Tissue-dependent regulation of miRNA activity
Ontology highlight
ABSTRACT:
ORGANISM(S): Zea mays
PROVIDER: GSE147576 | GEO | 2021/03/03
REPOSITORIES: GEO
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Project description:The wild grass Brachypodium distachyon has emerged as a model system for temperate grasses and biofuel plants. However, the global analysis of miRNAs, molecules known to be key for eukaryotic gene regulation, has been limited in B. distachyon to studies examining a few samples or that rely on computational predictions. Similarly an in-depth global analysis of miRNA-mediated target cleavage using Parallel Analysis of RNA Ends (PARE) data is lacking in B. distachyon. B. distachyon small RNAs were cloned and deeply sequenced from 17 libraries that represent different tissues and stresses. Using a computational pipeline, we identified 116 miRNAs including not only conserved miRNAs that have not been reported in B. distachyon, but also non-conserved miRNAs that were not found in other plants. To investigate miRNA-mediated cleavage function, four PARE libraries were constructed from key tissues and sequenced to a total depth of approximately 70 million sequences. The roughly 5 million distinct genome-matched sequences that resulted represent an extensive dataset to analyze small RNA-guided cleavage events. Analysis of the PARE and miRNA data provided experimental evidence for miRNA-mediated cleavage of 264 sites in predicted miRNA targets. In addition, PARE analysis revealed that differentially expressed miRNAs in the same family guide specific target RNA cleavage in a correspondingly tissue-preferential manner. B. distachyon miRNAs and target RNAs were experimentally identified and analyzed. Knowledge gained from this study should provide insights into the roles of miRNAs and the regulation of their targets in B. distachyon and related plants. Examination of various tissues and stresses in Brachypodium by high throughput sequencing for small RNA profiling and PARE (Parallel Analysis of RNA Ends)
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Project description:DCL1 and HYL1 are the core components of miRNA biogenesis machinery. hyl1-null mutants accumulate low levels of miRNAs and cause pleiotropic morphological phenotypes. Here, we reported that we identified 5 new alleles of DCL1 which are able to suppress the hyl1 morphological phenotypes and restore the miRNA accumulation level in hyl1 plants. These new alleles are located in the helicase and RNaseIIIa domains of DCL1, highlighting the critical functions of these domains. Biochemical analyses of these suppressors indicated that they process pri-miRNA more efficiently, with both higher Kcat and lower Km values. In addition, we investigated the functions of the DCL1 helicase domain. Our results indicated that the helicase domain is able to modulate the DCL1 processing activities. Based on these results, we proposed that HYL1 might exert its function through interaction with the DCL1 helicase domain. Small RNA sequencing from hyl1 and select hyl1/dcl1 suppressor mutants confirms thats suppression of the hyl1 phenotype is due to an increase in the accumulation, but not the precision, of miRNAs in the suppressor mutants.
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2012-04-24 | E-GEOD-29802 | biostudies-arrayexpress
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Project description:DICER-like 1 (DCL1) is a major player in micro RNA (miRNA) biogenesis and accordingly, its few known loss-of-function mutants are either lethal or display arrested seedling development. Consequently, generation of dcl1 mutants by reverse genetics and functional analysis of DCL1 in late-developing organs are challenging. Here, these challenges were resolved through the unique use of trans-activated RNA interference. Global, as well as organ-specific tomato DCL1 (SlDCL1) silencing was induced by crossing the generated responder line (OP:SlDCL1IR) with the appropriate driver line. Constitutive trans-activation knocked down SlDCL1 levels by ~95%, resulting in severe abnormalities including post-germination growth arrest accompanied by decreased miRNA and 21-nucleotide small RNA levels, but prominently elevated levels of 22- nucleotide small RNAs. The elevation in the 22- nucleotide small RNAs was correlated with specific upregulation of SlDCL2b and SlDCL2d, which are likely involved in their biogenesis. Leaf- and flower-specific OP:SlDCL1IR trans-activation inhibited blade outgrowth, induced premature bud senescence and produced pale petals, respectively, emphasizing the importance of SlDCL1-dependent small RNAs in these processes. Together, our results establish OP:SlDCL1IR as an efficient tool for analyzing processes regulated by SlDCL1-mediated gene regulation in tomato.
