems to be independent of endosomal sorting, because mutations in ESCRT-I and -III components do not affect the targeting of bicoid mRNA [58]. The first clear indications of endosomal RNA transport were provided while following the intracellular movement and assembly of retroviruses. Genomic RNA of HIV-1 is transported on endosomes and ESCRT-II components are needed for trafficking [59,60]. The idea of endosomal mRNA transport was further supported by the finding that kinesin-3-type motors such as Kif1B are involved in microtubule-dependent mRNA transport in neurons.

Trends in Genetics September 2014, Vol. 30, No. 9

Kinesin-3-type motors often carry membrane-binding domains and function in vesicular transport [61]. Finally, another intimate link between RNA biology and membrane trafficking was observed when studying RNA silencing in plants and animals [62]. During this mode of post-transcriptional regulation, the RNA-induced silencing complex (RISC) controls stability and translation of target mRNAs. The core components of RISC are Argonaute (Ago) proteins loaded with small RNAs (small interfering RNAs or microRNAs). Interestingly, Ago was initially identified as a membrane-associated protein with unknown function, GERp95 (Golgi–ER p95) [62]. Meanwhile, Ago and small RNAs were found to associate with the ER, Golgi, endosomes, multivesicular bodies (MVBs), and autophagosomes, and even to be secreted [62]. Functional studies in animal cells revealed that active RISC can be assembled at the cytoplasmic face of the ER [63] and that, in plants, ER-associated RISC regulates translation but not decay of target mRNAs [64]. Furthermore, interfering with MVB formation alters RISC assembly and function [65,66], suggesting regulation at several sites of the endomembrane system. A membrane-binding BAR (Bin–Amphiphysin–Rvs) domain protein was recently described as a possible adaptor to connect Ago with endosomes in neurons [67]. However, the exact functions of the various populations of membrane-associated RISC are still unclear. One intriguing possibility is that they recruit silenced mRNAs to endomembranes on which they would hitchhike to reach their proper location. In summary, evidence is accumulating that also in higher eukaryotes mRNA and membrane transport are more closely connected than previously anticipated. The examples of endosomal co-transport of viral RNA, the involvement of kinesin-3-type motors in neuronal mRNP trafficking, and the assembly of large ribonucleoprotein complexes on membranes suggest that basic concepts are most likely conserved between kingdoms. Concluding remarks Studying fungal model systems has revealed new mechanistic insights into mRNP and membrane trafficking. It has become clear that RNA-binding proteins co-operate extensively in recognizing RNA elements [14,15] and can function to directly connect mRNPs to membranes. Linking mRNPs to the cytoplasmic face of organelles such as the ER or endosomes provides an efficient means to coordinate local translation and membrane interaction of the translation products. Another advantage of this connection is the support of macromolecular complex assembly including retroviruses, RISC, and potentially also septins [40,60,68]. Importantly, endosome-coupled translation offers a novel mechanism to load the cytoplasmic face of endosomes with cargo proteins and thereby makes use of the sophisticated membrane trafficking system for transportation. Thus, endosome function is not restricted to mere recycling but also encompasses delivery of new material. This was first observed for polar growth of fungal hyphae, but Rab4and Rab5-positive endosomes have also been shown to be required for efficient axonal growth [69]. Based on these grounds, we envision that studying the tight molecular 415

Review links between mRNP and membrane traffic will foster the discovery of even more unexpected cell biological insights. Acknowledgments We are grateful to Drs K. Schipper, K. Zarnack, J. Bethune, and laboratory members for helpful comments on the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) to R-P.J. (DFG Ja696/7-1), to D.N. (FOR855, SFB646), and to M.F. (CEPLAS EXC 1028, FOR1334, DFG Fe448/7, DFG Fe448/8).

