Regulation of Commissureless by the Ubiquitin Ligase DNedd4 Is Required for Neuromuscular Synaptogenesis in Drosophila melanogaster

ABSTRACT Muscle synaptogenesis in Drosophila melanogaster requires endocytosis of Commissureless (Comm), a binding partner for the ubiquitin ligase dNedd4. We investigated whether dNedd4 and ubiquitination mediate this process. Here we show that Comm is expressed in intracellular vesicles in the muscle, whereas Comm bearing mutations in the two PY motifs (L/PPXY) responsible for dNedd4 binding [Comm(2PY→AY)], or bearing Lys→Arg mutations in all Lys residues that serve as ubiquitin acceptor sites [Comm(10K→R)], localize to the muscle surface, suggesting they cannot endocytose. Accordingly, aberrant muscle innervation is observed in the Comm(2PY→AY) and Comm(10K→R) mutants expressed early in muscle development. Similar muscle surface accumulation of Comm and innervation defects are observed when dNedd4 is knocked down by double-stranded RNA interference in the muscle, in dNedd4 heterozygote larvae, or in muscles overexpressing catalytically inactive dNedd4. Expression of the Comm mutants fused to a single ubiquitin that cannot be polyubiquitinated and mimics monoubiquitination [Comm(2PY→AY)-monoUb or Comm(10K→R)-monoUb] prevents the defects in both Comm endocytosis and synaptogenesis, suggesting that monoubiquitination is sufficient for Comm endocytosis in muscles. Expression of the Comm mutants later in muscle development, after synaptic innervation, has no effect. These results demonstrate that dNedd4 and ubiquitination are required for Commissureless endocytosis and proper neuromuscular synaptogenesis.

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The FBXO7 homologue nutcracker and binding partner PI31 in Drosophila melanogaster models of Parkinson’s disease

Genome ◽

10.1139/gen-2016-0087 ◽

2017 ◽

Vol 60 (1) ◽

pp. 46-54 ◽

Cited By ~ 5

Author(s):

Eric M. Merzetti ◽

Lindsay A. Dolomount ◽

Brian E. Staveley

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Drosophila Melanogaster ◽

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

Severe Form ◽

Altered Expression ◽

Binding Partner ◽

Locomotor Control ◽

Dependent Model

Parkinsonian-pyramidal syndrome (PPS) is an early onset form of Parkinson’s disease (PD) that shows degeneration of the extrapyramidal region of the brain to result in a severe form of PD. The toxic protein build-up has been implicated in the onset of PPS. Protein removal is mediated by an intracellular proteasome complex: an E3 ubiquitin ligase, the targeting component, is essential for function. FBXO7 encodes the F-box component of the SCF E3 ubiquitin ligase linked to familial forms of PPS. The Drosophila melanogaster homologue nutcracker (ntc) and a binding partner, PI31, have been shown to be active in proteasome function. We show that altered expression of either ntc or PI31 in dopaminergic neurons leads to a decrease in longevity and locomotor ability, phenotypes both associated with models of PD. Furthermore, expression of ntc-RNAi in an established α-synuclein-dependent model of PD rescues the phenotypes of diminished longevity and locomotor control.

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Binding of dsRNA by D. melanogaster Dicer-2 is substrate-dependent and regulated by Loquacious-PD

10.1101/2020.11.19.390161 ◽

2020 ◽

Author(s):

M. Jonely ◽

R. K. Singh ◽

B. L. Bass ◽

R. Noriega

Keyword(s):

