RESUMO
As photosynthetic organisms, plants need to prevent irreversible UV-induced DNA lesions. Through an unbiased, genome-wide approach, we have uncovered a previously unrecognized interplay between Global Genome Repair and small interfering RNAs (siRNAs) in the recognition of DNA photoproducts, prevalently in intergenic regions. Genetic and biochemical approaches indicate that, upon UV irradiation, the DNA DAMAGE-BINDING PROTEIN 2 (DDB2) and ARGONAUTE 1 (AGO1) of Arabidopsis thaliana form a chromatin-bound complex together with 21-nt siRNAs, which likely facilitates recognition of DNA damages in an RNA/DNA complementary strand-specific manner. The biogenesis of photoproduct-associated siRNAs involves the noncanonical, concerted action of RNA POLYMERASE IV, RNA-DEPENDENT RNA POLYMERASE-2, and DICER-LIKE-4. Furthermore, the chromatin association/dissociation of the DDB2-AGO1 complex is under the control of siRNA abundance and DNA damage signaling. These findings reveal unexpected nuclear functions for DCL4 and AGO1, and shed light on the interplay between small RNAs and DNA repair recognition factors at damaged sites.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas Argonautas/metabolismo , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , RNA de Plantas/metabolismo , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Proteínas Argonautas/genética , Cromatina/metabolismo , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Mutação , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/efeitos da radiação , Plantas Geneticamente Modificadas , Dímeros de Pirimidina/genética , Dímeros de Pirimidina/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo , Raios UltravioletaRESUMO
As obligate photosynthetic organisms plants are particularly exposed to the damaging effects of excess light and ultraviolet wavelengths, which can impact genome and epigenome dynamics by inducing DNA sequence and chromatin alterations. DNA DAMAGE-BINDING PROTEIN 2 (DDB2) is the main factor involved in the recognition of UV-induced DNA lesions during Global Genome Repair (GGR) in mammals and in plants. 1 In a recent study we reported that, in Arabidopsis, loss of DDB2 function alters DNA methylation patterns at many repeat loci and protein coding genes. We demonstrated that DDB2 acts in a complex with ARGONAUTE 4 (AGO4) to control de novo DNA methylation via the modulation of the local abundance of 24-nt small interfering RNAs (siRNAs). In addition, we found that DDB2 negatively regulates the expression of REPRESSOR OF SILENCING 1 (ROS1), a primary factor required for active DNA demethylation. Here we report that depletions of cognate GGR factors also lead to alterations of DNA methylation profiles at particular loci. Taken together, these findings reveal an interplay between GGR factors and DNA methylation patterns.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Metilação de DNA/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Reparo do DNA/genética , Reparo do DNA/fisiologia , Genoma de Planta/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , RNA Interferente Pequeno/genéticaRESUMO
In eukaryotes, DNA repair pathways help to maintain genome integrity and epigenomic patterns. However, the factors at the nexus of DNA repair and chromatin modification/remodeling remain poorly characterized. Here, we uncover a previously unrecognized interplay between the DNA repair factor DNA DAMAGE BINDING PROTEIN2 (DDB2) and the DNA methylation machinery in Arabidopsis thaliana Loss-of-function mutation in DDB2 leads to genome-wide DNA methylation alterations. Genetic and biochemical evidence indicate that at many repeat loci, DDB2 influences de novo DNA methylation by interacting with ARGONAUTE4 and by controlling the local abundance of 24-nucleotide short interfering RNAs (siRNAs). We also show that DDB2 regulates active DNA demethylation mediated by REPRESSOR OF SILENCING1 and DEMETER LIKE3. Together, these findings reveal a role for the DNA repair factor DDB2 in shaping the Arabidopsis DNA methylation landscape in the absence of applied genotoxic stress.
RESUMO
During infection, Beet necrotic yellow vein virus (BNYVV) particles localize transiently to the cytosolic surfaces of mitochondria. To understand the molecular basis and significance of this localization, we analyzed the targeting and membrane insertion properties of the viral proteins. ORF1 of BNYVV RNA-2 encodes the 21-kDa major coat protein, while ORF2 codes for a 75-kDa minor coat protein (P75) by readthrough of the ORF1 stop codon. Bioinformatic analysis highlighted a putative mitochondrial targeting sequence (MTS) as well as a major (TM1) and two minor (TM3 and TM4) transmembrane regions in the N-terminal part of the P75 readthrough domain. Deletion and gain-of-function analyses based on the localization of green fluorescent protein (GFP) fusions showed that the MTS was able to direct a reporter protein to mitochondria but that the protein was not persistently anchored to the organelles. GFP fused either to MTS and TM1 or to MTS and TM3-TM4 efficiently and specifically associated with mitochondria in vivo. The actual role of the individual domains in the interaction with the mitochondria seemed to be determined by the folding of P75. Anchoring assays to the outer membranes of isolated mitochondria, together with in vivo data, suggest that the TM3-TM4 domain is the membrane anchor in the context of full-length P75. All of the domains involved in mitochondrial targeting and anchoring were also indispensable for encapsidation, suggesting that the assembly of BNYVV particles occurs on mitochondria. Further data show that virions are subsequently released from mitochondria and accumulate in the cytosol.