RESUMEN
Genome topology is tied to R-loop formation and genome stability. However, the regulatory mechanism remains to be elucidated. By establishing a system to sense the connections between R-loops and genome topology states, we show that inhibiting DNA topoisomerase 1 (TOP1i) triggers the global increase of R-loops (called topoR-loops) and DNA damages, which are exacerbated in the DNA damage repair-compromised mutant atm. A suppressor screen identifies a mutation in POL2A, the catalytic subunit of DNA polymerase ε, rescuing the TOP1i-induced topoR-loop accumulation and genome instability in atm. Importantly we find that a highly conserved junction domain between the exonuclease and polymerase domains in POL2A is required for modulating topoR-loops near DNA replication origins and facilitating faithful DNA replication. Our results suggest that DNA replication acts in concert with genome topological states to fine-tune R-loops and thereby maintain genome integrity, revealing a likely conserved regulatory mechanism of TOP1i resistance in chemotherapy for ATM-deficient cancers.
Asunto(s)
Arabidopsis , Humanos , Arabidopsis/genética , Estructuras R-Loop/genética , ADN Polimerasa II/genética , Replicación del ADN/genética , Mutación , Daño del ADN , Inestabilidad Genómica/genéticaRESUMEN
Programmed constitutive heterochromatin silencing is essential for eukaryotic genome regulation, yet the initial step of this process is ambiguous. A large proportion of R-loops (RNA:DNA hybrids) had been unexpectedly identified within Arabidopsis pericentromeric heterochromatin with unknown functions. Through a genome-wide R-loop profiling screen, we find that DDM1 (decrease in DNA methylation 1) is the primary restrictor of pericentromeric R-loops via its RNA:DNA helicase activity. Low levels of pericentromeric R-loops resolved by DDM1 cotranscriptionally can facilitate constitutive heterochromatin silencing. Furthermore, we demonstrate that DDM1 physically excludes histone H2A variant H2A.Z and promotes H2A.W deposition for faithful heterochromatin initiation soon after R-loop clearance. The dual functions of DDM1 in R-loop resolution and H2A.Z eviction are essential for sperm nuclei structure maintenance in mature pollen. Our work unravels the cotranscriptional R-loop resolution coupled with accurate H2A variants deposition is the primary step of constitutive heterochromatin silencing in Arabidopsis, which might be conserved across eukaryotes.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Histonas/metabolismo , Heterocromatina/genética , Estructuras R-Loop , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Semillas/metabolismo , ARN , Proteínas de Unión al ADN/genética , Factores de Transcripción/metabolismoRESUMEN
An R-loop is a three-stranded chromatin structure that consists of a displaced single strand of DNA and an RNA:DNA hybrid duplex, which was thought to be a rare by-product of transcription. However, recent genome-wide data have shown that R-loops are widespread and pervasive in a variety of genomes, and a growing body of experimental evidence indicates that R-loops have both beneficial and harmful effects on an organism. To maximize benefit and avoid harm, organisms have evolved several means by which they tightly regulate R-loop levels. Here, we summarize our current understanding of the biogenesis and effects of R-loops, the mechanisms that regulate them, and methods of R-loop profiling, reviewing recent research advances on R-loops in plants. Furthermore, we provide perspectives on future research directions for R-loop biology in plants, which might lead to a more comprehensive understanding of R-loop functions in plant genome regulation and contribute to future agricultural improvements.
Asunto(s)
ADN , Estructuras R-Loop , ADN/química , ADN/genética , Cromatina , ARN/química , ARN/genética , Genoma de Planta/genéticaRESUMEN
Numerous RNAs are exported from the nucleus, abnormalities of which lead to cellular complications and diseases. How thousands of circular RNAs (circRNAs) are exported from the nucleus remains elusive. Here, we provide lines of evidence to demonstrate a link between the conserved Exportin 4 (XPO4) and nuclear export of a subset of circRNAs in metazoans. Exonic circRNAs (ecircRNAs) with higher expression levels, larger length, and lower GC content are more sensitive to XPO4 deficiency. Cellular insufficiency of XPO4 leads to nuclear circRNA accumulation, circRNA:DNA (ciR-loop) formation, linear RNA:DNA (liR-loop) buildup, and DNA damage. DDX39 known to modulate circRNA export can resolve ciR-loop, and splicing factors involved in the biogenesis of circRNAs can also affect the levels of ciR-loop. Testis and brain are two organs with high abundance of circRNAs, and insufficient XPO4 levels are detrimental, as Xpo4 heterozygous mice display male infertility and neural phenotypes. Increased levels of ciR-loop, R-loop, and DNA damage along with decreased cell numbers are observed in testis and hippocampus of Xpo4 heterozygotes. This study sheds light on the understandings of mechanism of circRNA export and reveals the significance of efficient nuclear export of circRNAs in cellular physiology.