Drosophila Melanogaster ◽

Rna Interference ◽

Molecular Recognition ◽

Rna Binding ◽

Regulatory Proteins ◽

Dependent Manner ◽

Biochemical Activity ◽

Double Stranded Rna ◽

Molecular Scale ◽

Local Binding

ABSTRACTDrosophila melanogaster Dicer-2 is a large, multidomain protein that cleaves double-stranded RNA (dsRNA) into small interfering RNAs in a terminus-dependent manner as part of the RNA interference pathway. We characterize the local binding environment involved in this substrate-selective molecular recognition event by monitoring the time-resolved photophysics of a cyanine dye linked to the dsRNA terminus. We observe substantial changes in the molecular rigidity and local freedom of motion of the probe as a function of distinct conformations of the biomolecular complex between Dicer-2 and dsRNA as a function of dsRNA termini, the presence of regulatory proteins, and the addition of a biochemical energy source (ATP) or a non-hydrolysable equivalent (ATP-γS). With a clustering analysis based solely on these molecular-scale measures of the local binding environment at the dsRNA terminus, we identify sub-populations of similar conformations that define distinct modes of molecular recognition which are correlated with biochemical activity. These observations reveal the important role of substrate-selective molecular recognition properties for proteins with multiple domains that can bind RNA, regulatory proteins, and cofactors.STATEMENT OF SIGNIFICANCEThe molecular-scale determinants of protein-RNA binding remain elusive, particularly when different subunits of a single protein confer specificity toward small structural differences of their RNA partners. An important case is that of Drosophila melanogaster Dicer-2, a critical component of the antiviral RNA interference response. Dicer-2 discriminates between double stranded RNA with blunt or 3’ overhang termini, a feature suggested to mediate recognition of “self” vs. “non-self” substrates. We study these interactions at the binding site with a fluorescent label at the RNA terminus, monitoring intramolecular and collective measures of flexibility to report on the local environment. Dicer-2 has distinct modes of molecular recognition which are regulated by accessory proteins and ATP, leading to different conformations and tuning biochemical activity.

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Optineurin links myosin VI to the Golgi complex and is involved in Golgi organization and exocytosis

The Journal of Cell Biology ◽

10.1083/jcb.200501162 ◽

2005 ◽

Vol 169 (2) ◽

pp. 285-295 ◽

Cited By ~ 275

Author(s):

Daniela A. Sahlender ◽

Rhys C. Roberts ◽

Susan D. Arden ◽

Giulietta Spudich ◽

Marcus J. Taylor ◽

...

Keyword(s):

Plasma Membrane ◽

Rna Interference ◽

Vesicular Stomatitis Virus ◽

Protein Interaction ◽

Golgi Complex ◽

Myosin Vi ◽

Binding Partner ◽

Binding Partners ◽

Vesicular Stomatitis ◽

Golgi Organization

Myosin VI plays a role in the maintenance of Golgi morphology and in exocytosis. In a yeast 2-hybrid screen we identified optineurin as a binding partner for myosin VI at the Golgi complex and confirmed this interaction in a range of protein interaction studies. Both proteins colocalize at the Golgi complex and in vesicles at the plasma membrane. When optineurin is depleted from cells using RNA interference, myosin VI is lost from the Golgi complex, the Golgi is fragmented and exocytosis of vesicular stomatitis virus G-protein to the plasma membrane is dramatically reduced. Two further binding partners for optineurin have been identified: huntingtin and Rab8. We show that myosin VI and Rab8 colocalize around the Golgi complex and in vesicles at the plasma membrane and overexpression of constitutively active Rab8-Q67L recruits myosin VI onto Rab8-positive structures. These results show that optineurin links myosin VI to the Golgi complex and plays a central role in Golgi ribbon formation and exocytosis.

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Viroids as a Tool to Study RNA-Directed DNA Methylation in Plants

Cells ◽

10.3390/cells10051187 ◽

2021 ◽

Vol 10 (5) ◽

pp. 1187

Author(s):

Michael Wassenegger ◽

Athanasios Dalakouras

Keyword(s):

