Molecular and functional characterization of Brassica BREVIPEDICELLUS orthologs involved in inflorescence architectureThis paper is one of a selection of papers published in a  Special Issue from the National Research Council of Canada – Plant Biotechnology Institute.

The diversity of inflorescence architecture in angiosperms relates to attracting pollinators and allowing the effective dispersal of seeds. Molecular understanding of the genetic factors regulating inflorescence architecture from the model system of Arabidopsis could provide critical insights for addressing this developmental process/pathway in a closely related crop species like Brassica napus L. Towards this objective, we have isolated and characterized the orthologs of the homeobox gene BREVIPEDICELLUS (BP) in three Brassica species, B. napus (BnBP), Brassica rapa L. (BrBP), and Brassica oleracea L. (BoBP). These Brassica orthologs show a high degree of conservation at the nucleotide and amino acid level, including the homeodomain and the intron positions. The B. napus ortholog complemented the Arabidopsis bp null allele and overexpression of BnBP in B. napus transgenic lines resulted in altered leaf phenotypes. Together, these results suggest that Brassica BP genes are functional orthologs of Arabidopsis BP. Ectopic expression of AS2, a negative regulator of BP encoding a LOB domain transcription factor, in transgenic B. napus produced compact inflorescence architecture reminiscent of the loss-of-function bp mutant phenotype described previously in Arabidopsis. These results provide evidence in support of BP as a potential target gene for modifying inflorescence architecture in Brassica crop species.

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Loss offlflTriggers JNK-Dependent Cell Death inDrosophila

BioMed Research International ◽

10.1155/2015/623573 ◽

2015 ◽

Vol 2015 ◽

pp. 1-8 ◽

Cited By ~ 7

Author(s):

Jiuhong Huang ◽

Lei Xue

Keyword(s):

Cell Death ◽

Function Analysis ◽

Ectopic Expression ◽

Negative Regulator ◽

Loss Of Function ◽

Jnk Pathway ◽

Dependent Cell ◽

Jnk Activation ◽

First Time

falafel(flfl) encodes aDrosophilahomolog of human SMEK whosein vivofunctions remain elusive. In this study, we performed gain-of-function and loss-of-function analysis inDrosophilaand identified flfl as a negative regulator of JNK pathway-mediated cell death. While ectopic expression offlflsuppresses TNF-triggered JNK-dependent cell death, loss offlflpromotes JNK activation and cell death in the developing eye and wing. These data report for the first time an essential physiological function offlflin maintaining tissue homeostasis and organ development. As the JNK signaling pathway has been evolutionary conserved from fly to human, a similar role of PP4R3 in JNK-mediated physiological process is speculated.

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Abnormal Expression of Human Mitoferrin (SLC25A37) Is Associated with a Variant of Erythropoietic Protoporphyria.

Blood ◽

10.1182/blood.v108.11.3.3 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3-3 ◽

Cited By ~ 2

Author(s):

George C. Shaw ◽

Nathaniel B. Langer ◽

Yongming Wang ◽

Liangtao Li ◽

Jerry Kaplan ◽

...

Keyword(s):

