Histone ubiquitylation/deubiquitylation plays a major role in the epigenetic regulation of gene e... more Histone ubiquitylation/deubiquitylation plays a major role in the epigenetic regulation of gene expression. In plants, OTLD1, a member of the ovarian tumor (OTU) deubiquitinase family, deubiquitylates histone 2B and represses the expression of genes involved in growth, cell expansion, and hormone signaling. OTLD1 lacks the intrinsic ability to bind DNA. How OTLD1, as well as most other known plant histone deubiquitinases, recognizes its target genes remains unknown. Here, we show that Arabidopsis transcription factor LSH10, a member of the ALOG protein family, interacts with OTLD1 in living plant cells. Loss-of-function LSH10 mutations relieve the OTLD1-promoted transcriptional repression of the target genes, resulting in their elevated expression, whereas recovery of the LSH10 function results in down-regulated transcription of the same genes. We show that LSH10 associates with the target gene chromatin as well as with DNA sequences in the promoter regions of the target genes. Furt...
Histone ubiquitylation/deubiquitylation plays a major role in the epigenetic regulation of gene e... more Histone ubiquitylation/deubiquitylation plays a major role in the epigenetic regulation of gene expression. In plants, OTLD1, a member of the ovarian tumor (OTU) deubiquitinase family, deubiquitylates monoubiquitylated histone 2B and represses the expression of genes involved in growth, cell expansion, and hormone signaling. Like many other histone-modifying enzymes, OTLD1 lacks the intrinsic ability to bind DNA. How OTLD1, as well as most other known plant histone deubiquitinases, is recruited specifically to the promoters of its target genes remains unknown. Here, we show that Arabidopsis transcription factor LSH10, a member of the ALOG protein family, interacts with OTLD1 in living plant cells. Loss-of-function LSH10 mutations relieve the OTLD1-promoted transcriptional repression of the target genes, resulting in their elevated expression, whereas recovery of the LSH10 function results in down-regulated transcription of the same genes. We then show that LSH10 associates directly ...
Histone monoubiquitination is associated with active chromatin and plays an important role in epi... more Histone monoubiquitination is associated with active chromatin and plays an important role in epigenetic regulation of gene expression in plants. Deubiquitinating enzymes remove the ubiquitin group from histones and thereby contribute to gene repression. The Arabidopsis thaliana genome encodes 50 deubiquitinases, yet only 2 of them-UBP26 and OTLD1, members of the USP/UBP (ubiquitin-specific protease and ubiquitin-binding protein) and OTU (ovarian tumor protease) deubiquitinase families-are known to target histones. Furthermore, UBP26 is the only plant histone deubiquitinase for which the functional role has been characterized in detail. We used gain- and loss-of-function alleles of OTLD1 to examine its role in the plant life cycle and showed that OTLD1 stimulates plant growth, increases cell size, and induces transcriptional repression of five major regulators of plant organ growth and development: GA20OX, WUS, OSR2, ARL, and ABI5 OTLD1 associated with chromatin at each of these tar...
As metabolic centers, plant organelles participate in maintenance, defense and signaling. MSH1 is... more As metabolic centers, plant organelles participate in maintenance, defense and signaling. MSH1 is a plant-specific protein involved in organellar genome stability in mitochondria and plastids. Plastid depletion of MSH1 causes heritable, non-genetic changes in development and DNA methylation. We investigated the msh1 phenotype using hemi-complementation mutants and transgene-null segregants from RNAi suppression lines to sub-compartmentalize MSH1 effects. We show that MSH1 expression is spatially regulated, specifically localizing to plastids within the epidermis and vascular parenchyma. The protein binds DNA and localizes to plastid and mitochondrial nucleoids, yet fractionation and protein:protein interactions data indicate that MSH1 also associates with the thylakoid membrane. Plastid MSH1 depletion results in variegation, abiotic stress tolerance, variable growth rate, and delayed maturity. Depletion from mitochondria results in 7-10% of plants altered in leaf morphology, heat to...
