Coilin Is Essential for Cajal Body Organization in Drosophila melanogaster

Cajal bodies (CBs) are nuclear organelles that occur in a variety of organisms, including vertebrates, insects, and plants. They are most often identified with antibodies against the marker protein coilin. Because the amino acid sequence of coilin is not strongly conserved evolutionarily, coilin orthologues have been difficult to recognize by homology search. Here, we report the identification of Drosophila melanogaster coilin and describe its distribution in tissues of the fly. Surprisingly, we found coilin not only in CBs but also in histone locus bodies (HLBs), calling into question the use of coilin as an exclusive marker for CBs. We analyzed two null mutants in the coilin gene and a piggyBac insertion mutant, which leads to specific loss of coilin from the germline. All three mutants are homozygous viable and fertile. Cells that lack coilin also lack distinct foci of other CB markers, including fibrillarin, the survival motor neuron (SMN) protein, U2 small nuclear RNA (snRNA), U5 snRNA, and the small CB-specific (sca) RNA U85. However, HLBs are not obviously affected in coilin-null flies. Thus, coilin is required for normal CB organization in Drosophila but is not essential for viability or production of functional gametes.

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The Drosophila melanogaster Cajal body

The Journal of Cell Biology ◽

10.1083/jcb.200511038 ◽

2006 ◽

Vol 172 (6) ◽

pp. 875-884 ◽

Cited By ~ 129

Author(s):

Ji-Long Liu ◽

Christine Murphy ◽

Michael Buszczak ◽

Sarah Clatterbuck ◽

Robyn Goodman ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Motor Neurons ◽

Nuclear Body ◽

Nuclear Bodies ◽

Cajal Body ◽

Small Nuclear Rna ◽

Nuclear Rna ◽

Survival Of Motor Neurons ◽

And Function ◽

Histone Locus

Cajal bodies (CBs) are nuclear organelles that are usually identified by the marker protein p80-coilin. Because no orthologue of coilin is known in Drosophila melanogaster, we identified D. melanogaster CBs using probes for other components that are relatively diagnostic for CBs in vertebrate cells. U85 small CB–specific RNA, U2 small nuclear RNA, the survival of motor neurons protein, and fibrillarin occur together in a nuclear body that is closely associated with the nucleolus. Based on its similarity to CBs in other organisms, we refer to this structure as the D. melanogaster CB. Surprisingly, the D. melanogaster U7 small nuclear RNP resides in a separate nuclear body, which we call the histone locus body (HLB). The HLB is invariably colocalized with the histone gene locus. Thus, canonical CB components are distributed into at least two nuclear bodies in D. melanogaster. The identification of these nuclear bodies now permits a broad range of questions to be asked about CB structure and function in a genetically tractable organism.

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RNA polymerase II condensate formation and association with Cajal and histone locus bodies in living human cells

10.1101/2020.12.25.424380 ◽

2020 ◽

Author(s):

Takashi Imada ◽

Takeshi Shimi ◽

Ai Kaiho ◽

Yasushi Saeki ◽

Hiroshi Kimura

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Gene Clusters ◽

Nuclear Bodies ◽

Histone Genes ◽

Small Nuclear Rna ◽

Pol Ii ◽

Histone Locus Bodies ◽

Dynamic Structures ◽

Histone Locus

ABSTRACTIn eukaryotic nuclei, a number of phase-separated nuclear bodies (NBs) are present. RNA polymerase II (Pol II) is the main player in transcription and forms large condensates in addition to localizing at numerous transcription foci. Cajal bodies (CBs) and histone locus bodies (HLBs) are NBs that are involved in transcriptional and post-transcriptional regulation of small nuclear RNA and histone genes. By live-cell imaging using human HCT116 cells, we here show that Pol II condensates (PCs) nucleated near CBs and HLBs, and the number of PCs increased during S phase concomitantly with the activation period of histone genes. Ternary PC–CB– HLB associates were formed via three pathways: nucleation of PCs and HLBs near CBs, interaction between preformed PC–HLBs with CBs, and nucleation of PCs near preformed CB– HLBs. Coilin knockout increased the co-localization rate between PCs and HLBs, whereas the number, nucleation timing, and phosphorylation status of PCs remained unchanged. Depletion of PCs did not affect CBs and HLBs. Treatment with 1,6-hexanediol revealed that PCs were more liquid-like than CBs and HLBs. Thus, PCs are dynamic structures often nucleated following the activation of gene clusters associated with other NBs. (187 words)

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Composition of the Survival Motor Neuron (SMN) complex in Drosophila melanogaster

10.1101/468223 ◽

2018 ◽

Author(s):

A. Gregory Matera ◽

Amanda C. Raimer ◽

Casey A. Schmidt ◽

Jo A. Kelly ◽

Gaith N. Droby ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Motor Neuron ◽