References 1 Jansen, R.P. (2001) mRNA localization: message on the move. Nat. Rev. Mol. Cell Biol. 2, 247–256 2 Eliscovich, C. et al. (2013) mRNA on the move: the road to its biological destiny. J. Biol. Chem. 288, 20361–20368 3 Martin, K.C. and Ephrussi, A. (2009) mRNA localization: gene expression in the spatial dimension. Cell 136, 719–730 4 Bullock, S.L. (2011) Messengers, motors and mysteries: sorting of eukaryotic mRNAs by cytoskeletal transport. Biochem. Soc. Trans. 39, 1161–1165 5 Marchand, V. et al. (2012) An intracellular transmission control protocol: assembly and transport of ribonucleoprotein complexes. Curr. Opin. Cell Biol. 24, 202–210 6 Doyle, M. and Kiebler, M.A. (2011) Mechanisms of dendritic mRNA transport and its role in synaptic tagging. EMBO J. 30, 3540–3552 7 Dreyfuss, G. et al. (2002) Messenger-RNA-binding proteins and the messages they carry. Nat. Rev. Mol. Cell Biol. 3, 195–205 8 Huotari, J. and Helenius, A. (2011) Endosome maturation. EMBO J. 30, 3481–3500 9 Akopian, D. et al. (2013) Signal recognition particle: an essential protein-targeting machine. Annu. Rev. Biochem. 82, 693–721 10 Heym, R.G. and Niessing, D. (2012) Principles of mRNA transport in yeast. Cell. Mol. Life Sci. 69, 1843–1853 11 Hermesh, O. and Jansen, R.P. (2013) Take the (RN)A-train: localization of mRNA to the endoplasmic reticulum. Biochim. Biophys. Acta 1833, 2519–2525 12 Shen, Z. et al. (2010) Cotranscriptional recruitment of She2p by RNA pol II elongation factor Spt4-Spt5/DSIF promotes mRNA localization to the yeast bud. Genes Dev. 24, 1914–1926 13 Mu¨ller, M. et al. (2009) Formation of She2p tetramers is required for mRNA binding, mRNP assembly, and localization. RNA 15, 2002–2012 14 Mu¨ller, M. et al. (2011) A cytoplasmic complex mediates specific mRNA recognition and localization in yeast. PLoS Biol. 9, e1000611 15 Niedner, A. et al. (2013) Role of Loc1p in assembly and reorganization of nuclear ASH1 messenger ribonucleoprotein particles in yeast. Proc. Natl. Acad. Sci. U.S.A. 110, E5049–E5058 16 Heym, R.G. et al. (2013) In vitro reconstitution of an mRNA-transport complex reveals mechanisms of assembly and motor activation. J. Cell Biol. 203, 971–984 17 Heuck, A. et al. (2010) The structure of the Myo4p globular tail and its function in ASH1 mRNA localization. J. Cell Biol. 189, 497–510 18 Shi, H. et al. (2014) Structure of a myosinbulletadaptor complex and pairing by cargo. Proc. Natl. Acad. Sci. U.S.A. 111, E1082–E1090 19 Sladewski, T.E. et al. (2013) Single-molecule reconstitution of mRNA transport by a class V myosin. Nat. Struct. Mol. Biol. 20, 952–957 20 Chung, S. and Takizawa, P.A. (2010) Multiple Myo4 motors enhance ASH1 mRNA transport in Saccharomyces cerevisiae. J. Cell Biol. 189, 755–767 21 Krementsova, E.B. et al. (2011) Two single-headed myosin V motors bound to a tetrameric adapter protein form a processive complex. J. Cell Biol. 195, 631–641 22 Bullock, S.L. et al. (2006) Guidance of bidirectional motor complexes by mRNA cargoes through control of dynein number and activity. Curr. Biol. 16, 1447–1452 23 Diehn, M. et al. (2000) Large-scale identification of secreted and membrane-associated gene products using DNA microarrays. Nat. Genet. 25, 58–62 24 Aronov, S. et al. (2007) mRNAs encoding polarity and exocytosis factors are co-transported with cortical ER to the incipient bud in yeast. Mol. Cell. Biol. 27, 3441–3455 416