ABSTRACT Ferredoxins (Fds) are small iron-sulfur proteins, which mediate electron transfer in a w... more ABSTRACT Ferredoxins (Fds) are small iron-sulfur proteins, which mediate electron transfer in a wide range of metabolic reactions. Several intriguing observations suggest that Fds may also directly associate with RNA, thus implicating a second role for these proteins in organellar RNA metabolism. Plants contain several closely-related Fd homologs, whose members are predicted to reside within the plastids. As strong mobile electron-carriers, able to partition between the stroma and the thylakoid membranes, Fds are therefore excellent candidates to regulate the expression of plastidic genes in a redox-dependant manner. Accordingly, the translation of D1 protein in the chloroplasts is mediated by a redox-poise involving the ferredoxin-thioredoxin system. Yet, despite these suggestive evidences, RNA binding activity has not been reported for an isolated Fd protein. Here, we established the intracellular locations of the six Fd paralogs in Arabidopsis to the plastids and demonstrated that one of these proteins, AtFd6, is associated with organellar transcripts in vivo. Biochemical analyses in vitro indicated that a re-combinant purified AtFd6-His protein binds with high affinity and specificity to psbA mRNA, in a redox-dependant manner.
Group II introns are large catalytic RNAs that are found in bacteria and organellar genomes of lo... more Group II introns are large catalytic RNAs that are found in bacteria and organellar genomes of lower eukaryotes, but are particularly prevalent within mitochondria in plants, where they are present in many critical genes. The excision of plant mitochondrial introns is essential for respiratory functions, and is facilitated in vivo by various protein cofactors. Typical group II introns are classified as mobile genetic elements, consisting of the self-splicing ribozyme and its own intron-encoded maturase protein. A hallmark of maturases is that they are intron-specific, acting as cofactors that bind their intron-containing pre-RNAs to facilitate splicing. However, the degeneracy of the mitochondrial introns in plants and the absence of cognate intron-encoded maturase open reading frames suggest that their splicing in vivo is assisted by 'trans'-acting protein factors. Interestingly, angiosperms harbor several nuclear-encoded maturase-related (nMat) genes that contain N-terminal mitochondrial localization signals. Recently, we established the roles of two of these paralogs in Arabidopsis, nMAT1 and nMAT2, in the splicing of mitochondrial introns. Here we show that nMAT4 (At1g74350) is required for RNA processing and maturation of nad1 introns 1, 3 and 4 in Arabidopsis mitochondria. Seed germination, seedling establishment and development are strongly affected in homozygous nmat4 mutants, which also show modified respiration phenotypes that are tightly associated with complex I defects.
Mitochondria (mt) in plants house about 20 group-II introns, which lie within protein-coding gene... more Mitochondria (mt) in plants house about 20 group-II introns, which lie within protein-coding genes required in both organellar genome expression and respiration activities. While in nonplant systems the splicing of group-II introns is mediated by proteins encoded within the introns themselves (known as “maturases”), only a single maturase ORF (matR) has retained in the mitochondrial genomes in plants; however, its putative role(s) in the splicing of organellar introns is yet to be established. Clues to other proteins are scarce, but these are likely encoded within the nucleus as there are no obvious candidates among the remaining ORFs within the mtDNA. Intriguingly, higher plants genomes contain four maturase-related genes, which exist in the nucleus as self-standing ORFs, out of the context of their evolutionary-related group-II introns “hosts.” These are all predicted to reside within mitochondria and may therefore act “in-trans” in the splicing of organellar-encoded introns. Here...
Transcriptome analyses enhance our understanding of RNAbased regulatory mechanisms in prokaryoti... more Transcriptome analyses enhance our understanding of RNAbased regulatory mechanisms in prokaryotic organ isms as well within the nuclearcytosolic compartments of eukaryotic cells. Higher plant organelles employ com plex posttranscriptional regulatory mechanisms, which are studied in a comprehensive fashion by different RNA methodologies. Northern blot hybridization provides with important data to the "steadystate" level of accu mulation of a given transcript in the sample, and thus remains as a standard method for detection and quanti tation of RNA levels, despite the advent of other powerful techniques. Here, we provide a modified method for orthern blotting which has been optimized for the anal n ysis of plant mitochondrial RNAs.
