Unmasking the role of the 3′ UTR in the cytoplasmic polyadenylation and translational regulation of maternal mRNAs

The poly(A)-dependent translational regulation of maternal mRNAs is an important mechanism to execute stage-specific programs of protein synthesis during early development. This control underlies many crucial developmental events including the meiotic maturation of oocytes and activation of the mitotic cell cycle at fertilization. A recent report⁽¹⁾ demonstrates that the 3′ untranslated region of the cyclin A1, B1, B2 and c-mos mRNAs determines the timing and extent of their cytoplasmic polyadenylation and translational activation during Xenopus oocyte maturation. These studies further establish that protein synthesis can be temporally and quantitatively controlled by developmentally regulated changes in the polyadenylation of maternal mRNAs.     

Fission yeast receptor of activated C kinase (RACK1) ortholog Cpc2 regulates mitotic commitment through Wee1 kinase

In the fission yeast Schizosaccharomyces pombe, Wee1-dependent inhibitory phosphorylation of the highly conserved Cdc2/Cdk1 kinase determines the mitotic onset when cells have reached a defined size. The receptor of activated C kinase (RACK1) is a scaffolding protein strongly conserved among eukaryotes which binds to other proteins to regulate multiple processes in mammalian cells, including the modulation of cell cycle progression during G(1)/S transition. We have recently described that Cpc2, the fission yeast ortholog to RACK1, controls from the ribosome the activation of MAPK cascades and the cellular defense against oxidative stress by positively regulating the translation of specific genes whose products participate in the above processes. Intriguingly, mutants lacking Cpc2 display an increased cell size at division, suggesting the existence of a specific cell cycle defect at the G(2)/M transition. In this work we show that protein levels of Wee1 mitotic inhibitor are increased in cells devoid of Cpc2, whereas the levels of Cdr2, a Wee1 inhibitor, are down-regulated in the above mutant. On the contrary, the kinetics of G(1)/S transition was virtually identical both in control and Cpc2-less strains. Thus, our results suggest that in fission yeast Cpc2/RACK1 positively regulates from the ribosome the mitotic onset by modulating both the protein levels and the activity of Wee1. This novel mechanism of translational control of cell cycle progression might be conserved in higher eukaryotes.     

A dual‐color marker system for in vivo visualization of cell cycle progression in Arabidopsis

Visualization of the spatiotemporal pattern of cell division is crucial to understand how multicellular organisms develop and how they modify their growth in response to varying environmental conditions. The mitotic cell cycle consists of four phases: S (DNA replication), M (mitosis and cytokinesis), and the intervening G1 and G2 phases; however, only G2/M‐specific markers are currently available in plants, making it difficult to measure cell cycle duration and to analyze changes in cell cycle progression in living tissues. Here, we developed another cell cycle marker that labels S‐phase cells by manipulating Arabidopsis CDT1a, which functions in DNA replication origin licensing. Truncations of the CDT1a coding sequence revealed that its carboxy‐terminal region is responsible for proteasome‐mediated degradation at late G2 or in early mitosis. We therefore expressed this region as a red fluorescent protein fusion protein under the S‐specific promoter of a histone 3.1‐type gene, HISTONE THREE RELATED2 (HTR2), to generate an S/G2 marker. Combining this marker with the G2/M‐specific CYCB1‐GFP marker enabled us to visualize both S to G2 and G2 to M cell cycle stages, and thus yielded an essential tool for time‐lapse imaging of cell cycle progression. The resultant dual‐color marker system, Cell Cycle Tracking in Plant Cells (Cytrap), also allowed us to identify root cells in the last mitotic cell cycle before they entered the endocycle. Our results demonstrate that Cytrap is a powerful tool for in vivo monitoring of the plant cell cycle, and thus for deepening our understanding of cell cycle regulation in particular cell types during organ development.     