Dna Methylation ◽

Rna Interference ◽

Gene Silencing ◽

Plant Cells ◽

Transcriptional Gene Silencing ◽

Rnai Machinery ◽

Double Stranded Rna ◽

Post Transcriptional Gene Silencing ◽

Cumulative Amount

Viroids are plant pathogenic, circular, non-coding, single-stranded RNAs (ssRNAs). Members of the Pospiviroidae family replicate in the nucleus of plant cells through double-stranded RNA (dsRNA) intermediates, thus triggering the host’s RNA interference (RNAi) machinery. In plants, the two RNAi pillars are Post-Transcriptional Gene Silencing (PTGS) and RNA-directed DNA Methylation (RdDM), and the latter has the potential to trigger Transcriptional Gene Silencing (TGS). Over the last three decades, the employment of viroid-based systems has immensely contributed to our understanding of both of these RNAi facets. In this review, we highlight the role of Pospiviroidae in the discovery of RdDM, expound the gradual elucidation through the years of the diverse array of RdDM’s mechanistic details and propose a revised RdDM model based on the cumulative amount of evidence from viroid and non-viroid systems.

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Analysis of maxillopedia Expression Pattern and Larval Cuticular Phenotype in Wild-Type and Mutant Tribolium

Genetics ◽

10.1093/genetics/155.2.721 ◽

2000 ◽

Vol 155 (2) ◽

pp. 721-731 ◽

Cited By ~ 7

Author(s):

Teresa D Shippy ◽

Jianhua Guo ◽

Susan J Brown ◽

Richard W Beeman ◽

Robin E Denell

Keyword(s):

Rna Interference ◽

Expression Pattern ◽

Tribolium Castaneum ◽

Expression Patterns ◽

Ectopic Expression ◽

Homeotic Gene ◽

Wild Type ◽

Double Stranded Rna ◽

Similar Expression ◽

Mutant Phenotypes

Abstract The Tribolium castaneum homeotic gene maxillopedia (mxp) is the ortholog of Drosophila proboscipedia (pb). Here we describe and classify available mxp alleles. Larvae lacking all mxp function die soon after hatching, exhibiting strong transformations of maxillary and labial palps to legs. Hypomorphic mxp alleles produce less severe transformations to leg. RNA interference with maxillopedia double-stranded RNA results in phenocopies of mxp mutant phenotypes ranging from partial to complete transformations. A number of gain-of-function (GOF) mxp alleles have been isolated based on transformations of adult antennae and/or legs toward palps. Finally, we have characterized the mxp expression pattern in wild-type and mutant embryos. In normal embryos, mxp is expressed in the maxillary and labial segments, whereas ectopic expression is observed in some GOF variants. Although mxp and Pb display very similar expression patterns, pb null embryos develop normally. The mxp mutant larval phenotype in Tribolium is consistent with the hypothesis that an ancestral pb-like gene had an embryonic function that was lost in the lineage leading to Drosophila.

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The kinase activity of human Rio1 is required for final steps of cytoplasmic maturation of 40S subunits

Molecular Biology of the Cell ◽

10.1091/mbc.e11-07-0639 ◽

2012 ◽

Vol 23 (1) ◽

pp. 22-35 ◽

Cited By ~ 65

Author(s):

Barbara Widmann ◽

Franziska Wandrey ◽

Lukas Badertscher ◽

Emanuel Wyler ◽

Jens Pfannstiel ◽

...

Keyword(s):

Rna Interference ◽

Protein Kinases ◽

18S Rrna ◽

Kinase Activity ◽

Ribosomal Biogenesis ◽

Ribosomal Subunits ◽

Binding Partner ◽

Rrna Maturation ◽

Cytoplasmic Maturation

RIO proteins form a conserved family of atypical protein kinases. Humans possess three distinct RIO kinases—hRio1, hRio2, and hRio3, of which only hRio2 has been characterized with respect to its role in ribosomal biogenesis. Here we show that both hRio1 and hRio3, like hRio2, are associated with precursors of 40S ribosomal subunits in human cells. Furthermore, we demonstrate that depletion of hRio1 by RNA interference affects the last step of 18S rRNA maturation and causes defects in the recycling of several trans-acting factors (hEnp1, hRio2, hLtv1, hDim2/PNO1, and hNob1) from pre-40S subunits in the cytoplasm. Although the effects of hRio1 and hRio2 depletion are similar, we show that the two kinases are not fully interchangeable. Moreover, rescue experiments with a kinase-dead mutant of hRio1 revealed that the kinase activity of hRio1 is essential for the recycling of the endonuclease hNob1 and its binding partner hDim2 from cytoplasmic pre-40S. Kinase-dead hRio1 is trapped on pre-40S particles containing hDim2 and hNob1 but devoid of hEnp1, hLtv1, and hRio2. These data reveal a role of hRio1 in the final stages of cytoplasmic pre-40S maturation.