Amino Acid ◽

Fetal Liver ◽

Amino Acid Level ◽

Ectopic Expression ◽

Disease Process ◽

Hypochromic Anemia ◽

K562 Cells ◽

Loss Of Function ◽

Erythropoietic Protoporphyria ◽

Iron Deficient

Abstract Mitoferrin (mfrn, slc25a37) is the high-affinity transporter of iron into mitochondria of developing erythroblasts and is essential for normal erythropoiesis. Recently, we described the cloning and characterization of mfrn in the zebrafish frascati (frs) mutant, in which homozygous null mutation leads to profound hypochromic anemia and embryonic lethality (Shaw GC, et al. 2006 Nature 440:96–100). Here we characterize the human MFRN orthologue and implicate a specific disease process purportedly caused by defects in MFRN expression. We studied human MFRN expression by a) analysis of human-tissue northern blots, b) fluorescent immunohistochemistry and confocal microscopy for subcellular localization, c) genetic complementation in MRS3/4 deficient yeast strains, and d) microinjection and rescue efficiency of frs mutant zebrafish. Human MFRN, mapping to chromosome 8p21, shows 82% and 64% similarity at the amino acid level to orthologous mouse and zebrafish Mfrn proteins respectively, and 60% similarity to a paralogous MFRN2 (SLC25A28). MFRN mRNA is highly expressed in fetal and adult hematopoietic tissues, including fetal liver and bone marrow, similar to zebrafish and mice. The MFRN protein localizes to the mitochondria like other SLC25 solute carriers. Ectopic expression of human MFRN capably restores hemoglobinized cells in anemic frs embryos, programs increased 55Fe-labeled heme synthesis in K562 cells, and rescues MRS3/4 mutant yeast for growth on iron-deficient media, demonstrating conservation of MFRN function. We evaluated 6 unrelated probands with a variant of erythropoietic protoporphyria (EPP) who harbor no mutations in ferrochelatase, the genetic cause of classic EPP. Five of the 6 index cases had severe hepatic disease necessitating liver transplantation. Our analysis of all 6 patients revealed the presence of an aberrantly spliced MFRN transcript, which was absent in normal individuals and patients with classic EPP. This aberrant spliceoform results in insertion of a 477 bp fragment from intron 2 and leads to an early nonsense codon after amino acid 156, prematurely truncating the MFRN protein. In contrast to normal MFRN cDNA, the variant MFRN cDNA from EPP patients fails to complement MRS3/4 mutant yeasts in low-iron media, demonstrating its loss-of-function activity. Our data demonstrate conserved function of human MFRN in erythroid mitochondrial iron metabolism and an association between a loss-of-function MFRN transcript and a variant of erythropoietic protoporphyria.

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An unexpected role for the conserved ADAM-family metalloprotease ADM-2 in Caenorhabditis elegans molting

10.1101/2021.12.15.472830 ◽

2021 ◽

Author(s):

Sarina M Bernazzani ◽

Braveen B Joseph ◽

Philli T Edeen ◽

Shaonil Binti ◽

David S Fay

Keyword(s):

Caenorhabditis Elegans ◽

Developmental Process ◽

Density Lipoprotein ◽

Matrix Metalloproteases ◽

Negative Regulator ◽

Binding Motif ◽

Loss Of Function ◽

Strong Suppression ◽

C Elegans ◽

Molting Process

Molting is a widespread developmental process in which the external extracellular matrix (ECM), the cuticle, is remodeled to allow for organismal growth and environmental adaptation. Studies in the nematode Caenorhabditis elegans have identified a diverse set of molting-associated factors including signaling molecules, intracellular trafficking regulators, ECM components, and ECM-modifying enzymes such as matrix metalloproteases. C. elegans NEKL-2 and NEKL-3, two conserved members of the NEK family of protein kinases, are essential for molting and promote the endocytosis of environmental steroid-hormone precursors by the epidermis. Steroids in turn drive the cyclic induction of many genes required for molting. Here we report a novel role for the sole C. elegans ADAM-meltrin metalloprotease family member, ADM-2, as a negative regulator of molting. Whereas loss of adm-2 led to strong suppression of molting defects in partial loss-of-function nekl mutants, overexpression of ADM-2 induced molting defects in wild-type animals. CRISPR genome editing implicated the Zn-binding motif within the metalloprotease domain as critical for mediating the effects of ADM-2 on molting. ADM-2 is expressed in the epidermis, and its trafficking through the endo-lysosomal network was disrupted after NEKL depletion. We also identified the epidermally expressed low-density lipoprotein receptor-related protein, LRP-1, as a candidate target of ADM-2 regulation. Whereas loss of ADM-2 activity led to the upregulation of LRP-1, ADM-2 overexpression caused a reduction in LRP-1 abundance, suggesting that ADM-2 may function as a sheddase for LRP-1. We propose that loss of adm-2 suppresses molting defects in nekl mutants by eliminating a negative regulator of LRP-1, thereby compensating for defects in the efficiency of LRP-1 and sterol uptake. Our findings emphasize the importance of endocytic trafficking for both the internalization of factors that promote molting and the removal of proteins that would otherwise be deleterious to the molting process.

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The conserved brassinosteroid-related transcription factor BIM1a negatively regulates fruit growth in tomato

Journal of Experimental Botany ◽

10.1093/jxb/eraa495 ◽

2020 ◽

Author(s):

Kentaro Mori ◽

Martine Lemaire-Chamley ◽

Joana Jorly ◽

Fernando Carrari ◽

Mariana Conte ◽

...