Autoregulation of Musashi1 mRNA translation during Xenopus oocyte maturation

The mRNA translational control protein, Musashi, plays a critical role in cell fate determination through sequence‐specific interactions with select target mRNAs. In proliferating stem cells, Musashi exerts repression of target mRNAs to promote cell cycle progression. During stem cell differentiation, Musashi target mRNAs are de‐repressed and translated. Recently, we have reported an obligatory requirement for Musashi to direct translational activation of target mRNAs during Xenopus oocyte meiotic cell cycle progression. Despite the importance of Musashi in cell cycle regulation, only a few target mRNAs have been fully characterized. In this study, we report the identification and characterization of a new Musashi target mRNA in Xenopus oocytes. We demonstrate that progesterone‐stimulated translational activation of the Xenopus Musashi1 mRNA is regulated through a functional Musashi binding element (MBE) in the Musashi1 mRNA 3′ untranslated region (3′ UTR). Mutational disruption of the MBE prevented translational activation of Musashi1 mRNA and its interaction with Musashi protein. Further, elimination of Musashi function through microinjection of inhibitory antisense oligonucleotides prevented progesterone‐induced polyadenylation and translation of the endogenous Musashi1 mRNA. Thus, Xenopus Musashi proteins regulate translation of the Musashi1 mRNA during oocyte maturation. Our results indicate that the hierarchy of sequential and dependent mRNA translational control programs involved in directing progression through meiosis are reinforced by an intricate series of nested, positive feedback loops, including Musashi mRNA translational autoregulation. These autoregulatory positive feedback loops serve to amplify a weak initiating signal into a robust commitment for the oocyte to progress through the cell cycle and become competent for fertilization.Mol. Reprod. Dev. 79: 553‐563, 2012. © 2012 Wiley Periodicals, Inc.     

Light-induced synchrony of cell division in the protonema of the fern,Pteris vittata

Summary In protonemata of Pteris vittata grown for 6 days under red light, which brings about a marked depression of mitotic activity, the first division of the cells was synchronously induced by irradiation with blue light, and subsequent cell divisions were also promoted. The peak of the mitotic index reached a maximum of about 70% at 11.5 hrs, and 90% of all protonemata divided between the 11th and 13th hour after exposure to blue light. When the protonemata were continuously irradiated with blue light, synchronism of the next cell division in the apical cells decreased to a mitotic index of about 30%, and further divisions occurred randomly.The synchronization of cell division was found to be a combined effect of red and blue light. Red light maintained the cells in the early G₁ phase of the cell cycle; blue light caused the cells to progress synchronously through the cell cycle, with an average duration of 12 hr. By using ³H-thymidine, the average duration of the G₁, S, G₂ and M phases was determined to be about 3.5, 5, 2.5 and 1 hr, respectively.Synchronous cell division could be induced in older protonemata grown for 6 to 12 days in red light and even in protonemata having two cells. It could be repeated in the same protonema by reexposure to red light for 24 hrs or more before another irradiation with blue light.     

Ribosomal slowdown mediates translational arrest during cellular division

Global mRNA translation is transiently inhibited during cellular division. We demonstrate that mitotic cells contain heavy polysomes, but these are significantly less translationally active than polysomes in cycling cells. Several observations indicate that mitotic translational attenuation occurs during the elongation stage: (i) in cycling nonsynchronized cultures, only mitotic cells fail to assemble stress granules when treated with agents that inhibit translational initiation; (ii) mitotic cells contain fewer free 80S complexes, which are less sensitive to high salt disassembly; (iii) mitotic polysomes are more resistant to enforced disassembly using puromycin; and (iv) ribosome transit time increases during mitosis. Elongation slowdown guarantees that polysomes are retained even if initiation is inhibited at the same time. Stalling translating ribosomes during mitosis may protect mRNAs and allow rapid resumption of translation immediately upon entry into the G(1) phase.     

