scholarly journals Involvement of the Cell Wall Integrity Pathway of Saccharomyces cerevisiae in Protection against Cadmium and Arsenate Stresses

2020 ◽  
Vol 86 (21) ◽  
Author(s):  
Todsapol Techo ◽  
Sirada Charoenpuntaweesin ◽  
Choowong Auesukaree
Keyword(s):  
Cell Cycle ◽  
Cell Wall ◽  
Molecular Mechanisms ◽  
Cell Cycle Control ◽  
Protective Role ◽  
Cell Wall Integrity ◽  
Cycle Phase ◽  
Cell Wall Biosynthesis ◽  
Cell Wall Modification ◽  
Content Type

ABSTRACT Contamination of soil and water with heavy metals and metalloids is a serious environmental problem. Cadmium and arsenic are major environmental contaminants that pose a serious threat to human health. Although toxicities of cadmium and arsenic to living organisms have been extensively studied, the molecular mechanisms of cellular responses to cadmium and arsenic remain poorly understood. In this study, we demonstrate that the cell wall integrity (CWI) pathway is involved in coping with cell wall stresses induced by cadmium and arsenate through its role in the regulation of cell wall modification. Interestingly, the Rlm1p and SBF (Swi4p-Swi6p) complex transcription factors of the CWI pathway were shown to be specifically required for tolerance to cadmium and arsenate, respectively. Furthermore, we found the PIR2 gene, encoding cell wall O-mannosylated heat shock protein, whose expression is under the control of the CWI pathway, is important for maintaining cell wall integrity during cadmium and arsenate stresses. In addition, our results revealed that the CWI pathway is involved in modulating the expression of genes involved in cell wall biosynthesis and cell cycle control in response to cadmium and arsenate via distinct sets of transcriptional regulators. IMPORTANCE Environmental pollution by metal/metalloids such as cadmium and arsenic has become a serious problem in many countries, especially in developing countries. This study shows that in the yeast S. cerevisiae, the CWI pathway plays a protective role against cadmium and arsenate through the upregulation of genes involved in cell wall biosynthesis and cell cycle control, possibly in order to modulate cell wall reconstruction and cell cycle phase transition, respectively. These data provide insights into molecular mechanisms underlying adaptive responses to cadmium and arsenate.

scholarly journals Critical Roles for Lipomannan and Lipoarabinomannan in Cell Wall Integrity of Mycobacteria and Pathogenesis of Tuberculosis

mBio ◽  
2013 ◽  
Vol 4 (1) ◽  
Author(s):  
Takeshi Fukuda ◽  
Takayuki Matsumura ◽  
Manabu Ato ◽  
Maho Hamasaki ◽  
Yukiko Nishiuchi ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Critical Role ◽  
Protective Role ◽  
Cell Wall Integrity ◽  
Structural Defects ◽  
Wall Structure ◽  
Content Type ◽  
Mycobacterial Cell ◽  
Mycobacterial Cell Wall

ABSTRACTLipomannan (LM) and lipoarabinomannan (LAM) are mycobacterial glycolipids containing a long mannose polymer. While they are implicated in immune modulations, the significance of LM and LAM as structural components of the mycobacterial cell wall remains unknown. We have previously reported that a branch-forming mannosyltransferase plays a critical role in controlling the sizes of LM and LAM and that deletion or overexpression of this enzyme results in gross changes in LM/LAM structures. Here, we show that such changes in LM/LAM structures have a significant impact on the cell wall integrity of mycobacteria. InMycobacterium smegmatis, structural defects in LM and LAM resulted in loss of acid-fast staining, increased sensitivity to β-lactam antibiotics, and faster killing by THP-1 macrophages. Furthermore, equivalentMycobacterium tuberculosismutants became more sensitive to β-lactams, and one mutant showed attenuated virulence in mice. Our results revealed previously unknown structural roles for LM and LAM and further demonstrated that they are important for the pathogenesis of tuberculosis.IMPORTANCETuberculosis (TB) is a global burden, affecting millions of people worldwide.Mycobacterium tuberculosisis a causative agent of TB, and understanding the biology ofM. tuberculosisis essential for tackling this devastating disease. The cell wall ofM. tuberculosisis highly impermeable and plays a protective role in establishing infection. Among the cell wall components, LM and LAM are major glycolipids found in allMycobacteriumspecies, show various immunomodulatory activities, and have been thought to play roles in TB pathogenesis. However, the roles of LM and LAM as integral parts of the cell wall structure have not been elucidated. Here we show that LM and LAM play critical roles in the integrity of mycobacterial cell wall and the pathogenesis of TB. These findings will now allow us to seek the possibility that the LM/LAM biosynthetic pathway is a chemotherapeutic target.

