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RNA (New York, N.Y.) May 2023Eukaryotic mRNAs are modified at the 5' end with a methylated guanosine (mG) that is attached to the transcription start site (TSS) nucleotide. The TSS nucleotide is...
Eukaryotic mRNAs are modified at the 5' end with a methylated guanosine (mG) that is attached to the transcription start site (TSS) nucleotide. The TSS nucleotide is 2'--methylated (Nm) by CMTR1 in organisms ranging from insects to human. In mammals, the TSS adenosine can be further -methylated by RNA polymerase II phosphorylated CTD-interacting factor 1 (PCIF1) to create mAm. Curiously, the fly ortholog of mammalian PCIF1 is demonstrated to be catalytic-dead, and its functions are not known. Here, we show that mutant flies display a reduced fertility which is particularly marked in females. Deep sequencing analysis of mutant ovaries revealed transcriptome changes with a notable increase in expression of genes belonging to the mitochondrial ATP synthetase complex. Furthermore, the Pcif1 protein is distributed along euchromatic regions of polytene chromosomes, and the mutation behaved as a modifier of position-effect-variegation (PEV) suppressing the heterochromatin-dependent silencing of the gene. Similar or stronger changes in the transcriptome and PEV phenotype were observed in flies that expressed a cytosolic version of Pcif1. These results point to a nuclear cotranscriptional gene regulatory role for the catalytic-dead fly Pcif1 that is probably based on its conserved ability to interact with the RNA polymerase II carboxy-terminal domain.
Topics: Female; Animals; Humans; Drosophila; RNA Polymerase II; Fertility; Transcriptome; Nucleotides; Drosophila melanogaster; Mammals; Nuclear Proteins; Adaptor Proteins, Signal Transducing
PubMed: 36754578
DOI: 10.1261/rna.079192.122 -
Cells Dec 2022The pericentromeric heterochromatin is largely composed of repetitive sequences, making it difficult to analyze with standard molecular biological methods. At the same...
The pericentromeric heterochromatin is largely composed of repetitive sequences, making it difficult to analyze with standard molecular biological methods. At the same time, it carries many functional elements with poorly understood mechanisms of action. The search for new experimental models for the analysis of heterochromatin is an urgent task. In this work, we used the mutation, which suppresses the underreplication of all types of repeated sequences, to analyze heterochromatin regions in polytene chromosomes of . In the background, we discovered and described in detail a new inversion, , which arose on a chromosome already carrying the inversion and transferred a large part of X chromosome heterochromatin, including the nucleolar organizer to a new euchromatic environment. Using nanopore sequencing and FISH, we have identified the eu- and heterochromatin breakpoints of . The combination of the new inversion and the mutation provides a promising tool for studies of X chromosome heterochromatin structure, nucleolar organization, and the nucleolar dominance phenomenon. In particular, we found that, with the complete polytenization of rDNA repeats, the nucleolus consists of a cloud-like structure corresponding to the classical nucleolus of polytene chromosomes, as well as an unusual intrachromosomal structure containing alternating transcriptionally active and inactive regions.
Topics: Animals; Drosophila melanogaster; Heterochromatin; X Chromosome; Repetitive Sequences, Nucleic Acid; Nucleolus Organizer Region; Carrier Proteins; Drosophila Proteins
PubMed: 36497131
DOI: 10.3390/cells11233872 -
ELife Dec 2022Asynchronous replication of chromosome domains during S phase is essential for eukaryotic genome function, but the mechanisms establishing which domains replicate early...
Asynchronous replication of chromosome domains during S phase is essential for eukaryotic genome function, but the mechanisms establishing which domains replicate early versus late in different cell types remain incompletely understood. Intercalary heterochromatin domains replicate very late in both diploid chromosomes of dividing cells and in endoreplicating polytene chromosomes where they are also underreplicated. SNF2-related factor SUUR imparts locus-specific underreplication of polytene chromosomes. SUUR negatively regulates DNA replication fork progression; however, its mechanism of action remains obscure. Here, we developed a novel method termed MS-Enabled Rapid protein Complex Identification (MERCI) to isolate a stable stoichiometric native complex SUMM4 that comprises SUUR and a chromatin boundary protein Mod(Mdg4)-67.2. Mod(Mdg4) stimulates SUUR ATPase activity and is required for a normal spatiotemporal distribution of SUUR in vivo. SUUR and Mod(Mdg4)-67.2 together mediate the activities of insulator that prevent certain enhancer-promoter interactions and establish euchromatin-heterochromatin barriers in the genome. Furthermore, or ) mutations reverse underreplication of intercalary heterochromatin. Thus, SUMM4 can impart late replication of intercalary heterochromatin by attenuating the progression of replication forks through euchromatin/heterochromatin boundaries. Our findings implicate a SNF2 family ATP-dependent motor protein SUUR in the insulator function, reveal that DNA replication can be delayed by a chromatin barrier, and uncover a critical role for architectural proteins in replication control. They suggest a mechanism for the establishment of late replication that does not depend on an asynchronous firing of late replication origins.