Keyword(s):

Target Genes ◽

Fruit Size ◽

Ectopic Expression ◽

Negative Regulator ◽

Crop Species ◽

Signalling Network ◽

Transgenic Lines ◽

Related Transcription Factor ◽

Fruit Pericarp

Abstract Brassinosteroids (BRs) are steroid hormones that play key roles in plant development and defense. Our goal is to harness the extensive knowledge of the Arabidopsis BR signalling network for improving productivity in crop species. This first requires identifying components of the conserved network and their function in the target species. Here, we investigated the function of SlBIM1a, the closest tomato homolog of AtBIM1, which is highly expressed in fruit. SlBIM1a overexpressing lines displayed severe plant and fruit dwarfism, and histological characterization of different transgenic lines revealed that SlBIM1a expression negatively correlated with fruit pericarp cell size, resulting in fruit size modifications. These growth phenotypes were in contrast to those found in Arabidopsis, and this was confirmed by the reciprocal ectopic expression of SlBIM1a/b in Arabidopsis and, AtBIM1 in tomato. These results determined that BIM1 function depends more on the recipient species than on its primary sequence. Yeast two-hybrid interaction studies and transcriptomic analyses of SlBIM1a overexpressing fruit, further suggested that SlBIM1a acts through its interaction with SlBZH1 to govern the transcriptional regulation of growth-related BRs target genes. Together, these results suggest that SlBIM1a is a negative regulator of pericarp cell expansion, possibly at the crossroad with auxin and light signalling.

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Functional Characterization of the Adenylyl Cyclase Genesgs-1by Analysis of a Mutational Spectrum inCaenorhabditis elegans

Genetics ◽

10.1093/genetics/161.1.133 ◽

2002 ◽

Vol 161 (1) ◽

pp. 133-142 ◽

Cited By ~ 4

Author(s):

Celine Moorman ◽

Ronald H A Plasterk

Keyword(s):

Life Span ◽

Adenylyl Cyclase ◽

Neuronal Degeneration ◽

Functional Characterization ◽

Adenylyl Cyclases ◽

Loss Of Function ◽

C Elegans ◽

Larval Stages ◽

Pharyngeal Pumping

AbstractThe sgs-1 (suppressor of activated Gαs) gene encodes one of the four adenylyl cyclases in the nematode C. elegans and is most similar to mammalian adenylyl cyclase type IX. We isolated a complete loss-of-function mutation in sgs-1 and found it to result in animals with retarded development that arrest in variable larval stages. sgs-1 mutant animals exhibit lethargic movement and pharyngeal pumping and (while not reaching adulthood) have a mean life span that is >50% extended compared to wild type. An extensive set of reduction-of-function mutations in sgs-1 was isolated in a screen for suppressors of a neuronal degeneration phenotype induced by the expression of a constitutively active version of the heterotrimeric Gαs subunit of C. elegans. Although most of these mutations change conserved residues within the catalytic domains of sgs-1, mutations in the less-conserved transmembrane domains are also found. The sgs-1 reduction-of-function mutants are viable and have reduced locomotion rates, but do not show defects in pharyngeal pumping or life span.

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Long noncoding RNA FER1L4 promotes the malignant processes of papillary thyroid cancer by targeting the miR-612/ Cadherin 4 axis

Cancer Cell International ◽

10.1186/s12935-021-02097-2 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Luyao Wu ◽

Yu Ding ◽

Houchao Tong ◽

Xi Zhuang ◽

Jingsheng Cai ◽

...

Keyword(s):

Thyroid Cancer ◽

Papillary Thyroid Cancer ◽

Noncoding Rna ◽

Animal Experiments ◽

Ectopic Expression ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Papillary Thyroid ◽

