The location of rRNA processing was analyzed by using in situ hybridization with ITS1 probe and immunolabeling of anti-fibrillarin mAb in pea (Pisum sativum) root pole cells. The results showed that rRNA processing si...The location of rRNA processing was analyzed by using in situ hybridization with ITS1 probe and immunolabeling of anti-fibrillarin mAb in pea (Pisum sativum) root pole cells. The results showed that rRNA processing sites were in dense fibrillar components (DFCs) and granular com-ponents (GCs), but not in fibrillar centers (FCs). Low doses of actinomycin D (AMD) treatment can selectively suppress pre-rRNA synthesis but cannot disturb the processing of preformed pre-rRNAs. With AMD treatment prolonged, the density of labeled signals gradually decreased, indicating the preformed pre-rRNAs were gradually processed.展开更多
Processing of pre-rRNA is one of the major events taking place In the nucleolus. U3 snoRNA, an rRNA spliceosomal factor, is suggested to be essential in the first cleavage step of the 5' ETS sequence in the proces...Processing of pre-rRNA is one of the major events taking place In the nucleolus. U3 snoRNA, an rRNA spliceosomal factor, is suggested to be essential in the first cleavage step of the 5' ETS sequence in the processing of pre-rRNA. Identification of U3 in the nucleolus provides a piece of indirect evidence for pre-rRNA processing site and transportation of processing products. In the present study, subnucleolar distribution of U3 snoRNA in the nucleolus of Pisum sativum L. was studied by in situ hybridization with a U3 snoRNA probe. The results showed that the U3 labeling signals were distributed throughout dense fibrillar components (DFCs) and granular components (GCs), while no signal was found in fibrillar centers (FCs). When treated with actinomycine D (AMD), the labeling signals were decreased. Along with the increase of the AMD treatment time, the labeling signals became fewer and they were found in the distal regions of DFC and GC. Our results indicated that pre-rRNA splicing took place in the regions of DFC and GC, and the transportation of pre-rRNA processing products was from the regions around FCs towards the distal regions.展开更多
Ribosome biogenesis,which takes place mainly in the nucleolus,involves coordinated expression of preribosomal RNAs(pre-rRNAs)and ribosomal proteins,pre-rRNA processing,and subunit assembly with the aid of numerous ass...Ribosome biogenesis,which takes place mainly in the nucleolus,involves coordinated expression of preribosomal RNAs(pre-rRNAs)and ribosomal proteins,pre-rRNA processing,and subunit assembly with the aid of numerous assembly factors.Our previous study showed that the Arabidopsis thaliana protein arginine methyltransferase AtPRMT3 regulates pre-rRNA processing;however,the underlying molecular mechanism remains unknown.Here,we report that AtPRMT3 interacts with Ribosomal Protein S2(RPS2),facilitating processing of the 90S/Small Subunit(SSU)processome and repressing nucleolar stress.We isolated an intragenic suppressor of atprmt3-2,which rescues the developmental defects of atprmt3-2 while produces a putative truncated AtPRMT3 protein bearing the entire N-terminus but lacking an intact enzymatic activity domain We further identified RPS2 as an interacting partner of AtPRMT3,and found that loss-of-function rps2a2b mutants were phenotypically reminiscent of atprmt3,showing pleiotropic developmental defects and aberrant pre-rRNA processing.RPS2B binds directly to pre-rRNAs in the nucleus,and such binding is enhanced in atprmt3-2.Consistently,multiple components of the 90S/SSU processome were more enriched by RPS2B in atprmt3-2,which accounts for early pre-rRNA processing defects and results in nucleolar stress.Collectively,our study uncovered a novel mechanism by which AtPRMT3 cooperates with RPS2B to facilitate the dynamic assembly/disassembly of the 90S/SSU processome during ribosome biogenesis and repress nucleolar stress.展开更多