Trends in Genetics September 2014, Vol. 30, No. 9

25 Trautwein, M. et al. (2004) Arf1p provides an unexpected link between COPI vesicles and mRNA in Saccharomyces cerevisiae. Mol. Biol. Cell 15, 5021–5037 26 Estrada, P. et al. (2003) Myo4p and She3p are required for cortical ER inheritance in Saccharomyces cerevisiae. J. Cell Biol. 163, 1255–1266 27 West, M. et al. (2011) A 3D analysis of yeast ER structure reveals how ER domains are organized by membrane curvature. J. Cell Biol. 193, 333–346 28 Schmid, M. et al. (2006) Coordination of endoplasmic reticulum and mRNA localization to the yeast bud. Curr. Biol. 16, 1538–1543 29 Genz, C. et al. (2013) Association of the yeast RNA-binding protein She2p with the tubular endoplasmic reticulum depends on membrane curvature. J. Biol. Chem. 288, 32384–32393 30 Fundakowski, J. et al. (2012) Localization of a subset of yeast mRNAs depends on inheritance of endoplasmic reticulum. Traffic 13, 1642–1652 31 Kraut-Cohen, J. and Gerst, J.E. (2010) Addressing mRNAs to the ER: cis sequences act up! Trends Biochem. Sci. 35, 459–469 32 Manford, A.G. et al. (2012) ER-to-plasma membrane tethering proteins regulate cell signaling and ER morphology. Dev. Cell 23, 1129–1140 33 Kraut-Cohen, J. et al. (2013) Translation- and SRP-independent mRNA targeting to the endoplasmic reticulum in the yeast Saccharomyces cerevisiae. Mol. Biol. Cell 24, 3069–3084 34 Gelin-Licht, R. et al. (2012) Scp160-dependent mRNA trafficking mediates pheromone gradient sensing and chemotropism in yeast. Cell Rep. 1, 483–494 35 Kilchert, C. and Spang, A. (2011) Cotranslational transport of ABP140 mRNA to the distal pole of S. cerevisiae. EMBO J. 30, 3567–3580 36 Houston, D.W. (2012) Cortical rotation and messenger RNA localization in Xenopus axis formation. Wiley Interdiscip. Rev. Dev. Biol. 1, 371–388 37 Cui, X.A. et al. (2012) p180 promotes the ribosome-independent localization of a subset of mRNA to the endoplasmic reticulum. PLoS Biol. 10, e1001336 38 Palacios, I.M. (2014) Hop-on hop-off: polysomes take a tour of the cell on endosomes. J. Cell Biol. 204, 287–289 39 Riquelme, M. (2013) Tip growth in filamentous fungi: a road trip to the apex. Annu. Rev. Microbiol. 67, 587–609 40 Baumann, S. et al. (2014) Endosomal transport of septin mRNA and protein indicates local translation on endosomes and is required for correct septin filamentation. EMBO Rep. 15, 94–102 41 Baumann, S. et al. (2012) Kinesin-3 and dynein mediate microtubuledependent co-transport of mRNPs and endosomes. J. Cell Sci. 125, 2740–2752 42 Vollmeister, E. et al. (2012) Microtubule-dependent mRNA transport in the model microorganism Ustilago maydis. RNA Biol. 9, 1–8 43 Go¨hre, V. et al. (2012) Microtubule-dependent membrane dynamics of Ustilago maydis: trafficking and function of Rab5a-positive endosomes. Commun. Integr. Biol. 5, 482–487 44 Steinberg, G. (2012) The transport machinery for motility of fungal endosomes. Fungal Genet. Biol. 49, 675–676 45 Schuster, M. et al. (2011) Kinesin-3 and dynein cooperate in long-range retrograde endosome motility along a non-uniform microtubule array. Mol. Biol. Cell 22, 3645–3657 46 Higuchi, Y. et al. (2014) Early endosome motility spatially organizes polysome distribution. J. Cell Biol. 204, 343–357 47 Ko¨nig, J. et al. (2009) The fungal RNA-binding protein Rrm4 mediates long-distance transport of ubi1 and rho3 mRNAs. EMBO J. 28, 1855–1866 48 Gould, G.W. and Lippincott-Schwartz, J. (2009) New roles for endosomes: from vesicular carriers to multi-purpose platforms. Nat. Rev. Mol. Cell Biol. 10, 287–292 49 Zarnack, K. and Feldbru¨gge, M. (2010) Microtubule-dependent mRNA transport in fungi. Eukaryot. Cell 9, 982–990 50 Koepke, J. et al. (2011) The RNA-binding protein Rrm4 is essential for efficient secretion of endochitinase Cts1. Mol. Cell. Proteomics 10, M111.011213 51 Stock, J. et al. (2012) Applying unconventional secretion of the endochitinase Cts1 to export heterologous proteins in Ustilago maydis. J. Biotechnol. 161, 80–91 52 Spiliotis, E.T. and Gladfelter, A.S. (2012) Spatial guidance of cell asymmetry: septin GTPases show the way. Traffic 13, 195–203