Cyclin A, cyclin B and stringlike are regulated separately in cell cycle arrested trochoblasts of Patella vulgata embryos

 Trochoblasts are the first cells to differentiate during the development of spiralian embryos. Differentiation is accompanied by a cell division arrest. In embryos of the limpet Patella vulgata, the participation of cell cycle-regulating factors in trochoblast arrest was analysed as a first step to unravel its cause. We determined the cell cycle phase in which the trochoblasts are arrested by analysing the subcellular locations of mitotic cyclins. The results show that the trochoblasts are most likely arrested in the G2 phase. This was supported by measurement of the DNA content in trochoblast nuclei after the last division. Trochoblasts complete their final division at the sixth mitotic cycle. This mitotic cycle resembles the first postblastoderm cell cycle of Drosophila, in which mitotic activity is controlled by expression of the string gene. As failure of string expression results in cell cycle arrest in the G2 phase, negative regulation of a Patella string homolog could be responsible for trochoblast arrest. Although Stl messengers disappeared from trochoblasts during their final division, expression was observed again 20 min later. Messengers remained present in all trochoblasts at low levels during further development. Thus, expression of the stringlike gene allows the cell cycle arrest of these cells, whereas in Drosophila cells arrested in division lack string messengers. Received: 10 February 1997 / Accepted: 23 November 1997     

Importance of PNO1 for growth and survival of urinary bladder carcinoma: Role in core‐regulatory circuitry

PNO1 (partner of Nob1) was known as a RNA‐binding protein in humans, and its ortholog PNO1 was reported to participate ribosome and proteasome biogenesis in yeasts. Yet there have been few studies about its functions in mammalian cells, and so far its role in human cells has never been reported, especially in urinary bladder cancer (UBC).We interrogated the cellular functions and clinical significance of PNO1 in, and its molecular mechanism through microarrays and bioinformatics analysis. Our findings support that PNO1 participates in promoting proliferation and colonogenesis, while reducing apoptosis of UBC cells, and is also predicted to be associated with the migration and metastasis of UBC PNO1 knockdown (KD) attenuated the tumorigenesis ability of UBC in mouse. PNO1 KD led to the altered expression of 1543 genes that are involved in a number of signalling pathways, biological functions and regulation networks. CD44, PTGS2, cyclin D1, CDK1, IL‐8, FRA1, as well as mTOR, p70 S6 kinase, p38 and Caspase‐3 proteins were all down‐regulated in PNO1 KD cells, suggesting the involvement of PNO1 in inflammatory responses, cell cycle regulation, chemotaxis, cell growth and proliferation, apoptosis, cell migration and invasiveness. This study will enhance our understanding of the molecular mechanism of UBC and may eventually provide novel targets for individualized cancer therapy.     

Starving for more: Nutrient sensing by LIN-28 in adult intestinal progenitor cells

ABSTRACT

In this Extra View, we extend our recent work on the protein LIN-28 and its role in adult stem cell divisions. LIN-28 is an mRNA- and microRNA-binding protein that is conserved from worms to humans. When expressed ectopically, it promotes the reprogramming of differentiated vertebrate cells into pluripotent stem cells as well as the regeneration of vertebrate tissues after injury. However, its endogenous function in stem cell populations is less clear. We recently reported that LIN-28 is specifically expressed in progenitor cells in the adult Drosophila intestine and enhances insulin signaling within this population. Loss of lin-28 alters the division patterns of these progenitor cells, limiting the growth of the intestinal epithelium that is ordinarily caused by feeding. Thus, LIN-28 is part of an uncharacterized circuit used to remodel a tissue in response to environmental cues like nutrition. Here, we extend this analysis by reporting that the levels of LIN-28 in progenitor cells are sensitive to nutrient availability. In addition, we speculate about the role of LIN-28 in the translational control of target mRNAs such as Insulin Receptor (InR) and how such translational control may be an important mechanism that underlies the stem cell dynamics needed for tissue homeostasis and growth.     