Topics: Animals; Drosophila; DNA-Binding Proteins; Heterochromatin; Drosophila melanogaster; Drosophila Proteins; Euchromatin; Chromatin; DNA Replication
PubMed: 36458689
DOI: 10.7554/eLife.81828 -
Insects Oct 2022One of the most popular tools for species discovery and resolution is the DNA barcode, typically based on the cytochrome oxidase I (COI) gene. However, other non-genic...
One of the most popular tools for species discovery and resolution is the DNA barcode, typically based on the cytochrome oxidase I (COI) gene. However, other non-genic barcodes are available for Diptera. The banding sequence of polytene chromosomes in some dipteran cells, particularly of the larval silk glands, can provide a unique species barcode. We used the sequence of bands to reveal a new species of black fly in the () species group from California, USA. To further characterize the species and provide more integrated taxonomy, we morphologically described all life stages above the egg, formally named the species n. sp., and provided a conventional COI barcode. The COI barcode confirmed the chromosomal and morphological evidence that the species is a new member of the group, and enabled identification of the larva and female, which are structurally similar to those of other species. The chromosomal barcode shows that this species has the most rearranged complement, compared with the eight other North American members of its species group, with up to 12 times the number of fixed rearrangements. Up to six chromosomal rearrangements, including autosomal polymorphisms and sex-linked phenomena, are shared with other members of the group. The most unique and conspicuous chromosomal feature of this new species is a large, pale-staining chromocenter from which the six chromosomal arms radiate. The distribution of this univoltine species in lowland rivers of California's Central Valley could make it vulnerable, given climate change and increasing land development.
PubMed: 36292851
DOI: 10.3390/insects13100903 -
PLoS Genetics Oct 2022Meiosis in males of higher dipterans is achiasmate. In their spermatocytes, pairing of homologs into bivalent chromosomes does not include synaptonemal complex and...
Meiosis in males of higher dipterans is achiasmate. In their spermatocytes, pairing of homologs into bivalent chromosomes does not include synaptonemal complex and crossover formation. While crossovers preserve homolog conjunction until anaphase I during canonical meiosis, an alternative system is used in dipteran males. Mutant screening in Drosophila melanogaster has identified teflon (tef) as being required specifically for alternative homolog conjunction (AHC) of autosomal bivalents. The additional known AHC genes, snm, uno and mnm, are needed for the conjunction of autosomal homologs and of sex chromosomes. Here, we have analyzed the pattern of TEF protein expression. TEF is present in early spermatocytes but cannot be detected on bivalents at the onset of the first meiotic division, in contrast to SNM, UNO and MNM (SUM). TEF binds to polytene chromosomes in larval salivary glands, recruits MNM by direct interaction and thereby, indirectly, also SNM and UNO. However, chromosomal SUM association is not entirely dependent on TEF, and residual autosome conjunction occurs in tef null mutant spermatocytes. The higher tef requirement for autosomal conjunction is likely linked to the quantitative difference in the amount of SUM protein that provides conjunction of autosomes and sex chromosomes, respectively. During normal meiosis, SUM proteins are far more abundant on sex chromosomes compared to autosomes. Beyond promoting SUM recruitment, TEF has a stabilizing effect on SUM proteins. Increased SUM causes excess conjunction and consequential chromosome missegregation during meiosis I after co-overexpression. Similarly, expression of SUM without TEF, and even more potently with TEF, interferes with chromosome segregation during anaphase of mitotic divisions in somatic cells, suggesting that the known AHC proteins are sufficient for establishment of ectopic chromosome conjunction. Overall, our findings suggest that TEF promotes alternative homolog conjunction during male meiosis without being part of the final physical linkage between chromosomes.
