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Biological function of RNA interference (RNAi) pathways in the moss Physcomitrella patens (Hedw.) Bruch & Schimp.

Basel Khraiwesh (UGent)
(2009)
Author
Promoter
Ralf Reski and Wolfgang Frank
Organization
Abstract
Developmental programs in animals and plants are controlled by RNA interference (RNAi) via small non-coding RNAs regulating mRNA stability and translation, and chromatin modifications. MiRNAs are a specific class of small RNAs, generated by Dicer proteins, that down-regulate the expression of target genes by base-pairing to their cognate mRNAs in the RNA-induced silencing complex (RISC). In addition, miRNAs initiate the release of ta-siRNAs from precursor molecules TASRNAs. In contrast to the seed plant Arabidopsis thaliana the genome of the moss Physcomitrella patens encodes two DICER-LIKE1 proteins, PpDCL1a and PpDCL1b. In this study we found that PpDCL1a is the functional equivalent of the Arabidopsis thaliana DICER-LIKE1 protein required for the biogenesis of miRNAs and ta-siRNAs. Unlike AtDCL1 and PpDCL1a, PpDCL1b is not required for miRNA biogenesis, but is essential for miRNA-directed targetRNA cleavage and subsequent generation of transitive siRNAs and ta-siRNAs. In PpDCL1b mutant lines miRNA accumulation was unaffected but cleavage of targetRNAs was abolished. Instead, miRNA:targetRNA duplexes, hypermethylation of genomic loci encoding targetRNAs, and reduced levels of targetRNAs occurred. In Physcomitrella patens wild type we observed epigenetic silencing of a miRNA target gene in response to the phytohormone abscisic acid indicating that miRNAs control their targets not only at the post-transcriptional but also at the transcriptional level. Based on these data from Physcomitrella patens a model is proposed for the gene-specific control of transcription factors upon dysfunctions of the RNAi machinery. We anticipate similar control mechanisms to exist in other eukaryotes as well. Artificial miRNAs (amiRNAs) have been mainly used to downregulate single or multiple protein coding genes.amiRNAs can be generated by exchanging the miRNA/miRNA* sequence within miRNA precursor genes, while maintaining the pattern of matches and mismatches in the foldback. Thus, for functional gene analysis, amiRNAs can be designed to target any gene of interest. The moss Physcomitrella patens exhibits the unique feature of a highly efficient homologous recombination mechanism, which allows for the generation of targeted gene knockout lines. However, the completion of the Physcomitrella genome sequence necessitates the development of alternative techniques to speed up such reverse genetics analyses, and to allow for more flexible inactivation of genes or groups of genes. To prove the adaptability of amiRNA expression in Physcomitrella two amiRNAs were designed, the first one targeting the gene PpFtsZ2-1, which is indispensable for chloroplast division, and the second one targeting the gene PpGNT1 encoding an N-acetylglucosaminyltransferase. The PpFtsZ2-1-amiRNA and PpGNT1-amiRNA were expressed from the Arabidopsis thaliana miR319a precursor fused to a constitutive promoter. Transgenic Physcomitrella lines harboring the overexpression constructs showed precise processing of the PpFtsZ2-1-amiRNA and PpGNT1-amiRNA, and an efficient knock-down of the PpFtsZ2-1 and PpGNT1 genes. Furthermore, chloroplast division was impeded in PpFtsZ2-1-amiRNA lines which phenocopied PpFtsZ2-1 knockout mutants. We also provide evidence for the amplification of the initial amiRNA signal by secondary transitive siRNAs, although these siRNAs do not seem to have a major effect on the sequence-related PpFtsZ2-2 mRNA, confirming specificity of the amiRNA approach. We provide evidence that amiRNAs can be used as an alternative tool for studying gene functions in Physcomitrella patens.
Keywords
Artificial MicroRNA, Transitive siRNA, MicroRNA, Small RNA, RNAi, Dicer, Pyscomitrella, Moss