Asunto(s)
ARN Circular , ARN , Animales , Carioferinas/genética , Carioferinas/metabolismo , Masculino , Ratones , ARN/genética , ARN/metabolismo , Empalme del ARN/genética , Factores de Empalme de ARN/metabolismo , ARN Circular/genéticaRESUMEN
R-loop is a three-stranded chromatin structure, comprising one single-stranded DNA and another DNA:RNA hybrid strand, plays various and essential biological functions in many organisms. Developing a precise, efficient, faithful, and unbiased genome-wide R-loop detection method with extensive adaptability in all organisms is at the top priority for R-loop biology. Here, we provide a straightforward and highly efficient protocol for genome-wide strand-specific R-loop profiling in various organisms. In brief, genomic DNA is extracted and fragmented by the cocktail of restriction enzymes, and then the DNA:RNA hybrids are immunoprecipitated, following by the single-stranded DNA adaptor ligation and next-generation sequencing (named as ssDRIP-seq). Coupling with a straightforward and step-by-step bioinformatic pipeline, this method can provide high resolution and comprehensive strand-specific information for R-loop formation. ssDRIP-seq has been successfully applied for detecting R-loops from prokaryotes such as E. coli, to eukaryotes such as S. cerevisiae, mammalian cell culture and tissues, as well as plants Arabidopsis and rice, with high reproducibility and sensitivity.
Asunto(s)
ADN de Cadena Simple , Estructuras R-Loop , Animales , ADN/genética , Escherichia coli/genética , Mamíferos/genética , ARN/genética , Reproducibilidad de los Resultados , Saccharomyces cerevisiae/genéticaRESUMEN
Intronic transposable elements (TEs) comprise a large proportion in eukaryotic genomes, but how they regulate the host genes remains to be explored. Our forward genetic screen disclosed the plant-specific RNA polymerases IV and V in suppressing intronic TE-mediated cryptic transcription initiation of a chimeric transcripts at FLC (FLCTE ). Initiation of FLCTE transcription is blocked by the locally formed intronic heterochromatin, which is directly associated with RNA Pol V to inhibit the entry of RNA Pol II and the occupancy of H3K4 methylation. Genome-wide Pol II Ser5p native elongation transcription sequencing revealed that a significant number of intronic heterochromatin-containing genes undergo this mechanism. This study sheds light on deeply understanding the function of intronic heterochromatin on host genes expression in eukaryotic genome.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Elementos Transponibles de ADN/genética , ARN Polimerasas Dirigidas por ADN/genética , Genoma de Planta/genética , Heterocromatina/genética , Intrones/genética , Proteínas de Dominio MADS/genética , Metilación de ADN , Proteínas de Plantas/genéticaRESUMEN
The R-loop, composed of a DNA-RNA hybrid and the displaced single-stranded DNA, regulates diverse cellular processes. However, how cellular R-loops are recognized remains poorly understood. Here, we report the discovery of the evolutionally conserved ALBA proteins (AtALBA1 and AtALBA2) functioning as the genic R-loop readers in Arabidopsis. While AtALBA1 binds to the DNA-RNA hybrid, AtALBA2 associates with single-stranded DNA in the R-loops in vitro. In vivo, these two proteins interact and colocalize in the nucleus, where they preferentially bind to genic regions with active epigenetic marks in an R-loop-dependent manner. Depletion of AtALBA1 or AtALBA2 results in hypersensitivity of plants to DNA damaging agents. The formation of DNA breaks in alba mutants originates from unprotected R-loops. Our results reveal that the AtALBA1 and AtALBA2 protein complex is the genic R-loop reader crucial for genome stability in Arabidopsis.