Arabidopsis thaliana L. yellow variegated （var2） mutant is defective in a chloroplast FtsH family metalloprotease, AtFtsH2/VAR2, and displays an intriguing green and white leaf variegation. This unique var2-mediated...Arabidopsis thaliana L. yellow variegated （var2） mutant is defective in a chloroplast FtsH family metalloprotease, AtFtsH2/VAR2, and displays an intriguing green and white leaf variegation. This unique var2-mediated leaf variegation offers a simple yet powerful tool for dissecting the genetic regulation of chloroplast development. Here, we report the isolation and characterization of a new var2 suppressor gene, SUPPRESSOR OF VARIEGA TION8 （SVR8）, which encodes a putative chloroplast ribosomal large subunit protein, L24. Mutations in SVR8 suppress var2 leaf variegation at ambient temperature and partially suppress the cold-induced chlorosis phenotype of var2. Loss of SVR8 causes unique chloroplast rRNA processing defects, particularly the 23S-4.5S dicistronic precursor. The recovery of the major abnormal processing site in svr823S-4.5S precursor indicate that it does not lie in the same position where SVR8/L24 binds on the ribosome. Surprisingly, we found that the loss of a chloroplast ribosomal small subunit protein, $21, results in aberrant chloroplast rRNA processing but not suppression of var2 variegation. These findings suggest that the disruption of specific aspects of chloroplast translation, rather than a general impairment in chloroplast translation, suppress var2variegation and the existence of complex genetic interactions in chloroplast development.展开更多
1A6/DRIM has been identified as UTP20, a small subunit processome component, functioning in 18S rRNA processing. In the present study, the maturation of 28S rRNA and 5.8S rRNA was inhibited when 1A6/DRIM was silenced ...1A6/DRIM has been identified as UTP20, a small subunit processome component, functioning in 18S rRNA processing. In the present study, the maturation of 28S rRNA and 5.8S rRNA was inhibited when 1A6/DRIM was silenced in HeLa cells; and coincidently, an accumulation of 32S rRNA precursor was observed. Immunoprecipitation was performed with the anti-1A6/DRIM antibody, followed by Northern blot with the ITS2 probe. The results showed that 1A6/DRIM was associated with both 32S and 12S rRNA precursors in vivo. The expression profile of 1A6/DRIM during rRNA processing was investigated by sucrose density gradient fractionation in combination with Western blot analysis. The results demonstrated that 1A6/DRIM was involved in the pre-60S particles in addition to the pre-40S particles and co-sediment with the 32S and 12S rRNA precursors in the nucleolus. Furthermore, the interaction of U8 snoRNA with 1A6/DRIM was revealed by immunoprecipitation. These results demonstrated that 1A6/DRIM interacted with both 32S rRNA and U8 snoRNA, being involved in 28S rRNA and 5.8S rRNA processing.展开更多
基金the Major State Basic Research Program (973) of China.
文摘The location of rRNA processing was analyzed by using in situ hybridization with ITS1 probe and immunolabeling of anti-fibrillarin mAb in pea (Pisum sativum) root pole cells. The results showed that rRNA processing sites were in dense fibrillar components (DFCs) and granular com-ponents (GCs), but not in fibrillar centers (FCs). Low doses of actinomycin D (AMD) treatment can selectively suppress pre-rRNA synthesis but cannot disturb the processing of preformed pre-rRNAs. With AMD treatment prolonged, the density of labeled signals gradually decreased, indicating the preformed pre-rRNAs were gradually processed.
文摘Processing of pre-rRNA is one of the major events taking place In the nucleolus. U3 snoRNA, an rRNA spliceosomal factor, is suggested to be essential in the first cleavage step of the 5' ETS sequence in the processing of pre-rRNA. Identification of U3 in the nucleolus provides a piece of indirect evidence for pre-rRNA processing site and transportation of processing products. In the present study, subnucleolar distribution of U3 snoRNA in the nucleolus of Pisum sativum L. was studied by in situ hybridization with a U3 snoRNA probe. The results showed that the U3 labeling signals were distributed throughout dense fibrillar components (DFCs) and granular components (GCs), while no signal was found in fibrillar centers (FCs). When treated with actinomycine D (AMD), the labeling signals were decreased. Along with the increase of the AMD treatment time, the labeling signals became fewer and they were found in the distal regions of DFC and GC. Our results indicated that pre-rRNA splicing took place in the regions of DFC and GC, and the transportation of pre-rRNA processing products was from the regions around FCs towards the distal regions.
基金This work was supported by grants from the National Natural Science Foundation of China(31788103 and 91540203 to X.Cao,31770874 to C.L.,31900932 to R.H.,and 31701096 to J.S.),Chinathe Strategic Priority Research Program of Chinese Academy of Sciences(XDB27030201 to X.Cao),China+1 种基金the Key Research Program of Frontier Sciences of Chinese Academy of Sciences(QYZDY-SSW-SMC022 to X.Cao),Chinathe State Key Laboratory of Plant Genomics,China.
