芽殖酵母ASH1 mRNA的定位机制

mRNA定位是一种基因转录后水平的重要调控机制,对细胞的生理活动和分化发育都有着极其重要的作用。在芽殖酵母有丝分裂中,ASH1 mRNA在子细胞芽尖因不对称定位表达抑制了子细胞交配类型的转换。本综述介绍了芽殖酵母ASH1 mRNA定位的分子机制。     

mRNA localization: motile RNA, asymmetric anchors

Techniques to label mRNA with green fluorescent protein (GFP) have provided the first real-time images of RNA motility in live yeast cells. Genetic screens for factors responsible for mRNA asymmetry (e. g. SHE genes) in yeast identified type V myosin among other proteins. Analysis of mRNA movement in various she mutants revealed the role of motor proteins in long-range transport, factors for particle formation, and cortical anchors for docking the mRNA.     

Conserved and clustered RNA recognition sequences are a critical feature of signals directing RNA localization in Xenopus oocytes

Although it is widely regarded that the targeting of RNA molecules to subcellular destinations depends upon the recognition of cis-elements found within their 3' untranslated regions (UTR), relatively little is known about the specific features of these cis-sequences that underlie their function. Interaction between specific repeated motifs within the 3' UTR and RNA-binding proteins has been proposed as a critical step in the localization of Vg1 RNA to the vegetal pole of Xenopus oocytes. To understand the relative contributions of repeated localization element (LE) sequences, we used comparative functional analysis of Vg1 LEs from two frog species, Xenopus laevis and Xenopus borealis. We show that clusters of repeated VM1 and E2 motifs are required for efficient localization. However, groups of either site alone are not sufficient for localization. In addition, we present evidence that the X. borealis Vg1 LE is recognized by the same set of RNA-binding proteins as the X. laevis Vg1 LE and is capable of productive interactions with the X. laevis transport machinery as it is sufficient to direct vegetal localization in X. laevis oocytes. These results suggest that clustered sets of cis-acting sites within the LE direct vegetal transport through specific interactions with the localization machinery.     

Ribonucleoprotein remodeling during RNA localization

Cytoplasmic RNA localization is a means to create polarity by restricting protein expression to a discrete subcellular location. RNA localization is a multistep process that begins with the recognition of cis-acting sequences within the RNA by specific trans-factors, and RNAs are localized in ribonucleoprotein (RNP) complexes that contain both the RNA and numerous protein components. Components of the localization machinery transport the RNP complex, usually in a translationally repressed state, to a distinct subcellular region, resulting in spatially restricted gene expression. Recent efforts to identify both the cis- and trans-factors required for RNA localization have elucidated RNA-protein interactions that are remodeled during localization.     

Ribonucleoprotein-dependent localization of the yeast class V myosin Myo4p

Class V myosins are motor proteins with functions in vesicle transport, organelle segregation, and RNA localization. Although they have been extensively studied, only little is known about the regulation of their spatial distribution. Here we demonstrate that a GFP fusion protein of the budding yeast class V myosin Myo4p accumulates at the bud cortex and is a component of highly dynamic cortical particles. Bud-specific enrichment depends on Myo4p's association with its cargo, a ribonucleoprotein complex containing the RNA-binding protein She2p. Cortical accumulation of Myo4p at the bud tip can be explained by a transient retention mechanism that requires SHE2 and, apparently, localized mRNAs bound to She2p. A mutant She2 protein that is unable to recognize its cognate target mRNA, ASH1, fails to localize Myo4p. Mutant She2p accumulates inside the nucleus, indicating that She2p shuttles between the nucleus and cytoplasm and is exported in an RNA-dependent manner. Consistently, inhibition of nuclear mRNA export results in nuclear accumulation of She2p and cytoplasmic Myo4p mislocalization. Loss of She2p can be complemented by direct targeting of a heterologous lacZ mRNA to a complex of Myo4p and its associated adaptor She3p, suggesting that She2p's function in Myo4p targeting is to link an mRNA to the motor complex.     

RNA localization and transport

RNA localization serves numerous purposes from controlling development and differentiation to supporting the physiological activities of cells and organisms. After a brief introduction into the history of the study of mRNA localization I will focus on animal systems, describing in which cellular compartments and in which cell types mRNA localization was observed and studied. In recent years numerous novel localization patterns have been described, and countless mRNAs have been documented to accumulate in specific subcellular compartments. These fascinating revelations prompted speculations about the purpose of localizing all these mRNAs. In recent years experimental evidence for an unexpected variety of different functions has started to emerge. Aside from focusing on the functional aspects, I will discuss various ways of localizing mRNAs with a focus on the mechanism of active and directed transport on cytoskeletal tracks. Structural studies combined with imaging of transport and biochemical studies have contributed to the enormous recent progress, particularly in understanding how dynein/dynactin/BicD (DDB) dependent transport on microtubules works. This transport process actively localizes diverse cargo in similar ways to the minus end of microtubules and, at least in flies, also individual mRNA molecules. A sophisticated mechanism ensures that cargo loading licenses processive transport.     