scholarly journals Characterization of theStreptomyces coelicolorGlycoproteome Reveals Glycoproteins Important for Cell Wall Biogenesis

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Tessa Keenan ◽  
Adam Dowle ◽  
Rachel Bates ◽  
Margaret C. M. Smith
Keyword(s):  
Cell Wall ◽  
Abc Transporters ◽  
Streptomyces Coelicolor ◽  
Cell Wall Integrity ◽  
Enzyme Function ◽  
Cell Physiology ◽  
Cell Wall Biosynthesis ◽  
Content Type ◽  
Glycosylation Sites

ABSTRACTThe physiological role of protein O-glycosylation in prokaryotes is poorly understood due to our limited knowledge of the extent of their glycoproteomes. InActinobacteria, defects in protein O-mannosyl transferase (Pmt)-mediated protein O-glycosylation have been shown to significantly retard growth (Mycobacterium tuberculosisandCorynebacterium glutamicum) or result in increased sensitivities to cell wall-targeting antibiotics (Streptomyces coelicolor), suggesting that protein O-glycosylation has an important role in cell physiology. Only a single glycoprotein (SCO4142, or PstS) has been identified to date inS. coelicolor. Combining biochemical and mass spectrometry-based approaches, we have isolated and characterized the membrane glycoproteome inS. coelicolor. A total of ninety-five high-confidence glycopeptides were identified which mapped to thirty-seven newS. coelicolorglycoproteins and a deeper understanding of glycosylation sites in PstS. Glycosylation sites were found to be modified with up to three hexose residues, consistent with what has been observed previously in otherActinobacteria.S. coelicolorglycoproteins have diverse roles and functions, including solute binding, polysaccharide hydrolases, ABC transporters, and cell wall biosynthesis, the latter being of potential relevance to the antibiotic-sensitive phenotype ofpmtmutants. Null mutants in genes encoding a putatived-Ala-d-Ala carboxypeptidase (SCO4847) and anl,d-transpeptidase (SCO4934) were hypersensitive to cell wall-targeting antibiotics. Additionally, thesco4847mutants displayed an increased susceptibility to lysozyme treatment. These findings strongly suggest that both glycoproteins are required for maintaining cell wall integrity and that glycosylation could be affecting enzyme function.IMPORTANCEIn prokaryotes, the role of protein glycosylation is poorly understood due to our limited understanding of their glycoproteomes. In someActinobacteria, defects in protein O-glycosylation have been shown to retard growth and result in hypersensitivity to cell wall-targeting antibiotics, suggesting that this modification is important for maintaining cell wall structure. Here, we have characterized the glycoproteome inStreptomyces coelicolorand shown that glycoproteins have diverse roles, including those related to solute binding, ABC transporters, and cell wall biosynthesis. We have generated mutants encoding two putative cell wall-active glycoproteins and shown them to be hypersensitive to cell wall-targeting antibiotics. These findings strongly suggest that both glycoproteins are required for maintaining cell wall integrity and that glycosylation affects enzyme function.

scholarly journals Gene Expression ofPneumocystis murinaafter Treatment with Anidulafungin Results in Strong Signals for Sexual Reproduction, Cell Wall Integrity, and Cell Cycle Arrest, Indicating a Requirement for Ascus Formation for Proliferation

2018 ◽  
Vol 62 (5) ◽  
Author(s):  
Melanie T. Cushion ◽  
Alan Ashbaugh ◽  
Keeley Hendrix ◽  
Michael J. Linke ◽  
Nikeya Tisdale ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Cell Wall ◽  
Life Cycle ◽  
Cell Cycle Arrest ◽  
Expression Profiles ◽  
Life Cycle Stage ◽  
Cell Wall Integrity ◽  
Content Type ◽  
Cycle Arrest