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Citation

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Chicago
Khraiwesh, Basel. 2009. “Biological Function of RNA Interference (RNAi) Pathways in the Moss Physcomitrella Patens (Hedw.) Bruch & Schimp.” Freiburg im Breisgau, Germany: Albert-Ludwigs-Universität. Fakultät für Biologie.
APA
Khraiwesh, B. (2009). Biological function of RNA interference (RNAi) pathways in the moss Physcomitrella patens (Hedw.) Bruch & Schimp. Albert-Ludwigs-Universität. Fakultät für Biologie, Freiburg im Breisgau, Germany.
Vancouver
1.
Khraiwesh B. Biological function of RNA interference (RNAi) pathways in the moss Physcomitrella patens (Hedw.) Bruch & Schimp. [Freiburg im Breisgau, Germany]: Albert-Ludwigs-Universität. Fakultät für Biologie; 2009.
MLA
Khraiwesh, Basel. “Biological Function of RNA Interference (RNAi) Pathways in the Moss Physcomitrella Patens (Hedw.) Bruch & Schimp.” 2009 : n. pag. Print.
@phdthesis{1057297,
  abstract     = {Developmental programs in animals and plants are controlled by RNA interference (RNAi) via small non-coding RNAs regulating mRNA stability and translation, and chromatin modifications. MiRNAs are a specific class of small RNAs, generated by Dicer proteins, that down-regulate the expression of target genes by base-pairing to their cognate mRNAs in the RNA-induced silencing complex (RISC). In addition, miRNAs initiate the release of ta-siRNAs from precursor molecules TASRNAs. In contrast to the seed plant Arabidopsis thaliana the genome of the moss Physcomitrella patens encodes two DICER-LIKE1 proteins, PpDCL1a and PpDCL1b. In this study we found that PpDCL1a is the functional equivalent of the Arabidopsis thaliana DICER-LIKE1 protein required for the biogenesis of miRNAs and ta-siRNAs. Unlike AtDCL1 and PpDCL1a, PpDCL1b is not required for miRNA biogenesis, but is essential for miRNA-directed targetRNA cleavage and subsequent generation of transitive siRNAs and ta-siRNAs. In PpDCL1b mutant lines miRNA accumulation was unaffected but cleavage of targetRNAs was abolished. Instead, miRNA:targetRNA duplexes, hypermethylation of genomic loci encoding targetRNAs, and reduced levels of targetRNAs occurred. In Physcomitrella patens wild type we observed epigenetic silencing of a miRNA target gene in response to the phytohormone abscisic acid indicating that miRNAs control their targets not only at the post-transcriptional but also at the transcriptional level. Based on these data from Physcomitrella patens a model is proposed for the gene-specific control of transcription factors upon dysfunctions of the RNAi machinery. We anticipate similar control mechanisms to exist in other eukaryotes as well.
Artificial miRNAs (amiRNAs) have been mainly used to downregulate single or multiple protein coding genes.amiRNAs can be generated by exchanging the miRNA/miRNA* sequence within miRNA precursor genes, while maintaining the pattern of matches and mismatches in the foldback. Thus, for functional gene analysis, amiRNAs can be designed to target any gene of interest. The moss Physcomitrella patens exhibits the unique feature of a highly efficient homologous recombination mechanism, which allows for the generation of targeted gene knockout lines. However, the completion of the Physcomitrella genome sequence necessitates the development of alternative techniques to speed up such reverse genetics analyses, and to allow for more flexible inactivation of genes or groups of genes. To prove the adaptability of amiRNA expression in Physcomitrella two amiRNAs were designed, the first one targeting the gene PpFtsZ2-1, which is indispensable for chloroplast division, and the second one targeting the gene PpGNT1 encoding an N-acetylglucosaminyltransferase. The PpFtsZ2-1-amiRNA and PpGNT1-amiRNA were expressed from the Arabidopsis thaliana miR319a precursor fused to a constitutive promoter. Transgenic Physcomitrella lines harboring the overexpression constructs showed precise processing of the PpFtsZ2-1-amiRNA and PpGNT1-amiRNA, and an efficient knock-down of the PpFtsZ2-1 and PpGNT1 genes. Furthermore, chloroplast division was impeded in PpFtsZ2-1-amiRNA lines which phenocopied PpFtsZ2-1 knockout mutants. We also provide evidence for the amplification of the initial amiRNA signal by secondary transitive siRNAs, although these siRNAs do not seem to have a major effect on the sequence-related PpFtsZ2-2 mRNA, confirming specificity of the amiRNA approach. We provide evidence that amiRNAs can be used as an alternative tool for studying gene functions in Physcomitrella patens.},
  author       = {Khraiwesh, Basel},
  keywords     = {Artificial MicroRNA,Transitive siRNA,MicroRNA,Small RNA,RNAi,Dicer,Pyscomitrella,Moss},
  language     = {eng},
  pages        = {II, 153},
  publisher    = {Albert-Ludwigs-Universität. Fakultät für Biologie},
  title        = {Biological function of RNA interference (RNAi) pathways in the moss Physcomitrella patens (Hedw.) Bruch & Schimp.},
  year         = {2009},
}