Widespread cytoplasmic mRNA transport in yeast: Identification of 22 bud-localized transcripts using DNA microarray analysis

Shepard, Kelly A.; Gerber, André P.; Jambhekar, Ashwini; Takizawa, Peter A.; Brown, Patrick O.; Herschlag, Daniel; DeRisi, Joe; Vale, Ronald D.

doi:10.1073/pnas.2033246100

Cited by 261 publications

(345 citation statements)

References 54 publications

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“…Ten of these RNAs were transcribed from cell cycle-regulated genes, whereas others appeared to participate in pathways related to sensing or responding to stress. The overlap between these previous studies and ours is high: we identified 41 mRNAs that co-isolated with She2p-PrA, including 90% and 65% of the transcripts previously identified, respectively 29,30 (Table 2 and Supplementary Table 3). Our results also suggest that the number of transcripts associated with She2p alone is greater than previously reported.…”

Section: Global Analyses Of Nop15p and She2p Associated Rnasmentioning

confidence: 61%

“…Our She2p RNP complex isolations form an excellent positive control, as in two recent microarray studies, 11 and 24 transcripts, respectively, were reported to be specifically associated with tagged trimeric She2p-She3p-Myo4p complexes 29,30 . Ten of these RNAs were transcribed from cell cycle-regulated genes, whereas others appeared to participate in pathways related to sensing or responding to stress.…”

Section: Global Analyses Of Nop15p and She2p Associated Rnasmentioning

confidence: 91%

“…The sixth component, the RNA-binding protein She2p, has been extensively studied by both proteomic and RNomic approaches [28][29][30] . She2p is involved in the localization of ASH1 mRNA to the distal tip of daughter cells during anaphase 28 , which is accomplished by She2p binding directly to the RNA and engaging it via the linker protein She3p to the myosin Myo4p that then transports the complex along actin cables to the bud tip 29 .…”

Section: T O T a L F T E Lu A T E T O T A L F T E Lu A T E T O T A L mentioning

confidence: 99%

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Comprehensive analysis of diverse ribonucleoprotein complexes

et al. 2007

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The study of the dynamic interactome of cellular ribonucleoprotein (RNP) particles has been hampered by severe methodological limitations. In particular, the affinity purification of intact RNP complexes from cell lysates suffers from RNA degradation, loss of interacting macromolecules and poor overall yields. Here we describe a rapid affinity-purification method for efficient isolation of the subcomplexes that dynamically organize different RNP biogenesis pathways in Saccharomyces cerevisiae. Our method overcomes many of the previous limitations to produce large RNP interactomes with almost no contamination.During and after eukaryotic transcription, RNA is packaged into RNP complexes, then modified, spliced, folded and exported into the cytoplasm through nuclear pore complexes (NPCs). There are two main forms of RNPs. Each contains a different type of RNA: mRNPs contain mRNA, and rRNPs contain ribosomal RNA (rRNA). Each also uses discrete processing factors, which associate with RNPs in a dynamic fashion to define the order of transcript maturation. The function and composition of many of these RNP complexes, however, is poorly understood.In recent years, there have been considerable efforts to study the composition of mRNPs and rRNPs in greater detail. A favored approach has been to purify these complexes from cell lysates [1][2][3][4] . But the limitations of presently available purification techniques pose a major hurdle to the study of RNA processing and export. Here we describe a method that aims to overcome these limitations and capture the dynamic interactome of maturing RNP complexes.We used this method to analyze complexes along both the ribosome and mRNA biogenesis pathways. RESULTSA strategy for isolation of RNP complexes Our strategy comprises seven steps that we optimized for efficient isolation of intact RNP complexes ( Fig. 1 and Supplementary Methods online). Collected cells are rapidly frozen in liquid nitrogen and broken open in the solid phase by milling 5 , using a planetary ball mill that produces particles of B1-2 mm in diameter (small enough for rapid and efficient RNP extraction). Because cell breakage occurs in the frozen state, we avoid damage to RNP complexes by released nucleases and proteases, as well as redistribution of proteins within the extract. The resulting frozen grindate is rapidly thawed into an RNP-compatible buffer (see Supplementary Methods), to produce an extract that is then immediately clarified by filtration. We isolate the tagged complexes using antibody-conjugated magnetic beads, rather than more commonly used antibody-conjugated resins such as Sepharose. The magnetic beads are small (2.8 mm) and so have a large surface area-to-volume ratio that increases the speed of binding. Moreover, the beads are impermeable, such that the antibodies are densely conjugated to their exposed surface; hence, there is no theoretical upper limit to the size of isolated complexes (unlike with permeable resins such as Sepharose, which are limited by their pore size). Incubation times...