ABSTRACTThe echinocandins are a class of antifungal agents that target β-1,3-d-glucan (BG) biosynthesis. In the ascigerousPneumocystisspecies, treatment with these drugs depletes the ascus life cycle stage, which contains BG, but large numbers of forms which do not express BG remain in the infected lungs. In the present study, the gene expression profiles ofPneumocystis murinawere compared between infected, untreated mice and mice treated with anidulafungin for 2 weeks to understand the metabolism of the persisting forms. Almost 80 genes were significantly up- or downregulated. Like other fungi exposed to echinocandins, genes associated with sexual replication, cell wall integrity, cell cycle arrest, and stress comprised the strongest upregulated signals inP. murinafrom the treated mice. The upregulation of theP. murinaβ-1,3-d-glucan endohydrolase and endo-1,3-glucanase was notable and may explain the disappearance of the existing asci in the lungs of treated mice since both enzymes can degrade BG. The biochemical measurement of BG in the lungs of treated mice and fluorescence microscopy with an anti-BG antibody supported the loss of BG. Downregulated signals included genes involved in cell replication, genome stability, and ribosomal biogenesis and function and thePneumocystis-specific genes encoding the major surface glycoproteins (Msg). These studies suggest thatP. murinaattempted to undergo sexual replication in response to a stressed environment and was halted in any type of proliferative cycle, likely due to a lack of BG. Asci appear to be a required part of the life cycle stage ofPneumocystis, and BG may be needed to facilitate progression through the life cycle via sexual replication.

scholarly journals Transcriptome and metabolome profiling provide insights into molecular mechanism of pseudostem elongation in banana

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Guiming Deng ◽  
Fangcheng Bi ◽  
Jing Liu ◽  
Weidi He ◽  
Chunyu Li ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Elongation ◽  
Molecular Mechanisms ◽  
Specific Gene ◽  
Wild Type ◽  
Banana Plant ◽  
Cell Wall Modification ◽  
Dwarf Cultivar ◽  
Important Trait ◽  
Metabolome Profiling

AbstractBackgroundBanana plant height is an important trait for horticultural practices and semi-dwarf cultivars show better resistance to damages by wind and rain. However, the molecular mechanisms controlling the pseudostem height remain poorly understood. Herein, we studied the molecular changes in the pseudostem of a semi-dwarf banana mutant Aifen No. 1 (Musaspp. Pisang Awak sub-group ABB) as compared to its wild-type dwarf cultivar using a combined transcriptome and metabolome approach.ResultsA total of 127 differentially expressed genes and 48 differentially accumulated metabolites were detected between the mutant and its wild type. Metabolites belonging to amino acid and its derivatives, flavonoids, lignans, coumarins, organic acids, and phenolic acids were up-regulated in the mutant. The transcriptome analysis showed the differential regulation of genes related to the gibberellin pathway, auxin transport, cell elongation, and cell wall modification. Based on the regulation of gibberellin and associated pathway-related genes, we discussed the involvement of gibberellins in pseudostem elongation in the mutant banana. Genes and metabolites associated with cell wall were explored and their involvement in cell extension is discussed.ConclusionsThe results suggest that gibberellins and associated pathways are possibly developing the observed semi-dwarf pseudostem phenotype together with cell elongation and cell wall modification. The findings increase the understanding of the mechanisms underlying banana stem height and provide new clues for further dissection of specific gene functions.

MODEL-BASED IDENTIFICATION AND ADAPTIVE CONTROL OF THE CORE MODULE IN A TYPICAL CELL CYCLE PATHWAY VIA NETWORK AND SYSTEM CONTROL THEORIES

2009 ◽  
Vol 12 (01) ◽  
pp. 21-43 ◽  
Author(s):  
BINHUA TANG ◽  
LI HE ◽  
QING JING ◽  
BAIRONG SHEN
Keyword(s):  
Cell Cycle ◽  
Adaptive Control ◽  
Molecular Mechanisms ◽  
Cell Cycle Control ◽  
System Control ◽  
Core Module ◽  
Typical Cell ◽  
And Performance ◽  
Control Theories

The loss of cell cycle control is often associated with cancers and other different diseases. With the accumulation of omics data, the network for molecule interactions in the cell cycle process will become much clearer. The identification of the crucial modules in a giant network and investigation of inherent control relations are very important to the understanding of the molecular mechanisms of diseases for new drug design. The paper proposes novel techniques in analyzing such core regulatory modules based on network and system control theories. We initially define the degree of participation (DOP) and the rate of activity (ROA) for indentifying core module components, and then the diverse contribution elasticity functions for quantifying pairwise regulatory or control activities between those components, thus facilitating the decomposition of expanded core modules and the formation of feedback loops within the control schema. Motivated by the inherent regulatory mechanisms, we expound a kind of multiphase nonlinear adaptive control algorithm in repelling abnormal genetic mutations, which directly and indirectly impact cancer development in biological cells and organs. Experimental predictions are also elucidated within the work, helping those in vivo design, verification and performance evaluation.