Loss Of Function ◽

Functional Roles

Abstract Background Long noncoding RNAs (lncRNAs) have emerged as crucial regulators in various cancers. However, the functional roles of most lncRNA in papillary thyroid cancer (PTC) are not detailly understood. This study aims to investigate the biological function and molecular mechanism of lncRNA Fer-1 like family member 4 (FER1L4) in PTC. Methods The expression of FER1L4 in PTC was determined via operating quantitative real-time PCR assays. Meanwhile, the clinical significance of FER1L4 in patients with PTC was described. The biological functions of FER1L4 on PTC cells were evaluated by gain and loss of function experiments. Moreover, animal experiments were performed to reveal the effect on tumor growth. Subcellular distribution of FER1L4 was determined by fluorescence in situ hybridization and subcellular localization assays. Luciferase reporter assay and RNA immunoprecipitation assay were applied to define the relationship between FER1L4, miR-612, and Cadherin 4 (CDH4). Results Upregulated expression of FER1L4 in PTC tissues was positively correlated with lymph node metastasis (P = 0.020), extrathyroidal extension (P = 0.013) and advanced TNM stages (P = 0.013). In addition, knockdown of FER1L4 suppressed PTC cell proliferation, migration, and invasion, whereas ectopic expression of FER1L4 inversely promoted these processes. Mechanistically, FER1L4 could competitively bind with miR-612 to prevent the degradation of its target gene CDH4. This condition was further confirmed in the rescue assays. Conclusions This study first demonstrates FER1L4 plays an oncogenic role in PTC via a FER1L4-miR-612-CDH4 axis and may provide new therapeutic and diagnostic targets for PTC.

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Partitioning of N-Ethylmaleimide-Sensitive Fusion (NSF) Protein Function in Drosophila melanogaster: dNSF1 Is Required in the Nervous System, and dNSF2 Is Required in Mesoderm

Genetics ◽

10.1093/genetics/158.1.265 ◽

2001 ◽

Vol 158 (1) ◽

pp. 265-278

Author(s):

Jessica A Golby ◽

Leigh Anna Tolar ◽

Leo Pallanck

Keyword(s):

Nervous System ◽

Protein Function ◽

Ectopic Expression ◽

Drosophila Genome ◽

Loss Of Function ◽

Stage Of Development ◽

Expression Studies ◽

Larval Nervous System ◽

Constitutive Secretion ◽

Temporal And Spatial

Abstract The N-ethylmaleimide-sensitive fusion protein (NSF) promotes the fusion of secretory vesicles with target membranes in both regulated and constitutive secretion. While it is thought that a single NSF may perform this function in many eukaryotes, previous work has shown that the Drosophila genome contains two distinct NSF genes, dNSF1 and dNSF2, raising the possibility that each plays a specific secretory role. To explore this possibility, we generated mutations in the dNSF2 gene and used these and novel dNSF1 loss-of-function mutations to analyze the temporal and spatial requirements and the degree of functional redundancy between dNSF1 and dNSF2. Results of this analysis indicate that dNSF1 function is required in the nervous system beginning at the adult stage of development and that dNSF2 function is required in mesoderm beginning at the first instar larval stage of development. Additional evidence suggests that dNSF1 and dNSF2 may play redundant roles during embryonic development and in the larval nervous system. Ectopic expression studies demonstrate that the dNSF1 and dNSF2 gene products can functionally substitute for one another. These results indicate that the Drosophila NSF proteins exhibit similar functional properties, but have evolved distinct tissue-specific roles.

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Genetic and Molecular Characterization of the Caenorhabditis elegans Gene, mel-26, a Postmeiotic Negative Regulator of MEI-1, a Meiotic-Specific Spindle Component

Genetics ◽

10.1093/genetics/150.1.119 ◽

1998 ◽

Vol 150 (1) ◽

pp. 119-128

Author(s):

M Rhys Dow ◽

Paul E Mains

Keyword(s):

Caenorhabditis Elegans ◽

Protein Interactions ◽

Negative Regulator ◽

Meiotic Spindle ◽

Loss Of Function ◽

Protein Protein Interactions ◽

Gain Of Function ◽

Recessive Mutations ◽

Female Germline

Abstract We have previously described the gene mei-1, which encodes an essential component of the Caenorhabditis elegans meiotic spindle. When ectopically expressed after the completion of meiosis, mei-1 protein disrupts the function of the mitotic cleavage spindles. In this article, we describe the cloning and the further genetic characterization of mel-26, a postmeiotic negative regulator of mei-1. mel-26 was originally identified by a gain-of-function mutation. We have reverted this mutation to a loss-of-function allele, which has recessive phenotypes identical to the dominant defects of its gain-of-function parent. Both the dominant and recessive mutations of mel-26 result in mei-1 protein ectopically localized in mitotic spindles and centrosomes, leading to small and misoriented cleavage spindles. The loss-of-function mutation was used to clone mel-26 by transformation rescue. As suggested by genetic results indicating that mel-26 is required only maternally, mel-26 mRNA was expressed predominantly in the female germline. The gene encodes a protein that includes the BTB motif, which is thought to play a role in protein-protein interactions.

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