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Section: Global Analyses Of Nop15p and She2p Associated Rnasmentioning

confidence: 61%

Section: Global Analyses Of Nop15p and She2p Associated Rnasmentioning

confidence: 91%

Section: T O T a L F T E Lu A T E T O T A L F T E Lu A T E T O T A L mentioning

confidence: 99%

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Comprehensive analysis of diverse ribonucleoprotein complexes

et al. 2007

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“…It has been reported that many mRNAs and RNA-binding proteins are localized to specific compartments, along with cytoskeletal elements, in a variety of eukaryotic cells (Jansen, 2001;Kloc et al, 2002). In S. cerevisiae, many mRNAs are localized to produce proteins in specific compartments such as the daughter cell or the mitochondria (Corral-Debrinski et al, 2000;Takizawa et al, 2000;Jansen, 2001;Kwon and Schnapp, 2001;Shepard et al, 2003). These observations suggest that individual mRNAs may be consistently transported to specific areas in the yeast cell.…”

Section: Discussionmentioning

confidence: 98%

Simultaneous yet Independent Regulation of Actin Cytoskeletal Organization and Translation Initiation by Glucose inSaccharomyces cerevisiae

Uesono

Ashe

Toh‐e

2004

MBoC

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Acute glucose deprivation rapidly but transiently depolarizes the actin cytoskeleton and inhibits translation initiation in Saccharomyces cerevisiae. Neither rapid actin depolarization nor translation inhibition upon glucose removal occurs in a reg1 disruptant, which is defective in glucose repression, or in the tpk1 w mutant, which has weak cAPK activity. In the absence of additional glucose, recovery of either actin polarization or translation initiation relies upon respiration, the Snf1p protein kinase, and the transcription factors Msn2p and Msn4p. The readdition of glucose to glucose-starved cells causes a rapid recovery of actin polarization as well as translation initiation without respiration. These results indicate that the simultaneous regulation of actin polarization and translation initiation is divided into three reactions: 1) rapid shutdown depending on Reg1p and cAPK after glucose removal, 2) slow adaptation depending on Snf1p and Msn2p/4p in the absence of glucose, and 3) rapid recovery upon readdition of glucose. On glucose removal, translation initiation is rapidly inhibited in a rom2 disruptant, which is defective in rapid actin depolarization, whereas rapid actin depolarization occurs in a pop2/caf1 disruptant, which is defective in rapid inhibition of translation initiation. Thus, translation initiation and actin polarization seem to be simultaneously but independently regulated by glucose deprivation. INTRODUCTIONThe actin cytoskeleton provides the structural basis for cell polarity in the yeast Saccharomyces cerevisiae and is organized primarily into two morphologically distinct structures: cortical patches and cables (Botstein et al., 1997). Cortical patches are discrete cytoskeletal bodies at the plasma membrane, whereas actin cables are long bundles of actin filaments that are oriented along the mother-bud axis. Both structures are polarized during the budding phase of the cell cycle. During early G1 phase, cortical patches and cables are randomly distributed. As a bud emerges, cortical patches cluster at the growing tip and cables extend from the mother cell into the bud. Near the end of bud growth, the patches and cables redistribute randomly, disrupting the asymmetric distribution of the actin cytoskeleton. At the end of mitosis, patches accumulate in and cables reorient toward the mother-bud neck in connection with cytokinesis and septum formation (Botstein et al., 1997).The actin cytoskeleton is also regulated in response to environmental stimuli. Mating pheromone causes polarization of the actin cytoskeleton toward the tip of the mating projection via the Cdc42p GTPase (Read et al., 1992;Leberer et al., 1997). In contrast, stresses such as osmotic shock, heat shock, and glucose starvation depolarize the actin cytoskeleton in budded cells as shown in Figure 1A (Novick et al., 1989;Chowdhury et al., 1992;Delley and Hall, 1999). In particular, the depolarization by heat or osmotic stress is known to be a rapid and transient reaction. A recent report has indicated that the rapid depol...