Muscular Atrophy ◽

Structural Similarity ◽

Survival Motor Neuron ◽

Sm Proteins ◽

Native Promoter ◽

Smn Complex ◽

Biological Studies ◽

Smn Protein

AbstractSpinal Muscular Atrophy (SMA) is caused by homozygous mutations in the human survival motor neuron 1 (SMN1) gene. SMN protein has a well-characterized role in the biogenesis of small nuclear ribonucleoproteins (snRNPs), core components of the spliceosome. SMN is part of an oligomeric complex with core binding partners, collectively called Gemins. Biochemical and cell biological studies demonstrate that certain Gemins are required for proper snRNP assembly and transport. However, the precise functions of most Gemins are unknown. To gain a deeper understanding of the SMN complex in the context of metazoan evolution, we investigated the composition of the SMN complex in Drosophila melanogaster. Using a stable transgenic line that exclusively expresses Flag-tagged SMN from its native promoter, we previously found that Gemin2, Gemin3, Gemin5, and all nine classical Sm proteins, including Lsm10 and Lsm11, co-purify with SMN. Here, we show that CG2941 is also highly enriched in the pulldown. Reciprocal co-immunoprecipitation reveals that epitope-tagged CG2941 interacts with endogenous SMN in Schneider2 cells. Bioinformatic comparisons show that CG2941 shares sequence and structural similarity with metazoan Gemin4. Additional analysis shows that three other genes (CG14164, CG31950 and CG2371) are not orthologous to Gemins 6-7-8, respectively, as previously suggested. In D.melanogaster, CG2941 is located within an evolutionarily recent genomic triplication with two other nearly identical paralogous genes (CG32783 and CG32786). RNAi-mediated knockdown of CG2941 and its two close paralogs reveals that Gemin4 is essential for organismal viability.

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Integrator Subunit 4 is a ‘Symplekin-like’ Scaffold that Associates with INTS9/11 to Form the Integrator Cleavage Module

10.1101/172635 ◽

2017 ◽

Author(s):

Todd R. Albrecht ◽

Sergey P. Shevtsov ◽

Lauren G. Mascibroda ◽

Natoya J. Peart ◽

Iain A. Sawyer ◽

...

Keyword(s):

Rna Polymerase Ii ◽

Cajal Body ◽

Catalytic Core ◽

Histone Locus Bodies ◽

Transcriptional Regulatory ◽

Significant Homology ◽

Regulatory Complex ◽

Cleavage And Polyadenylation ◽

Histone Locus ◽

Β Sheet

AbstractIntegrator (INT) is a transcriptional regulatory complex associated with RNA polymerase II that is required for the 3’-end processing of both UsnRNAs and enhancer RNAs. Integrator subunits 9 (INTS9) and INTS11 constitute the catalytic core of INT and are paralogues of the cleavage and polyadenylation specificity factors CPSF100 and CPSF73. While CPSF73/100 are known to associate with a third protein called Symplekin, there is no paralog of Symplekin within INT raising the question of how INTS9/11 associate with the other INT subunits. Here, we have identified that INTS4 is a specific and conserved interaction partner of INTS9/11 that does not interact with either subunit individually. Although INTS4 has no significant homology with Symplekin, it possesses N-terminal HEAT repeats similar to Symplekin but also contains a β-sheet rich C-terminal region, both of which are important to bind INTS9/11. We assess three functions of INT including UsnRNA 3’-end processing, maintenance of Cajal body integrity, and formation of histone locus bodies to conclude that INTS4/9/11 are the most critical of the INT subunits for UsnRNA biogenesis. Altogether, these results indicate that INTS4/9/11 compose a heterotrimeric complex that likely represents the Integrator ‘cleavage module’ responsible for its endonucleolytic activity.

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Drosophila CG2469 Encodes a Homolog of Human CTR9 and Is Essential for Development

G3 Genes|Genome|Genetics ◽

10.1534/g3.116.035196 ◽

2016 ◽

Vol 6 (12) ◽

pp. 3849-3857 ◽

Cited By ~ 5

Author(s):

Dhananjay Chaturvedi ◽

Mayu Inaba ◽

Shane Scoggin ◽

Michael Buszczak

Keyword(s):

Germ Cell ◽

Sequence Similarity ◽

Cell Loss ◽

Germline Stem Cell ◽

Cell Nuclei ◽

Transcriptional Initiation ◽

Histone Locus Bodies ◽

Paf1 Complex ◽

Histone Locus ◽

Drosophila Cells

Abstract Conserved from yeast to humans, the Paf1 complex participates in a number of diverse processes including transcriptional initiation and polyadenylation. This complex typically includes five proteins: Paf1, Rtf1, Cdc73, Leo1, and Ctr9. Previous efforts identified clear Drosophila homologs of Paf1, Rtf1, and Cdc73 based on sequence similarity. Further work showed that these proteins help to regulate gene expression and are required for viability. To date, a Drosophila homolog of Ctr9 has remained uncharacterized. Here, we show that the gene CG2469 encodes a functional Drosophila Ctr9 homolog. Both human and Drosophila Ctr9 localize to the nuclei of Drosophila cells and appear enriched in histone locus bodies. RNAi knockdown of Drosophila Ctr9 results in a germline stem cell loss phenotype marked by defects in the morphology of germ cell nuclei. A molecular null mutation of Drosophila Ctr9 results in lethality and a human cDNA CTR9 transgene rescues this phenotype. Clonal analysis in the ovary using this null allele reveals that loss of Drosophila Ctr9 results in a reduction of global levels of histone H3 trimethylation of lysine 4 (H3K4me3), but does not compromise the maintenance of stem cells in ovaries. Given the differences between the null mutant and RNAi knockdown phenotypes, the germ cell defects caused by RNAi likely result from the combined loss of Drosophila Ctr9 and other unidentified genes. These data provide further evidence that the function of this Paf1 complex component is conserved across species.