An exclusively nuclear RNA-binding protein affects asymmetric localization of ASH1 mRNA and Ash1p in yeast

The localization of ASH1 mRNA to the distal tip of budding yeast cells is essential for the proper regulation of mating type switching in Saccharomyces cerevisiae. A localization element that is predominantly in the 3'-untranslated region (UTR) can direct this mRNA to the bud. Using this element in the three-hybrid in vivo RNA-binding assay, we identified a protein, Loc1p, that binds in vitro directly to the wild-type ASH1 3'-UTR RNA, but not to a mutant RNA incapable of localizing to the bud nor to several other mRNAs. LOC1 codes for a novel protein that recognizes double-stranded RNA structures and is required for efficient localization of ASH1 mRNA. Accordingly, Ash1p gets symmetrically distributed between daughter and mother cells in a loc1 strain. Surprisingly, Loc1p was found to be strictly nuclear, unlike other known RNA-binding proteins involved in mRNA localization which shuttle between the nucleus and the cytoplasm. We propose that efficient cytoplasmic ASH1 mRNA localization requires a previous interaction with specific nuclear factors.     

Sea urchin small RNA ribonucleoprotein particles: identification, synthesis, and subcellular localization during early embryonic development.

Small RNAs in sea urchins were examined in order to characterize developmental changes in their level, subcellular localization, synthesis, and association with proteins and other RNAs. Small RNAs such as the U snRNAs, 5S and 5.8S rRNAs, and 7S RNAs were identified by their mobility on highly cross-linked acrylamide gels. In addition, 7SL and U1 RNAs were identified by northern blot hybridization to cloned human and sea urchin probes, respectively. The level, subcellular localization, and association with proteins or RNA do not change for most small RNAs from fertilization to blastula, even though this is the time when the stored maternal pool of many small RNAs is being supplemented and replaced by embryonically synthesized RNAs. New embryonic synthesis of small RNAs was first detected at the 8-12 hr blastula stage. Although the predicted subsets of the total small RNA pool can be found in the appropriate subcellular compartments, newly synthesized small RNAs have a predominantly cytoplasmic localization: All of the newly synthesized small RNAs were found to be constituents of small RNPs. The RNPs containing newly synthesized small RNAs had sedimentation rates indistinguishable from their maternal counterparts. Thus, on the basis of sedimentation rate, no gross differences could be detected between maternal and embryonic small RNP pools. These small RNPs include a cytoplasmic RNP containing newly synthesized U1 snRNA and the sea urchin signal recognition particle (SRP) containing the 7SL, RNA. We have also identified a small RNP bearing the 5S rRNA which is present in both eggs and embryos. The presence of multiple, abundant, small RNAs and RNPs that are maintained at constant levels in particular subcellular fractions throughout development suggests that small RNAs may be involved in many more cellular activities than have so far been described.     

A second essential function of the Est1-binding arm of yeast telomerase RNA

The enzymatic ribonucleoprotein telomerase maintains telomeres in many eukaryotes, including humans, and plays a central role in aging and cancer. Saccharomyces cerevisiae telomerase RNA, TLC1, is a flexible scaffold that tethers telomerase holoenzyme protein subunits to the complex. Here we test the hypothesis that a lengthy conserved region of the Est1-binding TLC1 arm contributes more than simply Est1-binding function. We separated Est1 binding from potential other functions by tethering TLC1 to Est1 via a heterologous RNA-protein binding module. We find that Est1-tethering rescues in vivo function of telomerase RNA alleles missing nucleotides specifically required for Est1 binding, but not those missing the entire conserved region. Notably, however, telomerase function is restored for this condition by expressing the arm of TLC1 in trans. Mutational analysis shows that the Second Essential Est1-arm Domain (SEED) maps to an internal loop of the arm, which SHAPE chemical mapping and 3D modeling suggest could be regulated by conformational change. Finally, we find that the SEED has an essential, Est1-independent role in telomerase function after telomerase recruitment to the telomere. The SEED may be required for establishing telomere extendibility or promoting telomerase RNP holoenzyme activity.     