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“…Similarly, the cortical ER is moved by Myo4p, the other yeast myosin-V, which forms a complex with She3p (Estrada et al, 2003). Several mRNAs are also moved by Myo4p and require both She3p and She2p, proteins whose roles are not well established (Bohl et al, 2000;Long et al, 2000;Shepard et al, 2003;Heuck et al, 2007;Hodges et al, 2008). Moreover, rather than attachment to the globular tail, She3p binds the rod of Myo4p.…”

Section: Introductionmentioning

confidence: 99%

PTC1Is Required for Vacuole Inheritance and Promotes the Association of the Myosin-V Vacuole-specific Receptor Complex

Jin

Eves

Tang

et al. 2009

MBoC

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Organelle inheritance occurs during cell division. In Saccharomyces cerevisiae, inheritance of the vacuole, and the distribution of mitochondria and cortical endoplasmic reticulum are regulated by Ptc1p, a type 2C protein phosphatase. Here we show that PTC1/VAC10 controls the distribution of additional cargoes moved by a myosin-V motor. These include peroxisomes, secretory vesicles, cargoes of Myo2p, and ASH1 mRNA, a cargo of Myo4p. We find that Ptc1p is required for the proper distribution of both Myo2p and Myo4p. Surprisingly, PTC1 is also required to maintain the steady-state levels of organelle-specific receptors, including Vac17p, Inp2p, and Mmr1p, which attach Myo2p to the vacuole, peroxisomes, and mitochondria, respectively. Furthermore, Vac17p fused to the cargo-binding domain of Myo2p suppressed the vacuole inheritance defect in ptc1⌬ cells. These findings suggest that PTC1 promotes the association of myosin-V with its organelle-specific adaptor proteins. Moreover, these observations suggest that despite the existence of organelle-specific receptors, there is a higher order regulation that coordinates the movement of diverse cellular components. INTRODUCTIONDuring each cell cycle, cytoplasmic organelles are actively distributed between dividing cells to maintain organelle copy number and volume. The yeast Saccharomyces cerevisiae is an excellent model system for studying the spatial and temporal control of organelle inheritance. In yeast, several organelles are transmitted from mother to daughter cells. These include the vacuole, mitochondria, the endoplasmic reticulum (ER), late-Golgi elements and peroxisomes (reviewed in Weisman, 2006).Early in the cell cycle, a portion of each organelle is transported into the emerging bud. The polarized transport of most organelles from the mother to the bud is an active process that requires the actin cytoskeleton, myosin-V motors, and receptor proteins, which physically connect the motor to organelle cargoes (Beach et al., 2000;Yin et al., 2000;Boldogh et al., 2001;Barr, 2002;Bretscher, 2003;Du et al., 2004;Fagarasanu et al., 2006bFagarasanu et al., , 2007Weisman, 2006). Thus, formation of a complex between the motor and receptor protein is important for polarized organelle transport.Similar to yeast, in vertebrates, myosin-V motors move cargoes along the actin cytoskeleton. The best studied cargo of vertebrate myosin-V are melanosomes, which are moved in melanocytes by myosin-Va. Melanosomes attach to myosin-Va through Rab27a and melanophilin (Fukuda and Kuroda, 2002;Wu et al., 2002). Rab27a, through geranylgeranylation, attaches to the melanosome membrane, and melanophilin connects myosin-Va and Rab27a. Myosin-V-based intracellular movement has been analyzed in many other eukaryotes, including frogs, fish, mammals, and plants; plant myosin-XI is the functional homologue of yeast and vertebrate myosin-V ( Kinkema and Schiefelbein, 1994;Provance and Mercer, 1999;Tuma and Gelfand, 1999;Desnos et al., 2007;Li and Nebenfuhr, 2007;Sheets et al., 2007;Shimmen, 2007).Ge...

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Widespread cytoplasmic mRNA transport in yeast: Identification of 22 bud-localized transcripts using DNA microarray analysis

Cited by 261 publications

References 54 publications

Comprehensive analysis of diverse ribonucleoprotein complexes

Comprehensive analysis of diverse ribonucleoprotein complexes

Simultaneous yet Independent Regulation of Actin Cytoskeletal Organization and Translation Initiation by Glucose inSaccharomyces cerevisiae

PTC1Is Required for Vacuole Inheritance and Promotes the Association of the Myosin-V Vacuole-specific Receptor Complex

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