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The influence of storage parameters on measurement of survival motor neuron (SMN) protein levels: Implications for pre-clinical studies and clinical trials for spinal muscular atrophy

Neuromuscular Disorders ◽

10.1016/j.nmd.2014.05.013 ◽

2014 ◽

Vol 24 (11) ◽

pp. 973-977 ◽

Cited By ~ 4

Author(s):

Gillian Hunter ◽

Sarah L. Roche ◽

Eilidh Somers ◽

Heidi R. Fuller ◽

Thomas H. Gillingwater

Keyword(s):

Clinical Trials ◽

Spinal Muscular Atrophy ◽

Motor Neuron ◽

Clinical Studies ◽

Muscular Atrophy ◽

Survival Motor Neuron ◽

Protein Levels ◽

Smn Protein

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Composition of the Survival Motor Neuron (SMN) Complex in Drosophila melanogaster

G3 Genes|Genome|Genetics ◽

10.1534/g3.118.200874 ◽

2018 ◽

pp. g3.200874.2018 ◽

Cited By ~ 4

Author(s):

A. Gregory Matera ◽

Amanda C. Raimer ◽

Casey A. Schmidt ◽

Jo A. Kelly ◽

Gaith N. Droby ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Motor Neuron ◽

Survival Motor Neuron ◽

Smn Complex

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Survival motor neuron (SMN) protein in the spinal anterior horn cells of patients with sporadic amyotrophic lateral sclerosis

Brain Research ◽

10.1016/j.brainres.2010.11.070 ◽

2011 ◽

Vol 1372 ◽

pp. 152-159 ◽

Cited By ~ 6

Author(s):

Yingshan Piao ◽

Tomoyo Hashimoto ◽

Sachiko Takahama ◽

Akiyoshi Kakita ◽

Takashi Komori ◽

...

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Motor Neuron ◽

Survival Motor Neuron ◽

Anterior Horn ◽

Sporadic Amyotrophic Lateral Sclerosis ◽

Anterior Horn Cells ◽

Smn Protein ◽

Lateral Sclerosis

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Mitochondrial defects in the respiratory complex I contribute to impaired translational initiation via ROS and energy homeostasis in SMA motor neurons

Acta Neuropathologica Communications ◽

10.1186/s40478-020-01101-6 ◽

2020 ◽

Vol 8 (1) ◽

Author(s):

Maximilian Paul Thelen ◽

Brunhilde Wirth ◽

Min Jeong Kye

Keyword(s):

Protein Synthesis ◽

Motor Neuron ◽

Motor Neurons ◽

Complex I ◽

Energy Homeostasis ◽

Muscular Atrophy ◽

Survival Motor Neuron ◽

Translational Initiation ◽

Protein Levels ◽

Smn Protein

AbstractSpinal muscular atrophy (SMA) is a neuromuscular disease characterized by loss of lower motor neurons, which leads to proximal muscle weakness and atrophy. SMA is caused by reduced survival motor neuron (SMN) protein levels due to biallelic deletions or mutations in the SMN1 gene. When SMN levels fall under a certain threshold, a plethora of cellular pathways are disturbed, including RNA processing, protein synthesis, metabolic defects, and mitochondrial function. Dysfunctional mitochondria can harm cells by decreased ATP production and increased oxidative stress due to elevated cellular levels of reactive oxygen species (ROS). Since neurons mainly produce energy via mitochondrial oxidative phosphorylation, restoring metabolic/oxidative homeostasis might rescue SMA pathology. Here, we report, based on proteome analysis, that SMA motor neurons show disturbed energy homeostasis due to dysfunction of mitochondrial complex I. This results in a lower basal ATP concentration and higher ROS production that causes an increase of protein carbonylation and impaired protein synthesis in SMA motor neurons. Counteracting these cellular impairments with pyruvate reduces elevated ROS levels, increases ATP and SMN protein levels in SMA motor neurons. Furthermore, we found that pyruvate-mediated SMN protein synthesis is mTOR-dependent. Most importantly, we showed that ROS regulates protein synthesis at the translational initiation step, which is impaired in SMA. As many neuropathies share pathological phenotypes such as dysfunctional mitochondria, excessive ROS, and impaired protein synthesis, our findings suggest new molecular interactions among these pathways. Additionally, counteracting these impairments by reducing ROS and increasing ATP might be beneficial for motor neuron survival in SMA patients.

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