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1.
Genes Dev ; 35(7-8): 483-488, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33664058

RESUMO

It is unknown how ribosomal gene (rDNA) arrays from multiple chromosomal nucleolar organizers (NORs) partition within human nucleoli. Exploration of this paradigm for chromosomal organization is complicated by the shared DNA sequence composition of five NOR-bearing acrocentric chromosome p-arms. Here, we devise a methodology for genetic manipulation of individual NORs. Efficient "scarless" genome editing of rDNA repeats is achieved on "poised" human NORs held within monochromosomal cell hybrids. Subsequent transfer to human cells introduces "active" NORs yielding readily discernible functional customized ribosomes. We reveal that ribosome biogenesis occurs entirely within constrained territories, tethered to individual NORs inside a larger nucleolus.


Assuntos
Nucléolo Celular/metabolismo , Região Organizadora do Nucléolo/genética , Região Organizadora do Nucléolo/metabolismo , Ribossomos/metabolismo , Sequência de Bases , Linhagem Celular , Nucléolo Celular/genética , Cromossomos/metabolismo , Edição de Genes , Humanos , Ribossomos/genética
2.
Genes Dev ; 33(23-24): 1688-1701, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31727772

RESUMO

Human nucleolar organizer regions (NORs), containing ribosomal gene (rDNA) arrays, are located on the p-arms of acrocentric chromosomes (HSA13-15, 21, and 22). Absence of these p-arms from genome references has hampered research on nucleolar formation. Previously, we assembled a distal junction (DJ) DNA sequence contig that abuts rDNA arrays on their telomeric side, revealing that it is shared among the acrocentrics and impacts nucleolar organization. To facilitate inclusion into genome references, we describe sequencing the DJ from all acrocentrics, including three versions of HSA21, ∼3 Mb of novel sequence. This was achieved by exploiting monochromosomal somatic cell hybrids containing single human acrocentric chromosomes with NORs that retain functional potential. Analyses revealed remarkable DJ sequence and functional conservation among human acrocentrics. Exploring chimpanzee acrocentrics, we show that "DJ-like" sequences and abutting rDNA arrays are inverted as a unit in comparison to humans. Thus, rDNA arrays and linked DJs represent a conserved functional locus. We provide direct evidence for exchanges between heterologous human acrocentric p-arms, and uncover extensive structural variation between chromosomes and among individuals. These findings lead us to revaluate the molecular definition of NORs, identify novel genomic structural variation, and provide a rationale for the distinctive chromosomal organization of NORs.


Assuntos
Cromossomos/química , Cromossomos/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Região Organizadora do Nucléolo/química , Região Organizadora do Nucléolo/genética , Animais , Sequência de Bases , Linhagem Celular , Sequência Conservada/genética , Estruturas Genéticas/genética , Variação Genética , Humanos , Células Híbridas , Camundongos , Pan troglodytes/genética
3.
Proc Natl Acad Sci U S A ; 117(19): 10368-10377, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32332163

RESUMO

Nucleoli, the sites of ribosome biogenesis and the largest structures in human nuclei, form around nucleolar organizer regions (NORs) comprising ribosomal DNA (rDNA) arrays. NORs are located on the p-arms of the five human acrocentric chromosomes. Defining the rules of engagement between these p-arms and nucleoli takes on added significance as describing the three-dimensional organization of the human genome represents a major research goal. Here we used fluorescent in situ hybridization (FISH) and immuno-FISH on metaphase chromosomes from karyotypically normal primary and hTERT-immortalized human cell lines to catalog NORs in terms of their relative rDNA content and activity status. We demonstrate that a proportion of acrocentric p-arms in cell lines and from normal human donors have no detectable rDNA. Surprisingly, we found that all NORs with detectable rDNA are active, as defined by upstream binding factor loading. We determined the nucleolar association status of all NORs during interphase, and found that nucleolar association of acrocentric p-arms can occur independently of rDNA content, suggesting that sequences elsewhere on these chromosome arms drive nucleolar association. In established cancer lines, we characterize a variety of chromosomal rearrangements involving acrocentric p-arms and observe silent, rDNA-containing NORs that are dissociated from nucleoli. In conclusion, our findings indicate that within human nuclei, positioning of all 10 acrocentric chromosomes is dictated by nucleolar association. Furthermore, these nucleolar associations are buffered against interindividual variation in the distribution of rDNA.