Topics: Animals; Male; Drosophila melanogaster; Drosophila; Drosophila Proteins; Polytetrafluoroethylene; Chromosome Segregation; Meiosis; Sex Chromosomes; Chromosome Pairing
PubMed: 36251690
DOI: 10.1371/journal.pgen.1010469 -
PLoS Genetics Oct 2022Chromatin insulators are responsible for orchestrating long-range interactions between enhancers and promoters throughout the genome and align with the boundaries of...
Chromatin insulators are responsible for orchestrating long-range interactions between enhancers and promoters throughout the genome and align with the boundaries of Topologically Associating Domains (TADs). Here, we demonstrate an association between gypsy insulator proteins and the phosphorylated histone variant H2Av (γH2Av), normally a marker of DNA double strand breaks. Gypsy insulator components colocalize with γH2Av throughout the genome, in polytene chromosomes and in diploid cells in which Chromatin IP data shows it is enriched at TAD boundaries. Mutation of insulator components su(Hw) and Cp190 results in a significant reduction in γH2Av levels in chromatin and phosphatase inhibition strengthens the association between insulator components and γH2Av and rescues γH2Av localization in insulator mutants. We also show that γH2Av, but not H2Av, is a component of insulator bodies, which are protein condensates that form during osmotic stress. Phosphatase activity is required for insulator body dissolution after stress recovery. Together, our results implicate the H2A variant with a novel mechanism of insulator function and boundary formation.
Topics: Animals; Chromatin; DNA; Drosophila; Drosophila Proteins; Drosophila melanogaster; Histones; Insulator Elements; Microtubule-Associated Proteins; Nuclear Proteins; Phosphoric Monoester Hydrolases; Polytene Chromosomes
PubMed: 36197938
DOI: 10.1371/journal.pgen.1010396 -
Biology Sep 2022The ultrastructure of antipodal cells of the embryo sac was studied at different stages of differentiation and programmed cell death. The importance of cell function in...
The ultrastructure of antipodal cells of the embryo sac was studied at different stages of differentiation and programmed cell death. The importance of cell function in the antipodal complex is evidenced by the fact that it is fully formed before double fertilization, past the stages of proliferation of three initial cells, and several rounds of genome endoreduplication during differentiation. In this study, we showed that the actively synthesizing organelles, the granular reticulum, and Golgi apparatus, alter their structure during differentiation and death. The polymorphism of the shape of the mitochondria and plastids was demonstrated. For the first time, the actin filaments of the cytoskeleton and numerous multivesicular bodies associated with the plasma membrane were detected in the cytoplasm. The transfer of cytoplasm and organelles between antipodal cells and into the coenocyte of the endosperm was confirmed. DNA breaks and the release of cytochrome c at various stages of death were revealed. To understand the function of the antipodal cells, a quantitative PCR analysis of the expression of wheat genes involved in protective, antistress, and metabolic processes was carried out. We found that gene expression in the antipodal cell fraction was increased compared with that in the whole embryo sac. On the basis of the data, we assume that antipodal cells produce both nutrients and numerous antistress factors that ensure the normal development of the endosperm of the grain, which, in turn, further ensures the development of the embryo.
PubMed: 36138819
DOI: 10.3390/biology11091340 -
PloS One 2022The ribosomal protein uL11 is located at the basis of the ribosome P-stalk and plays a paramount role in translational efficiency. In addition, no mutant for uL11 is...
The ribosomal protein uL11 is located at the basis of the ribosome P-stalk and plays a paramount role in translational efficiency. In addition, no mutant for uL11 is available suggesting that this gene is haplo-insufficient as many other Ribosomal Protein Genes (RPGs). We have previously shown that overexpression of Drosophila melanogaster uL11 enhances the transcription of many RPGs and Ribosomal Biogenesis genes (RiBis) suggesting that uL11 might globally regulate the level of translation through its transcriptional activity. Moreover, uL11 trimethylated on lysine 3 (uL11K3me3) interacts with the chromodomain of the Enhancer of Polycomb and Trithorax Corto, and both proteins co-localize with RNA Polymerase II at many sites on polytene chromosomes. These data have led to the hypothesis that the N-terminal end of uL11, and more particularly the trimethylation of lysine 3, supports the extra-ribosomal activity of uL11 in transcription. To address this question, we mutated the lysine 3 codon using a CRISPR/Cas9 strategy and obtained several lysine 3 mutants. We describe here the first mutants of D. melanogaster uL11. Unexpectedly, the uL11K3A mutant, in which the lysine 3 codon is replaced by an alanine, displays a genuine Minute phenotype known to be characteristic of RPG deletions (longer development, low fertility, high lethality, thin and short bristles) whereas the uL11K3Y mutant, in which the lysine 3 codon is replaced by a tyrosine, is unaffected. In agreement, the rate of translation decreases in uL11K3A but not in uL11K3Y. Co-immunoprecipitation experiments show that the interaction between uL11 and the Corto chromodomain is impaired by both mutations. However, Histone Association Assays indicate that the mutant proteins still bind chromatin. RNA-seq analyses from wing imaginal discs show that Corto represses RPG expression whereas very few genes are deregulated in uL11 mutants. We propose that Corto, by repressing RPG expression, ensures that all ribosomal proteins are present at the correct stoichiometry, and that uL11 fine-tunes its transcriptional regulation of RPGs.