scholarly journals Cardiolipin Alters Rhodobacter sphaeroides Cell Shape by Affecting Peptidoglycan Precursor Biosynthesis

mBio ◽  
2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Ti-Yu Lin ◽  
William S. Gross ◽  
George K. Auer ◽  
Douglas B. Weibel
Keyword(s):  
Cell Wall ◽  
Rhodobacter Sphaeroides ◽  
Cell Morphology ◽  
Cell Shape ◽  
Molecular Mechanisms ◽  
Wild Type ◽  
Anionic Phospholipid ◽  
Content Type ◽  
Lipid Ii ◽  
Topological Features

ABSTRACT Cardiolipin (CL) is an anionic phospholipid that plays an important role in regulating protein biochemistry in bacteria and mitochondria. Deleting the CL synthase gene (Δcls) in Rhodobacter sphaeroides depletes CL and decreases cell length by 20%. Using a chemical biology approach, we found that a CL deficiency does not impair the function of the cell wall elongasome in R. sphaeroides; instead, biosynthesis of the peptidoglycan (PG) precursor lipid II is decreased. Treating R. sphaeroides cells with fosfomycin and d-cycloserine inhibits lipid II biosynthesis and creates phenotypes in cell shape, PG composition, and spatial PG assembly that are strikingly similar to those seen with R. sphaeroides Δcls cells, suggesting that CL deficiency alters the elongation of R. sphaeroides cells by reducing lipid II biosynthesis. We found that MurG—a glycosyltransferase that performs the last step of lipid II biosynthesis—interacts with anionic phospholipids in native (i.e., R. sphaeroides) and artificial membranes. Lipid II production decreases 25% in R. sphaeroides Δcls cells compared to wild-type cells, and overexpression of MurG in R. sphaeroides Δcls cells restores their rod shape, indicating that CL deficiency decreases MurG activity and alters cell shape. The R. sphaeroides Δcls mutant is more sensitive than the wild-type strain to antibiotics targeting PG synthesis, including fosfomycin, d-cycloserine, S-(3,4-dichlorobenzyl)isothiourea (A22), mecillinam, and ampicillin, suggesting that CL biosynthesis may be a potential target for combination chemotherapies that block the bacterial cell wall. IMPORTANCE The phospholipid composition of the cell membrane influences the spatial and temporal biochemistry of cells. We studied molecular mechanisms connecting membrane composition to cell morphology in the model bacterium Rhodobacter sphaeroides. The peptidoglycan (PG) layer of the cell wall is a dominant component of cell mechanical properties; consequently, it has been an important antibiotic target. We found that the anionic phospholipid cardiolipin (CL) plays a role in determination of the shape of R. sphaeroides cells by affecting PG precursor biosynthesis. Removing CL in R. sphaeroides alters cell morphology and increases its sensitivity to antibiotics targeting proteins synthesizing PG. These studies provide a connection to spatial biochemical control in mitochondria, which contain an inner membrane with topological features in common with R. sphaeroides.

scholarly journals Aspergillus fumigatus G-Protein Coupled Receptors GprM and GprJ Are Important for the Regulation of the Cell Wall Integrity Pathway, Secondary Metabolite Production, and Virulence

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Aílton Pereira da Costa Filho ◽  
Guilherme Thomaz Pereira Brancini ◽  
Patrícia Alves de Castro ◽  
Clara Valero ◽  
Jaire Alves Ferreira Filho ◽  
...  
Keyword(s):  
Cell Wall ◽  
G Protein ◽  
Galleria Mellonella ◽  
G Protein Coupled Receptors ◽  
Cell Wall Integrity ◽  
Fungal Development ◽  
Content Type ◽  
Cell Wall Integrity Pathway ◽  
Mycotoxin Biosynthesis ◽  
G Protein Coupled