Life Cycle of the pseudocolonial dinoflagellate Polykrikos kofoidii (Gymnodiniales,Dinoflagellata)

The athecate, pseudocolonial polykrikoid dinoflag‐ellates show a greater morphological complexity than many other dinoflagellate cells and contain not only elaborate extrusomes but sulci, cinguli, flagellar pairs, and nuclei in multiple copies. Among polykrikoids, Polykrikos kofoidii is a common species that plays an important role as a grazer of toxic planktonic algae but whose life cycle is poorly known. In this study, the main life cycle stages of P. kofoidii were examined and documented for the first time. The formation of gametes, 2‐zooid‐1‐nucleus stages very different from vegetative cells, was observed and the process of gamete fusion, isogamy, was recorded. Karyogamy followed shortly after completed plasmogamy. A complex reorganization of furrows (cinguli and sulci) and flagella followed zygote formation, resulting in a 4‐zooid zygote with one nucleus. The fate of zygotes under different nutritional conditions was also investigated; well‐fed zygotes were able to reenter the vegetative cycle via meiotic divisions as indicated by nuclear cyclosis. However, nuclear cyclosis was preceded by a presumably mitotic division of the primary zygote nucleus which by definition would imply that P. kofoidii has a diplohaplontic life cycle. Nuclear cyclosis in germlings hatched from spiny resting cysts indicate that these cysts are of zygote origin (hypnozygotes). Hypnozygote formation, cyst hatching, the morphology of the germling (a 1‐zooid cell), and its development into a normal pseudocolony are documented here for the first time. There is evidence that P. kofoidii has a system of complex heterothallism.     

Analysis of gemini pollen 3 mutant suggests a broad function of AUGMIN in microtubule organization during sexual reproduction in Arabidopsis

In flowering plants, male gametes arise via meiosis of diploid pollen mother cells followed by two rounds of mitotic division. Haploid microspores undergo polar nuclear migration and asymmetric division at pollen mitosis I to segregate the male germline, followed by division of the germ cell to generate a pair of sperm cells. We previously reported two gemini pollen (gem) mutants that produced twin‐celled pollen arising from polarity and cytokinesis defects at pollen mitosis I in Arabidopsis. Here, we report an independent mutant, gem3, with a similar division phenotype and severe genetic transmission defects through pollen. Cytological analyses revealed that gem3 disrupts cell division during male meiosis, at pollen mitosis I and during female gametophyte development. We show that gem3 is a hypomorphic allele (aug6‐1) of AUGMIN subunit 6, encoding a conserved component in the augmin complex, which mediates microtubule (MT)‐dependent MT nucleation in acentrosomal cells. We show that MT arrays are disturbed in gem3/aug6‐1 during male meiosis and pollen mitosis I using fluorescent MT‐markers. Our results demonstrate a broad role for the augmin complex in MT organization during sexual reproduction, and highlight gem3/aug6‐1 mutants as a valuable tool for the investigation of augmin‐dependent MT nucleation and dynamics in plant cells.     

PPR polyadenylation factor defines mitochondrial mRNA identity and stability in trypanosomes

In Trypanosoma brucei, most mitochondrial mRNAs undergo internal changes by RNA editing and 3′ end modifications. The temporally separated and functionally distinct modifications are manifested by adenylation prior to editing, and by post‐editing extension of a short A‐tail into a long A/U‐heteropolymer. The A‐tail stabilizes partially and fully edited mRNAs, while the A/U‐tail enables mRNA binding to the ribosome. Here, we identify an essential pentatricopeptide repeat‐containing RNA binding protein, kinetoplast polyadenylation factor 3 (KPAF3), and demonstrate its role in protecting pre‐mRNA against degradation by the processome. We show that KPAF3 recruits KPAP1 poly(A) polymerase to the 3′ terminus, thus leading to pre‐mRNA stabilization, or decay depending on the occurrence and extent of editing. In vitro, KPAF3 stimulates KPAP1 activity and inhibits mRNA uridylation by RET1 TUTase. Our findings indicate that KPAF3 selectively directs pre‐mRNA toward adenylation rather than uridylation, which is a default post‐trimming modification characteristic of ribosomal and guide RNAs. As a quality control mechanism, KPAF3 binding ensures that mRNAs entering the editing pathway are adenylated and, therefore, competent for post‐editing A/U‐tailing and translational activation.