Histone ubiquitylation/deubiquitylation plays a major role in the epigenetic regulation of gene e... more Histone ubiquitylation/deubiquitylation plays a major role in the epigenetic regulation of gene expression. In plants, OTLD1, a member of the ovarian tumor (OTU) deubiquitinase family, deubiquitylates histone 2B and represses the expression of genes involved in growth, cell expansion, and hormone signaling. OTLD1 lacks the intrinsic ability to bind DNA. How OTLD1, as well as most other known plant histone deubiquitinases, recognizes its target genes remains unknown. Here, we show that Arabidopsis transcription factor LSH10, a member of the ALOG protein family, interacts with OTLD1 in living plant cells. Loss-of-function LSH10 mutations relieve the OTLD1-promoted transcriptional repression of the target genes, resulting in their elevated expression, whereas recovery of the LSH10 function results in down-regulated transcription of the same genes. We show that LSH10 associates with the target gene chromatin as well as with DNA sequences in the promoter regions of the target genes. Furt...
Histone ubiquitylation/deubiquitylation plays a major role in the epigenetic regulation of gene e... more Histone ubiquitylation/deubiquitylation plays a major role in the epigenetic regulation of gene expression. In plants, OTLD1, a member of the ovarian tumor (OTU) deubiquitinase family, deubiquitylates monoubiquitylated histone 2B and represses the expression of genes involved in growth, cell expansion, and hormone signaling. Like many other histone-modifying enzymes, OTLD1 lacks the intrinsic ability to bind DNA. How OTLD1, as well as most other known plant histone deubiquitinases, is recruited specifically to the promoters of its target genes remains unknown. Here, we show that Arabidopsis transcription factor LSH10, a member of the ALOG protein family, interacts with OTLD1 in living plant cells. Loss-of-function LSH10 mutations relieve the OTLD1-promoted transcriptional repression of the target genes, resulting in their elevated expression, whereas recovery of the LSH10 function results in down-regulated transcription of the same genes. We then show that LSH10 associates directly ...
Histone monoubiquitination is associated with active chromatin and plays an important role in epi... more Histone monoubiquitination is associated with active chromatin and plays an important role in epigenetic regulation of gene expression in plants. Deubiquitinating enzymes remove the ubiquitin group from histones and thereby contribute to gene repression. The Arabidopsis thaliana genome encodes 50 deubiquitinases, yet only 2 of them-UBP26 and OTLD1, members of the USP/UBP (ubiquitin-specific protease and ubiquitin-binding protein) and OTU (ovarian tumor protease) deubiquitinase families-are known to target histones. Furthermore, UBP26 is the only plant histone deubiquitinase for which the functional role has been characterized in detail. We used gain- and loss-of-function alleles of OTLD1 to examine its role in the plant life cycle and showed that OTLD1 stimulates plant growth, increases cell size, and induces transcriptional repression of five major regulators of plant organ growth and development: GA20OX, WUS, OSR2, ARL, and ABI5 OTLD1 associated with chromatin at each of these tar...
As metabolic centers, plant organelles participate in maintenance, defense and signaling. MSH1 is... more As metabolic centers, plant organelles participate in maintenance, defense and signaling. MSH1 is a plant-specific protein involved in organellar genome stability in mitochondria and plastids. Plastid depletion of MSH1 causes heritable, non-genetic changes in development and DNA methylation. We investigated the msh1 phenotype using hemi-complementation mutants and transgene-null segregants from RNAi suppression lines to sub-compartmentalize MSH1 effects. We show that MSH1 expression is spatially regulated, specifically localizing to plastids within the epidermis and vascular parenchyma. The protein binds DNA and localizes to plastid and mitochondrial nucleoids, yet fractionation and protein:protein interactions data indicate that MSH1 also associates with the thylakoid membrane. Plastid MSH1 depletion results in variegation, abiotic stress tolerance, variable growth rate, and delayed maturity. Depletion from mitochondria results in 7-10% of plants altered in leaf morphology, heat to...