ABSTRACT G-protein coupled receptors (GPCRs) are extracellular signaling receptors that sense environmental cues. Fungi sense their environment primarily through GPCR-mediated signaling pathways, which, in turn, regulate fungal development, metabolism, virulence, and mycotoxin biosynthesis. Aspergillus fumigatus is an important human pathogen that causes aspergillosis, a heterogeneous group of diseases that present a wide range of clinical manifestations. Here, we investigate in detail the role of the GPCRs GprM and GprJ in growth and gene expression. GprM and GprJ are important for melanin production and the regulation of the cell wall integrity (CWI) pathway. Overexpression of gprM and gprJ causes a 20 and 50% reduction in growth rate compared to the wild-type (WT) strain and increases sensitivity to cell wall-damaging agents. Phosphorylation of the CWI protein kinase MpkA is increased in the ΔgprM and ΔgprJ strains and decreased in the overexpression mutants compared to the WT strain. Furthermore, differences in cell wall polysaccharide concentrations and organization were observed in these strains. Transcriptome sequencing suggests that GprM and GprJ negatively regulate genes encoding secondary metabolites (SMs). Mass spectrometry analysis confirmed that the production of fumagillin, pyripyropene, fumigaclavine C, fumiquinazoline, and fumitremorgin is reduced in the ΔgprM and ΔgprJ strains, at least partially through the activation of MpkA. Overexpression of grpM also resulted in the regulation of many transcription factors, with AsgA predicted to function downstream of GprM and MpkA signaling. Finally, we show that the ΔgprM and ΔgprJ mutants are reduced in virulence in the Galleria mellonella insect model of invasive aspergillosis. IMPORTANCE A. fumigatus is the main etiological agent of invasive pulmonary aspergillosis, a life-threatening fungal disease that occurs in severely immunocompromised humans. Withstanding the host environment is essential for A. fumigatus virulence, and sensing of extracellular cues occurs primarily through G-protein coupled receptors (GPCRs) that activate signal transduction pathways, which, in turn, regulate fungal development, metabolism, virulence, and mycotoxin biosynthesis. The A. fumigatus genome encodes 15 putative classical GPCRs, with only three having been functionally characterized to date. In this work, we show that the two GPCRs GprM and GprJ regulate the phosphorylation of the mitogen-activated protein kinase MpkA and thus control the regulation of the cell wall integrity pathway. GprM and GprJ are also involved in the regulation of the production of the secondary metabolites fumagillin, pyripyropene, fumigaclavine C, fumiquinazoline, melanin, and fumitremorgin, and this regulation partially occurs through the activation of MpkA. Furthermore, GprM and GprJ are important for virulence in the insect model Galleria mellonella. This work therefore functionally characterizes two GPCRs and shows how they regulate several intracellular pathways that have been shown to be crucial for A. fumigatus virulence.

scholarly journals Sugar Beet (Beta vulgaris) Guard Cells Responses to Salinity Stress: A Proteomic Analysis

2020 ◽  
Vol 21 (7) ◽  
pp. 2331
Author(s):  
Fatemeh Rasouli ◽  
Ali Kiani-Pouya ◽  
Leiting Li ◽  
Heng Zhang ◽  
Zhonghua Chen ◽  
...  
Keyword(s):  
Cell Wall ◽  
Sugar Beet ◽  
Molecular Mechanisms ◽  
Stress Proteins ◽  
Lipid Transfer Protein ◽  
Guard Cells ◽  
Ascorbate Oxidase ◽  
Carbon Gain ◽  
Inorganic Pyrophosphatase ◽  
Cell Wall Modification

Soil salinity is a major environmental constraint affecting crop growth and threatening global food security. Plants adapt to salinity by optimizing the performance of stomata. Stomata are formed by two guard cells (GCs) that are morphologically and functionally distinct from the other leaf cells. These microscopic sphincters inserted into the wax-covered epidermis of the shoot balance CO2 intake for photosynthetic carbon gain and concomitant water loss. In order to better understand the molecular mechanisms underlying stomatal function under saline conditions, we used proteomics approach to study isolated GCs from the salt-tolerant sugar beet species. Of the 2088 proteins identified in sugar beet GCs, 82 were differentially regulated by salt treatment. According to bioinformatics analysis (GO enrichment analysis and protein classification), these proteins were involved in lipid metabolism, cell wall modification, ATP biosynthesis, and signaling. Among the significant differentially abundant proteins, several proteins classified as “stress proteins” were upregulated, including non-specific lipid transfer protein, chaperone proteins, heat shock proteins, inorganic pyrophosphatase 2, responsible for energized vacuole membrane for ion transportation. Moreover, several antioxidant enzymes (peroxide, superoxidase dismutase) were highly upregulated. Furthermore, cell wall proteins detected in GCs provided some evidence that GC walls were more flexible in response to salt stress. Proteins such as L-ascorbate oxidase that were constitutively high under both control and high salinity conditions may contribute to the ability of sugar beet GCs to adapt to salinity by mitigating salinity-induced oxidative stress.

scholarly journals EslB is required for cell wall biosynthesis and modification in Listeria monocytogenes