Identification and nuclear localization of yeast pre-messenger RNA processing components: RNA2 and RNA3 proteins

Temperature-sensitive mutations in the RNA2 through RNA11 genes of yeast prevent the processing of nuclear pre-mRNAs. We have raised antisera that detect the RNA2 and RNA3 proteins in immunoblots of extracts of yeast containing high copy number RNA2 and RNA3 plasmids. Subcellular fractionation of yeast cells that overproduce the RNA2 and RNA3 proteins has revealed that these proteins are enriched in nuclear fractions. Indirect immunofluorescence results have indicated that these proteins are localized in yeast nuclei. These localization results are consistent with the fact that these genes have a role in processing yeast pre-mRNA.     

Phylogeny of decapods: moving towards a consensus

Although the recognition of four broad groups within Decapoda – natantians, macrurans, anomurans and brachyurans – has long been a staple of textbooks and even the primary taxonomic literature, a precise resolution of phylogenetic relationships within the order has proved more difficult. Indeed, there have been as many schemes of decapod taxonomy and phylogeny as there were experts who wished to offer an opinion. In this decade, utilization of explicit cladistic methods of analysis and the application of molecular techniques have produced a series of clear hypotheses concerning the relationships within many of the groups of Decapoda. It is apparent that earlier conflicts of opinion can be related in part to the implicit problems of dealing with paraphyletic groups near the base of the tree that are too broadly defined by only general or plesiomorphic features. Comprehensive morphological analyses of both fossil and living forms, with attention being paid to defining synapomorphies, can lead to resolution of old controversies. Molecular techniques hold great promise towards providing further resolution, but currently suffer from insufficiencies of sampling. Nevertheless, where once there was chaos and vexation, there is now some enlightenment. The situation can only improve, but the broad outlines of decapod deep history are already emerging.     

酵母甾醇转运蛋白研究进展

甾醇是一类广泛存在于生物体内的环戊烷骈多氢菲衍生物,其不仅是细胞膜的重要组成成分,还具有重要的生理和药理活性。随着合成生物学和代谢工程技术的发展,近些年来应用酵母细胞异源合成甾醇的研究不断深入。但由于甾醇是疏水性大分子,倾向于积累在酵母的膜结构中而引发细胞毒性,一定程度上限制了甾醇产量的进一步提升。因此,揭示酵母中甾醇转运机制,特别是与甾醇转运相关的转运蛋白的工作原理,有助于设计新的策略,解除酵母细胞工厂中的甾醇积累毒性、实现甾醇增产。酵母中甾醇转运主要通过蛋白质介导的非囊泡运输机制来完成,本文归纳了酵母中已报道的5类甾醇转运相关蛋白,即OSBP/ORPs家族蛋白、LAM家族蛋白、NPC样甾醇转运蛋白、ABC转运家族蛋白和CAP超家族蛋白,汇总了这些蛋白对细胞内甾醇梯度分布和稳态维持所起的重要作用。此外,本文还综述了甾醇转运蛋白在酵母细胞工厂中的应用现状。     

Nuclear retention of RNA as a mechanism for localization.

Two mutant RNAs, one derived from tRNA(imet), the second from U1 snRNA, that are defective in export from the nucleus to the cytoplasm have been studied. In both cases, the RNAs are shown to be transport competent but prevented from leaving the nucleus by interaction with saturable binding sites. This contradicts previous hypotheses to explain the behavior of the tRNA mutant, and highlights a general problem in using mutant RNAs to study nuclear export. In the case of these mutants, it is argued that nuclear retention is likely to be artifactual. However, the additional example of U6 snRNA is described. In this case, nuclear retention appears to be a physiological mechanism by which intranuclear localization is achieved. Evidence that the site of interaction with the La protein in U6 snRNA is important for its nuclear retention is presented.     

Photosynthetic water oxidation: towards a mechanism

This mini-review outlines the current theories on the mechanism of electron transfer from water to P680, the location and structure of the water oxidising complex and the role of the manganese cluster. We discuss how our data fit in with current theories and put forward our ideas on the location and mechanism of water oxidation.     

Absorption by a moving spherical organelle in a heterogeneous cytoplasm: implications for the role of trafficking in a symplast

An organelle which absorbs (or secretes) a particular factor will find its mass transfer rate diffusion-limited if it is stationary with respect to its ambient cytoplasm; but organellar motion will raise that limit as a non-decreasing function of the Peclet number P. It is shown analytically that (i) no Whitehead paradox need be encountered in the creeping flow regime and (ii) the flux of the factor will be an even function of the Peclet number, P. By a novel analytic solution method, the flux is shown numerically to increase as P2 for P < or = 1. For P > or = 10, a quasi-planar approximating geometry yields analytically a flux which increases as P1/3. These two solutions overlap smoothly in the range 1 < or = P > or = 10. For P approximately 1, convection should increase the mass flux by roughly 100%.