ABSTRACT Ferredoxins (Fds) are small iron-sulfur proteins, which mediate electron transfer in a w... more ABSTRACT Ferredoxins (Fds) are small iron-sulfur proteins, which mediate electron transfer in a wide range of metabolic reactions. Several intriguing observations suggest that Fds may also directly associate with RNA, thus implicating a second role for these proteins in organellar RNA metabolism. Plants contain several closely-related Fd homologs, whose members are predicted to reside within the plastids. As strong mobile electron-carriers, able to partition between the stroma and the thylakoid membranes, Fds are therefore excellent candidates to regulate the expression of plastidic genes in a redox-dependant manner. Accordingly, the translation of D1 protein in the chloroplasts is mediated by a redox-poise involving the ferredoxin-thioredoxin system. Yet, despite these suggestive evidences, RNA binding activity has not been reported for an isolated Fd protein. Here, we established the intracellular locations of the six Fd paralogs in Arabidopsis to the plastids and demonstrated that one of these proteins, AtFd6, is associated with organellar transcripts in vivo. Biochemical analyses in vitro indicated that a re-combinant purified AtFd6-His protein binds with high affinity and specificity to psbA mRNA, in a redox-dependant manner.
Group II introns are large catalytic RNAs that are found in bacteria and organellar genomes of lo... more Group II introns are large catalytic RNAs that are found in bacteria and organellar genomes of lower eukaryotes, but are particularly prevalent within mitochondria in plants, where they are present in many critical genes. The excision of plant mitochondrial introns is essential for respiratory functions, and is facilitated in vivo by various protein cofactors. Typical group II introns are classified as mobile genetic elements, consisting of the self-splicing ribozyme and its own intron-encoded maturase protein. A hallmark of maturases is that they are intron-specific, acting as cofactors that bind their intron-containing pre-RNAs to facilitate splicing. However, the degeneracy of the mitochondrial introns in plants and the absence of cognate intron-encoded maturase open reading frames suggest that their splicing in vivo is assisted by 'trans'-acting protein factors. Interestingly, angiosperms harbor several nuclear-encoded maturase-related (nMat) genes that contain N-terminal mitochondrial localization signals. Recently, we established the roles of two of these paralogs in Arabidopsis, nMAT1 and nMAT2, in the splicing of mitochondrial introns. Here we show that nMAT4 (At1g74350) is required for RNA processing and maturation of nad1 introns 1, 3 and 4 in Arabidopsis mitochondria. Seed germination, seedling establishment and development are strongly affected in homozygous nmat4 mutants, which also show modified respiration phenotypes that are tightly associated with complex I defects.
Mitochondria (mt) in plants house about 20 group-II introns, which lie within protein-coding gene... more Mitochondria (mt) in plants house about 20 group-II introns, which lie within protein-coding genes required in both organellar genome expression and respiration activities. While in nonplant systems the splicing of group-II introns is mediated by proteins encoded within the introns themselves (known as “maturases”), only a single maturase ORF (matR) has retained in the mitochondrial genomes in plants; however, its putative role(s) in the splicing of organellar introns is yet to be established. Clues to other proteins are scarce, but these are likely encoded within the nucleus as there are no obvious candidates among the remaining ORFs within the mtDNA. Intriguingly, higher plants genomes contain four maturase-related genes, which exist in the nucleus as self-standing ORFs, out of the context of their evolutionary-related group-II introns “hosts.” These are all predicted to reside within mitochondria and may therefore act “in-trans” in the splicing of organellar-encoded introns. Here...
Transcriptome analyses enhance our understanding of RNAbased regulatory mechanisms in prokaryoti... more Transcriptome analyses enhance our understanding of RNAbased regulatory mechanisms in prokaryotic organ isms as well within the nuclearcytosolic compartments of eukaryotic cells. Higher plant organelles employ com plex posttranscriptional regulatory mechanisms, which are studied in a comprehensive fashion by different RNA methodologies. Northern blot hybridization provides with important data to the "steadystate" level of accu mulation of a given transcript in the sample, and thus remains as a standard method for detection and quanti tation of RNA levels, despite the advent of other powerful techniques. Here, we provide a modified method for orthern blotting which has been optimized for the anal n ysis of plant mitochondrial RNAs.
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