文摘Ribosome biogenesis,which takes place mainly in the nucleolus,involves coordinated expression of preribosomal RNAs(pre-rRNAs)and ribosomal proteins,pre-rRNA processing,and subunit assembly with the aid of numerous assembly factors.Our previous study showed that the Arabidopsis thaliana protein arginine methyltransferase AtPRMT3 regulates pre-rRNA processing;however,the underlying molecular mechanism remains unknown.Here,we report that AtPRMT3 interacts with Ribosomal Protein S2(RPS2),facilitating processing of the 90S/Small Subunit(SSU)processome and repressing nucleolar stress.We isolated an intragenic suppressor of atprmt3-2,which rescues the developmental defects of atprmt3-2 while produces a putative truncated AtPRMT3 protein bearing the entire N-terminus but lacking an intact enzymatic activity domain We further identified RPS2 as an interacting partner of AtPRMT3,and found that loss-of-function rps2a2b mutants were phenotypically reminiscent of atprmt3,showing pleiotropic developmental defects and aberrant pre-rRNA processing.RPS2B binds directly to pre-rRNAs in the nucleus,and such binding is enhanced in atprmt3-2.Consistently,multiple components of the 90S/SSU processome were more enriched by RPS2B in atprmt3-2,which accounts for early pre-rRNA processing defects and results in nucleolar stress.Collectively,our study uncovered a novel mechanism by which AtPRMT3 cooperates with RPS2B to facilitate the dynamic assembly/disassembly of the 90S/SSU processome during ribosome biogenesis and repress nucleolar stress.
基金supported by funds from National Natural Science Foundation of China to F.Y. (31071073 31170219) and to X.L. (31100864)+2 种基金by Chinese Ministry of Education Program of New Century Excellent Talents in University (NCET 09 0657) to F.Yby funding to S.R. from the US Department of Energy Energy Biosciences panel (DE FG02 94ER20147)
文摘Arabidopsis thaliana L. yellow variegated （var2） mutant is defective in a chloroplast FtsH family metalloprotease, AtFtsH2/VAR2, and displays an intriguing green and white leaf variegation. This unique var2-mediated leaf variegation offers a simple yet powerful tool for dissecting the genetic regulation of chloroplast development. Here, we report the isolation and characterization of a new var2 suppressor gene, SUPPRESSOR OF VARIEGA TION8 （SVR8）, which encodes a putative chloroplast ribosomal large subunit protein, L24. Mutations in SVR8 suppress var2 leaf variegation at ambient temperature and partially suppress the cold-induced chlorosis phenotype of var2. Loss of SVR8 causes unique chloroplast rRNA processing defects, particularly the 23S-4.5S dicistronic precursor. The recovery of the major abnormal processing site in svr823S-4.5S precursor indicate that it does not lie in the same position where SVR8/L24 binds on the ribosome. Surprisingly, we found that the loss of a chloroplast ribosomal small subunit protein, $21, results in aberrant chloroplast rRNA processing but not suppression of var2 variegation. These findings suggest that the disruption of specific aspects of chloroplast translation, rather than a general impairment in chloroplast translation, suppress var2variegation and the existence of complex genetic interactions in chloroplast development.
基金supported by the National High-Tech Research and Development Program of China (2006AA02A402 and 2008AA02Z131)the National Natural Science Foundation of China (30771224)+1 种基金the Ministry of Education 985 project of China (985-2-016-24)National Key Basic Research Program of China (2006CB943603)
文摘1A6/DRIM has been identified as UTP20, a small subunit processome component, functioning in 18S rRNA processing. In the present study, the maturation of 28S rRNA and 5.8S rRNA was inhibited when 1A6/DRIM was silenced in HeLa cells; and coincidently, an accumulation of 32S rRNA precursor was observed. Immunoprecipitation was performed with the anti-1A6/DRIM antibody, followed by Northern blot with the ITS2 probe. The results showed that 1A6/DRIM was associated with both 32S and 12S rRNA precursors in vivo. The expression profile of 1A6/DRIM during rRNA processing was investigated by sucrose density gradient fractionation in combination with Western blot analysis. The results demonstrated that 1A6/DRIM was involved in the pre-60S particles in addition to the pre-40S particles and co-sediment with the 32S and 12S rRNA precursors in the nucleolus. Furthermore, the interaction of U8 snoRNA with 1A6/DRIM was revealed by immunoprecipitation. These results demonstrated that 1A6/DRIM interacted with both 32S rRNA and U8 snoRNA, being involved in 28S rRNA and 5.8S rRNA processing.