Topics: Animals; Drosophila Proteins; Lysine; Mutation; Ribosomal Proteins; Transcriptional Activation
PubMed: 35981051
DOI: 10.1371/journal.pone.0273198 -
Genetics Sep 2022In Drosophila chromosomal rearrangements can be maintained and are associated with karyotypic variability among populations from different geographic localities. The...
In Drosophila chromosomal rearrangements can be maintained and are associated with karyotypic variability among populations from different geographic localities. The abundance of variability in gene arrangements among chromosomal arms is even greater when comparing more distantly related species and the study of these chromosomal changes has provided insights into the evolutionary history of species in the genus. In addition, the sequencing of genomes of several Drosophila species has offered the opportunity to establish the global pattern of genomic evolution, at both genetic and chromosomal level. The combined approaches of comparative analysis of syntenic blocks and direct physical maps on polytene chromosomes have elucidated changes in the orientation of genomic sequences and the difference between heterochromatic and euchromatic regions. Unfortunately, the centromeric heterochromatic regions cannot be studied using the cytological maps of polytene chromosomes because they are underreplicated and therefore reside in the chromocenter. In Drosophila melanogaster, a cytological map of the heterochromatin has been elaborated using mitotic chromosomes from larval neuroblasts. In the current work, we have expanded on that mapping by producing cytological maps of the mitotic heterochromatin in an additional 10 sequenced Drosophila species. These maps highlight 2 apparently different paths, for the evolution of the pericentric heterochromatin between the subgenera Sophophora and Drosophila. One path leads toward a progressive complexity of the pericentric heterochromatin (Sophophora) and the other toward a progressive simplification (Drosophila). These maps are also useful for a better understanding how karyotypes have been altered by chromosome arm reshuffling during evolution.
Topics: Animals; Drosophila; Drosophila Proteins; Drosophila melanogaster; Heterochromatin; Polytene Chromosomes
PubMed: 35946576
DOI: 10.1093/genetics/iyac119 -
Proceedings of the National Academy of... Jun 2022Cryoelectron tomography of the cell nucleus using scanning transmission electron microscopy and deconvolution processing technology has highlighted a large-scale, 100-...
Cryoelectron tomography of the cell nucleus using scanning transmission electron microscopy and deconvolution processing technology has highlighted a large-scale, 100- to 300-nm interphase chromosome structure, which is present throughout the nucleus. This study further documents and analyzes these chromosome structures. The paper is divided into four parts: 1) evidence (preliminary) for a unified interphase chromosome structure; 2) a proposed unified interphase chromosome architecture; 3) organization as chromosome territories (e.g., fitting the 46 human chromosomes into a 10-μm-diameter nucleus); and 4) structure unification into a polytene chromosome architecture and lampbrush chromosomes. Finally, the paper concludes with a living light microscopy cell study showing that the G1 nucleus contains very similar structures throughout. The main finding is that this chromosome structure appears to coil the 11-nm nucleosome fiber into a defined hollow structure, analogous to a Slinky helical spring [https://en.wikipedia.org/wiki/Slinky; motif used in Bowerman , 10, e65587 (2021)]. This Slinky architecture can be used to build chromosome territories, extended to the polytene chromosome structure, as well as to the structure of lampbrush chromosomes.
Topics: Cell Nucleus; Chromatin; Chromosomes, Human; Humans; Interphase; Nucleosomes
PubMed: 35749363
DOI: 10.1073/pnas.2119101119