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Abnormal muscle development in the heldup3 mutant of Drosophila melanogaster is caused by a splicing defect affecting selected troponin I isoforms

Molecular and Cellular Biology ◽

10.1128/mcb.13.3.1433-1439.1993 ◽

1993 ◽

Vol 13 (3) ◽

pp. 1433-1439

Author(s):

J A Barbas ◽

J Galceran ◽

L Torroja ◽

A Prado ◽

A Ferrús

Keyword(s):

Drosophila Melanogaster ◽

Splice Site ◽

Muscle Development ◽

Troponin I ◽

Differential Splicing ◽

Thin Filaments ◽

Molecular Changes ◽

Splicing Defect ◽

Visible Effect ◽

Thoracic Muscles

The troponin I (TnI) gene of Drosophila melanogaster encodes a family of 10 isoforms resulting from the differential splicing of 13 exons. Four of these exons (6a1, 6a2, 6b1, and 6b2) are mutually exclusive and very similar in sequence. TnI isoforms show qualitative specificity whereby each muscle expresses a selected repertoire of them. In addition, TnI isoforms show quantitative specificity whereby each muscle expresses characteristic amounts of each isoform. In the mutant heldup3, the development of the thoracic muscles DLM, DVM, and TDT is aborted. The mutation consists of a one-nucleotide displacement of the 3' AG splice site at the intron preceding exon 6b1, resulting in the failure to produce all exon 6b1-containing TnI isoforms. These molecular changes in a constituent of the thin filaments cause the selective failure to develop the DLM, DVM, and TDT muscles while having no visible effect on other muscles wherein exon 6b1 expression is minor.

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tarsal-less is expressed as a gap gene but has no gap gene phenotype in the moth midge Clogmia albipunctata

Royal Society Open Science ◽

10.1098/rsos.180458 ◽

2018 ◽

Vol 5 (8) ◽

pp. 180458 ◽

Cited By ~ 2

Author(s):

Eva Jiménez-Guri ◽

Karl R. Wotton ◽

Johannes Jaeger

Keyword(s):

Gene Expression ◽

Drosophila Melanogaster ◽

Rna Interference ◽

Gap Gene ◽

Gap Genes ◽

Subtle Effect ◽

Bona Fide ◽

Vinegar Fly ◽

Clogmia Albipunctata ◽

Overlapping Domains

Gap genes are involved in segment determination during early development of the vinegar fly Drosophila melanogaster and other dipteran insects (flies, midges and mosquitoes). They are expressed in overlapping domains along the antero-posterior (A–P) axis of the blastoderm embryo. While gap domains cover the entire length of the A–P axis in Drosophila, there is a region in the blastoderm of the moth midge Clogmia albipunctata , which lacks canonical gap gene expression. Is a non-canonical gap gene functioning in this area? Here, we characterize tarsal-less ( tal ) in C. albipunctata . The homologue of tal in the flour beetle Tribolium castaneum (called milles-pattes, mlpt ) is a bona fide gap gene. We find that Ca-tal is expressed in the region previously reported as lacking gap gene expression. Using RNA interference, we study the interaction of Ca-tal with gap genes. We show that Ca-tal is regulated by gap genes, but only has a very subtle effect on tailless (Ca-tll), while not affecting other gap genes at all. Moreover, cuticle phenotypes of Ca-tal depleted embryos do not show any gap phenotype. We conclude that Ca-tal is expressed and regulated like a gap gene, but does not function as a gap gene in C. albipunctata .

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