2013-11-01 | GSE51959 | GEO
Project description:DICER-like 1 (DCL1) is a major player in micro RNA (miRNA) biogenesis and accordingly, its few known loss-of-function mutants are either lethal or display arrested seedling development. Consequently, generation of dcl1 mutants by reverse genetics and functional analysis of DCL1 in late-developing organs are challenging. Here, these challenges were resolved through the unique use of trans-activated RNA interference. Global, as well as organ-specific tomato DCL1 (SlDCL1) silencing was induced by crossing the generated responder line (OP:SlDCL1IR) with the appropriate driver line. Constitutive trans-activation knocked down SlDCL1 levels by ~95%, resulting in severe abnormalities including post-germination growth arrest accompanied by decreased miRNA and 21-nucleotide small RNA levels, but prominently elevated levels of 22- nucleotide small RNAs. The elevation in the 22- nucleotide small RNAs was correlated with specific upregulation of SlDCL2b and SlDCL2d, which are likely involved in their biogenesis. Leaf- and flower-specific OP:SlDCL1IR trans-activation inhibited blade outgrowth, induced premature bud senescence and produced pale petals, respectively, emphasizing the importance of SlDCL1-dependent small RNAs in these processes. Together, our results establish OP:SlDCL1IR as an efficient tool for analyzing processes regulated by SlDCL1-mediated gene regulation in tomato. Examination of small RNA populations in control (35S:LhG4) and SlDCL1 silenced seedlings (35S>>SlDCL1IR) 2 biological replicates each
2013-11-01 | E-GEOD-51959 | biostudies-arrayexpress
Project description:The wild grass Brachypodium distachyon has emerged as a model system for temperate grasses and biofuel plants. However, the global analysis of miRNAs, molecules known to be key for eukaryotic gene regulation, has been limited in B. distachyon to studies examining a few samples or that rely on computational predictions. Similarly an in-depth global analysis of miRNA-mediated target cleavage using Parallel Analysis of RNA Ends (PARE) data is lacking in B. distachyon. B. distachyon small RNAs were cloned and deeply sequenced from 17 libraries that represent different tissues and stresses. Using a computational pipeline, we identified 116 miRNAs including not only conserved miRNAs that have not been reported in B. distachyon, but also non-conserved miRNAs that were not found in other plants. To investigate miRNA-mediated cleavage function, four PARE libraries were constructed from key tissues and sequenced to a total depth of approximately 70 million sequences. The roughly 5 million distinct genome-matched sequences that resulted represent an extensive dataset to analyze small RNA-guided cleavage events. Analysis of the PARE and miRNA data provided experimental evidence for miRNA-mediated cleavage of 264 sites in predicted miRNA targets. In addition, PARE analysis revealed that differentially expressed miRNAs in the same family guide specific target RNA cleavage in a correspondingly tissue-preferential manner. B. distachyon miRNAs and target RNAs were experimentally identified and analyzed. Knowledge gained from this study should provide insights into the roles of miRNAs and the regulation of their targets in B. distachyon and related plants.
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Project description:MicroRNAs (miRNAs) are a class of small RNAs which typically function by guiding cleavage of target messenger RNAs. They have been shown to play major roles in a variety of plant processes including development, and responses to pathogens and environmental stresses. To identify new miRNAs and regulation in Arabidopsis thaliana, 27 small RNA libraries were constructed and sequenced from various tissues, stresses and small RNA biogenesis mutants, resulting in 95 million genome-matched sequences. The use of rdr2 to enrich the miRNA population greatly enhanced this analysis and led to the discovery of 44 new miRNAs arising from both known and new precursors. Parallel Analysis of RNA Ends (PARE) data provide evidence that the majority guide target cleavage. The inclusion of novel stress/tissue conditions, such as submergence-stressed flowers, enabled identification of new stress regulation of both miRNAs and their targets, all of which were validated in wild type plants. By combining small RNA expression analysis with ARGONAUTE (AGO) immunoprecipitation data and global target cleavage data from PARE, a much more complete picture of Arabidopsis miRNAs was obtained. This combinatorial approach led to the discovery of AGO loading and target cleavage biases, which gave important insights into tissue-specific expression patterns, pathogen responses and the role of sequence variation among closely related miRNA family members. Examination of various tissues, stresses and small RNA biogenesis mutants in Arabidopsis by high-throughput sequencing for small RNA profiling. We have used AGO-IP and PARE data from the published data, which were downloaded from NCBI GEO with the following accession number. AGO-IP from GSM253622, GSM707682, GSM642335, GSM642336, GSM512703, GSM512702, GSM707683, GSM707684, GSM707685, GSM149080, GSM253623, GSM304283, GSM642337, GSM642338, GSM253624, GSM415788, GSM707686, GSM707687, GSM707688, GSM707689, GSM415787, GSM149081, GSM253625, GSM415789, GSM415790, GSM304285, GSM415791, GSM415792 PARE sequencing data from xrn4 flowers were obtained from Gene Expression Omnibus with accession number GSM280227.
2013-12-31 | E-GEOD-44622 | biostudies-arrayexpress