Assuntos
DNA Ribossômico/genética , Região Organizadora do Nucléolo/metabolismo , Região Organizadora do Nucléolo/fisiologia , Linhagem Celular , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Centrômero/fisiologia , Cromossomos Humanos/metabolismo , DNA Ribossômico/metabolismo , Genoma Humano/genética , Genoma Humano/fisiologia , Humanos , Hibridização in Situ Fluorescente/métodos , Região Organizadora do Nucléolo/genética , Ribossomos/metabolismo
4.
Elife ; 132024 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-38240312

RESUMO

Out of the several hundred copies of rRNA genes arranged in the nucleolar organizing regions (NOR) of the five human acrocentric chromosomes, ~50% remain transcriptionally inactive. NOR-associated sequences and epigenetic modifications contribute to the differential expression of rRNAs. However, the mechanism(s) controlling the dosage of active versus inactive rRNA genes within each NOR in mammals is yet to be determined. We have discovered a family of ncRNAs, SNULs (Single NUcleolus Localized RNA), which form constrained sub-nucleolar territories on individual NORs and influence rRNA expression. Individual members of the SNULs monoallelically associate with specific NOR-containing chromosomes. SNULs share sequence similarity to pre-rRNA and localize in the sub-nucleolar compartment with pre-rRNA. Finally, SNULs control rRNA expression by influencing pre-rRNA sorting to the DFC compartment and pre-rRNA processing. Our study discovered a novel class of ncRNAs influencing rRNA expression by forming constrained nucleolar territories on individual NORs.


Assuntos
Região Organizadora do Nucléolo , Precursores de RNA , Humanos , Animais , Região Organizadora do Nucléolo/genética , Região Organizadora do Nucléolo/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Cromossomos Humanos/metabolismo , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Mamíferos/genética
5.
FEBS J ; 284(23): 3977-3985, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28500793

RESUMO

Nucleoli are the sites of ribosome biogenesis and the largest membraneless subnuclear structures. They are intimately linked with growth and proliferation control and function as sensors of cellular stress. Nucleoli form around arrays of ribosomal gene (rDNA) repeats also called nucleolar organizer regions (NORs). In humans, NORs are located on the short arms of all five human acrocentric chromosomes. Multiple NORs contribute to the formation of large heterochromatin-surrounded nucleoli observed in most human cells. Here we will review recent findings about their genomic architecture. The dynamic nature of nucleoli began to be appreciated with the advent of photodynamic experiments using fluorescent protein fusions. We review more recent data on nucleoli in Xenopus germinal vesicles (GVs) which has revealed a liquid droplet-like behavior that facilitates nucleolar fusion. Further analysis in both XenopusGVs and Drosophila embryos indicates that the internal organization of nucleoli is generated by a combination of liquid-liquid phase separation and active processes involving rDNA. We will attempt to integrate these recent findings with the genomic architecture of human NORs to advance our understanding of how nucleoli form and respond to stress in human cells.


Assuntos
Nucléolo Celular/genética , DNA Ribossômico/genética , Genoma Humano/genética , Região Organizadora do Nucléolo/genética , Ribossomos/genética , Animais , Fenômenos Biofísicos , Nucléolo Celular/metabolismo , DNA Ribossômico/metabolismo , Humanos , Modelos Genéticos , Região Organizadora do Nucléolo/química , Ribossomos/metabolismo
6.
PLoS One ; 9(2): e88385, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24586322

RESUMO

Regulation of DNMT1 is critical for epigenetic control of many genes and for genome stability. Using phylogenetic analysis we characterized a block of 27 nucleotides in the 3'UTR of Dnmt1 mRNA identical between humans and Xenopus and investigated the role of the individual elements contained within it. This region contains a cytoplasmic polyadenylation element (CPE) and a Musashi binding element (MBE), with CPE binding protein 1 (CPEB1) known to bind to the former in mouse oocytes. The presence of these elements usually indicates translational control by elongation and shortening of the poly(A) tail in the cytoplasm of the oocyte and in some somatic cell types. We demonstrate for the first time cytoplasmic polyadenylation of Dnmt1 during periods of oocyte growth in mouse and during oocyte activation in Xenopus. Furthermore we show by RNA immunoprecipitation that Musashi1 (MSI1) binds to the MBE and that this element is required for polyadenylation in oocytes. As well as a role in oocytes, site-directed mutagenesis and reporter assays confirm that mutation of either the MBE or CPE reduce DNMT1 translation in somatic cells, but likely act in the same pathway: deletion of the whole conserved region has more severe effects on translation in both ES and differentiated cells. In adult cells lacking MSI1 there is a greater dependency on the CPE, with depletion of CPEB1 or CPEB4 by RNAi resulting in substantially reduced levels of endogenous DNMT1 protein and concurrent upregulation of the well characterised CPEB target mRNA cyclin B1. Our findings demonstrate that CPE- and MBE-mediated translation regulate DNMT1 expression, representing a novel mechanism of post-transcriptional control for this gene.


Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Proteínas do Tecido Nervoso/genética , Filogenia , Poliadenilação/genética , Biossíntese de Proteínas/genética , Proteínas de Ligação a RNA/genética , Animais , Sequência de Bases , Southern Blotting , Western Blotting , Galinhas , Citoplasma/metabolismo , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/metabolismo , Primers do DNA/genética , Vetores Genéticos/genética , Células HeLa , Humanos , Imunoprecipitação , Camundongos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/metabolismo , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Xenopus , Peixe-Zebra
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