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. Wild-type, hyl1-2, hyl1-2/dcl1-20, and hyl1-2/dcl1-21 plants were sampled from both rosette leaves and flowers.
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.
Project description:HYL1 (DRB1) is a double-stranded RNA-binding protein involved in miRNA processing in plants. It is a core component of the Microprocessor complex and enhances the efficiency and precision of miRNA processing by the DCL1 protein. In this work, we report a novel function of HYL1 protein in the transcription of MIR genes. DRB1 co-localizes with RNA Polymerase II and affects its distribution along MIR genes. Moreover, proteomic experiments revealed that HYL1 protein interacts with many transcription factors. Finally, we show that the action of DRB1 is not limited to MIR genes as it impacts the expression of many other genes, the majority of which are involved in plant response to light. These discoveries add DRB1 as another player in gene regulation at the transcriptional level, independent of its role in miRNA biogenesis.
Project description:1, Using mRNA-Seq to get expression profiling of rrp6l1-2 mutant and Col-0 wild-type (WT); 2,Using MethylC-Seq to provide single-base resolution of DNA methylation status in rrp6l1-2 mutant; 3, Using small RNA-Seq(sRNA-Seq) to get small RNA profiling of rrp6l1-2 and WT mRNA-Seq: 2 samples examined, WT and rrp6l1-2 mutant; MethylC-Seq: 1 sample examined, rrp6l1-2 mutant; small RNA-Seq: 2 samples examined, WT and rrp6l1-2 mutant
Project description:In Arabidopsis thaliana, four different DICER-LIKE (DCL) proteins have distinct, but partially overlapping functions in the biogenesis of microRNAs (miRNAs) and small interfering RNAs (siRNAs) from longer, non-coding precursor RNAs. To analyze the impact of different components of the small RNA (sRNA) biogenesis machinery on the transcriptome, we subjected dcl and other mutants impaired in sRNA biogenesis to whole-genome tiling array analysis. We compared both protein-coding genes and noncoding transcripts, including most pri-miRNAs, in two tissues and several stress conditions. We discovered distinct effects of dcl1, hyl1 and se mutations on the transcriptome, as well as a number of common genes affected in dcl1 and dcl2 dcl3 dcl4 triple mutants. Our results furthermore suggest that the DCL1 is not only involved in miRNA action, but can also contribute to silencing of certain transposons, apparently through an effect on DNA methylation. Together, our findings contribute to the knowledge of both specialization and overlap between different RNA silencing pathways.
Project description:1, Using mRNA-Seq to get expression profiling of rrp6l1-2 mutant and Col-0 wild-type (WT); 2,Using MethylC-Seq to provide single-base resolution of DNA methylation status in rrp6l1-2 mutant; 3, Using small RNA-Seq(sRNA-Seq) to get small RNA profiling of rrp6l1-2 and WT
Project description:Endogenous small RNAs (sRNAs) contribute to gene regulation and genome homeostasis but their activities and functions are incompletely known. The maize genome has a high number of transposable elements (TEs; almost 85%), some of which spawn abundant sRNAs. We performed sRNA and total RNA sequencing from control and abiotically stressed B73 wild-type (wt) plants and rmr6-1 mutants. RMR6 encodes the largest subunit of the RNA polymerase IV (Pol IV) complex, and is responsible for accumulation of most 24 nucleotide (nt) small interfering RNA (siRNAs). We identified novel MIRNA loci and verified miR399 target conservation in maize. RMR6-dependent 23-24 nt siRNA loci were specifically enriched in the upstream region of the most highly expressed genes. Most genes mis-regulated in rmr6-1 did not show a significant correlation with loss of flanking siRNAs, but we identified one gene supporting existing models of direct gene regulation by TE-derived siRNAs. Long-term drought correlated with changes of miRNA and sRNA accumulation, in particular inducing down-regulation of a set of sRNA loci in the wt leaf. sRNA profile of maize leaf and shoot apical meristematic area, of wt and rmr6-1 mutant plants grown under 1) control conditions 2) salt stress 3) drought stress 4) salt+drought stress. Each condition was replicated two/three times, after 10 days of treatment and after 7 days of recovery.