Review 53 Barral, Y. (2010) Cell biology. Septins at the nexus. Science 329, 1289–1290 54 Bridges, A.A. et al. (2014) Septin assemblies form by diffusion-driven annealing on membranes. Proc. Natl. Acad. Sci. U.S.A. 111, 2146–2151 55 Penalva, M.A. et al. (2012) Searching for gold beyond mitosis: mining intracellular membrane traffic in Aspergillus nidulans. Cell. Logist. 2, 2–14 56 Zimyanin, V.L. et al. (2008) In vivo imaging of oskar mRNA transport reveals the mechanism of posterior localization. Cell 134, 843–853 57 Cohen, R.S. (2005) The role of membranes and membrane trafficking in RNA localization. Biol. Cell 97, 5–18 58 Irion, U. and St Johnston, D. (2007) bicoid RNA localization requires specific binding of an endosomal sorting complex. Nature 445, 554–558 59 Molle, D. et al. (2009) Endosomal trafficking of HIV-1 gag and genomic RNAs regulates viral egress. J. Biol. Chem. 284, 19727–19743 60 Ghoujal, B. et al. (2012) ESCRT-II’s involvement in HIV-1 genomic RNA trafficking and assembly. Biol. Cell 104, 706–721 61 Lyons, D.A. et al. (2009) Kif1b is essential for mRNA localization in oligodendrocytes and development of myelinated axons. Nat. Genet. 41, 854–858 62 Kim, Y.J. et al. (2014) Traffic into silence: endomembranes and posttranscriptional RNA silencing. EMBO J. 33, 968–980 63 Stalder, L. et al. (2013) The rough endoplasmatic reticulum is a central nucleation site of siRNA-mediated RNA silencing. EMBO J. 32, 1115–1127 64 Li, S. et al. (2013) MicroRNAs inhibit the translation of target mRNAs on the endoplasmic reticulum in Arabidopsis. Cell 153, 562–574 65 Gibbings, D.J. et al. (2009) Multivesicular bodies associate with components of miRNA effector complexes and modulate miRNA activity. Nat. Cell Biol. 11, 1143–1149 66 Lee, Y.S. et al. (2009) Silencing by small RNAs is linked to endosomal trafficking. Nat. Cell Biol. 11, 1150–1156 67 Antoniou, A. et al. (2014) PICK1 links Argonaute 2 to endosomes in neuronal dendrites and regulates miRNA activity. EMBO Rep. 15, 548–556 68 Gibbings, D. and Voinnet, O. (2010) Control of RNA silencing and localization by endolysosomes. Trends Cell Biol. 20, 491–501 69 Falk, J. et al. (2014) Rab5 and Rab4 regulate axon elongation in the Xenopus visual system. J. Neurosci. 34, 373–391 70 Martin, S.G. and Arkowitz, R.A. (2014) Cell polarization in budding and fission yeasts. FEMS Microbiol. Rev. 38, 228–253 71 Hammer, J.A., 3rd and Sellers, J.R. (2012) Walking to work: roles for class V myosins as cargo transporters. Nat. Rev. Mol. Cell Biol. 13, 13–26

Trends in Genetics September 2014, Vol. 30, No. 9

72 Fagarasanu, A. and Rachubinski, R.A. (2007) Orchestrating organelle inheritance in Saccharomyces cerevisiae. Curr. Opin. Microbiol. 10, 528–538 73 Merlini, L. et al. (2013) Mate and fuse: how yeast cells do it. Open Biol. 3, 130008 74 Friedman, J.R. and Voeltz, G.K. (2011) The ER in 3D: a multifunctional dynamic membrane network. Trends Cell Biol. 21, 709–717 75 Tavassoli, S. et al. (2013) Plasma membrane–endoplasmic reticulum contact sites regulate phosphatidylcholine synthesis. EMBO Rep. 14, 434–440 76 Fox, P.D. et al. (2013) Plasma membrane domains enriched in cortical endoplasmic reticulum function as membrane protein trafficking hubs. Mol. Biol. Cell 24, 2703–2713 77 Reid, D.W. and Nicchitta, C.V. (2012) Primary role for endoplasmic reticulum-bound ribosomes in cellular translation identified by ribosome profiling. J. Biol. Chem. 287, 5518–5527 78 Prilusky, J. and Bibi, E. (2009) Studying membrane proteins through the eyes of the genetic code revealed a strong uracil bias in their coding mRNAs. Proc. Natl. Acad. Sci. U.S.A. 106, 6662–6666 79 Djamei, A. and Kahmann, R. (2012) Ustilago maydis: dissecting the molecular interface between pathogen and plant. PLoS Pathog. 8, e1002955 80 Vollmeister, E. et al. (2012) Fungal development of the plant pathogen Ustilago maydis. FEMS Microbiol. Rev. 36, 59–77 81 Yu, E.Y. et al. (2013) Brh2 and Rad51 promote telomere maintenance in Ustilago maydis, a new model system of DNA repair proteins at telomeres. DNA Repair 12, 472–479 82 Heimel, K. et al. (2013) Crosstalk between the unfolded protein response and pathways that regulate pathogenic development in Ustilago maydis. Plant Cell 25, 4262–4277 83 Freitag, J. et al. (2012) Cryptic peroxisomal targeting via alternative splicing and stop codon read-through in fungi. Nature 485, 522–525 84 Terfru¨chte, M. et al. (2014) Establishing a versatile Golden Gate cloning system for genetic engineering in fungi. Fungal Genet. Biol. 62, 1–10 85 Dagdas, Y.F. et al. (2012) Septin-mediated plant cell invasion by the rice blast fungus, Magnaporthe oryzae. Science 336, 1590–1595 86 Mostowy, S. and Cossart, P. (2012) Septins: the fourth component of the cytoskeleton. Nat. Rev. Mol. Cell Biol. 13, 183–194 87 Gladfelter, A.S. (2010) Guides to the final frontier of the cytoskeleton: septins in filamentous fungi. Curr. Opin. Microbiol. 13, 720–726 88 McMurray, M.A. et al. (2011) Septin filament formation is essential in budding yeast. Dev. Cell 20, 540–549

417