2020 ◽  
Author(s):  
Jeanine Rismondo ◽  
Lisa M. Schulz ◽  
Maria Yacoub ◽  
Ashima Wadhawan ◽  
Michael Hoppert ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Division ◽  
Listeria Monocytogenes ◽  
Abc Transporter ◽  
Cell Lysis ◽  
Growth Conditions ◽  
Cell Wall Integrity ◽  
Growth Defect ◽  
Cell Wall Biosynthesis ◽  
High Concentrations

Lysozyme is an important component of the innate immune system. It functions by hydrolysing the peptidoglycan (PG) layer of bacteria. The human pathogen Listeria monocytogenes is intrinsically lysozyme resistant. The peptidoglycan N-deacetylase PgdA and O-acetyltransferase OatA are two known factors contributing to its lysozyme resistance. Furthermore, it was shown that the absence of components of an ABC transporter, here referred to as EslABC, leads to reduced lysozyme resistance. How its activity is linked to lysozyme resistance is still unknown. To investigate this further, a strain with a deletion in eslB, coding for a membrane component of the ABC transporter, was constructed in L. monocytogenes strain 10403S. The eslB mutant showed a 40-fold reduction in the minimal inhibitory concentration to lysozyme. Analysis of the PG structure revealed that the eslB mutant produced PG with reduced levels of O-acetylation. Using growth and autolysis assays, we show that the absence of EslB manifests in a growth defect in media containing high concentrations of sugars and increased endogenous cell lysis. A thinner PG layer produced by the eslB mutant under these growth conditions might explain these phenotypes. Furthermore, the eslB mutant had a noticeable cell division defect and formed elongated cells. Microscopy analysis revealed that an early cell division protein still localized in the eslB mutant indicating that a downstream process is perturbed. Based on our results, we hypothesize that EslB affects the biosynthesis and modification of the cell wall in L. monocytogenes and is thus important for the maintenance of cell wall integrity. IMPORTANCE The ABC transporter EslABC is associated with the intrinsic lysozyme resistance of Listeria monocytogenes. However, the exact role of the transporter in this process and in the physiology of L. monocytogenes is unknown. Using different assays to characterize an eslB deletion strain, we found that the absence of EslB not only affects lysozyme resistance, but also endogenous cell lysis, cell wall biosynthesis, cell division and the ability of the bacterium to grow in media containing high concentrations of sugars. Our results indicate that EslB is by a yet unknown mechanism an important determinant for cell wall integrity in L. monocytogenes.

scholarly journals Cell Cycle Kinase Polo Is Controlled by a Widespread 3′ Untranslated Region Regulatory Sequence inDrosophila melanogaster

2019 ◽  
Vol 39 (15) ◽  
Author(s):  
Marta S. Oliveira ◽  
Jaime Freitas ◽  
Pedro A. B. Pinto ◽  
Ana de Jesus ◽  
Joana Tavares ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Drosophila Melanogaster ◽  
Untranslated Region ◽  
Molecular Mechanisms ◽  
Alternative Polyadenylation ◽  
Upstream Sequence ◽  
Sequence Element ◽  
Content Type ◽  
Cell Cycle Kinase

ABSTRACTAlternative polyadenylation generates transcriptomic diversity, although the physiological impact and regulatory mechanisms involved are still poorly understood. The cell cycle kinase Polo is controlled by alternative polyadenylation in the 3′ untranslated region (3′UTR), with critical physiological consequences. Here, we characterized the molecular mechanisms required forpoloalternative polyadenylation. We identified a conserved upstream sequence element (USE) close to thepoloproximal poly(A) signal. Transgenic flies without this sequence show incorrect selection ofpolopoly(A) signals with consequent downregulation of Polo expression levels and insufficient/defective activation of Polo kinetochore targets Mps1 and Aurora B. Deletion of the USE results in abnormal mitoses in neuroblasts, revealing a role for this sequencein vivo. We found that Hephaestus binds to the USE RNA and thathephaestusmutants display defects inpoloalternative polyadenylation concomitant with a striking reduction in Polo protein levels, leading to mitotic errors and aneuploidy. Bioinformatic analyses show that the USE is preferentially localized upstream of noncanonical polyadenylation signals inDrosophila melanogastergenes. Taken together, our results revealed the molecular mechanisms involved inpoloalternative polyadenylation, with remarkable physiological functions in Polo expression and activity at the kinetochores, and disclosed a newin vivofunction for USEs inDrosophila melanogaster.

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