Project description:The processing of Arabidopsis thaliana microRNAs (miRNAs) from longer primary transcripts (pri-miRNAs) requires the activity of several proteins, including DICER-LIKE1 (DCL1), the double stranded RNA binding protein HYPONASTIC LEAVES1 (HYL1), and the zinc finger protein SERRATE (SE). It has been noted before that the morphological appearance of weak se mutants is reminiscent of plants with mutations in ABH1/CBP80 and CBP20, which encode the two subunits of the nuclear cap-binding complex. We report that, like SE, the cap-binding complex is necessary for proper processing of pri-miRNAs. Inactivation of either ABH1/CBP80 or CBP20 results in decreased levels of mature miRNAs accompanied by increased levels of pri-miRNAs. Whole genome tiling array analyses reveal that se, abh1/cbp80 and cbp20 mutants also share similar pre-mRNA splicing defects, leading to the accumulation of many partially spliced transcripts. This is unlikely to be an indirect consequence of improper miRNA processing or other mRNA turnover pathways, since introns retained in se, abh1/cbp80 and cbp20 mutants are not affected by mutations in other genes required for miRNA processing or for non-sense-mediated mRNA decay. Taken together, our results uncover dual roles in splicing and miRNA processing that distinguish SE and the cap-binding complex from specialized miRNA processing factors such as DCL1 and HYL1. Keywords: Tiling array analysis of RNA populations from wild type, se, abh1 and cbp20 mutants
Project description:This dataset includes the transcriptomes of plants bearing mutations in genes enconding for three related Arabidopsis proteins: SERRATE (SE), CAP-BINDING PROTEIN 20 (CBP20) and CAP-BINDING PROTEIN 80 (CBP80). The transcriptomes were obtained by mRNA sequencing. Comparison of the RNA accumulation patterns will enable the idenfication of common patterns and uncover possible common functions of SE, CBP20 and CBP80 in regulating gene expression.
Project description:Long intergenic noncoding RNAs (lincRNAs) transcribed from intergenic regions play important roles in key biological processes.Analysis of published transcriptom datasets suggested some lincRNAs may be regulated by SERRATE,CBP20 and CBP80 in Arabidopsis. To further investigate the regulation, we use Arabidopsis lincRNA arrays v1 to detect lincRNA expression in se-2 and cbp20/80 double mutants.We found the expression levels of 940 lincRNAs (20%) out of the 4,634 lincRNAs with probes in array were significantly alerted in all mutants (P-value of eBays ANOVA < 0.05 & fold change of signal intensity > 2). This group included 525 up-regulated and 415 down-regulated lincRNAs. Seedlings(Col-0), Seedlings(se-2), Seedlings(cbp20,80 double mutant) x 3 biological replicates
Project description:Long intergenic noncoding RNAs (lincRNAs) transcribed from intergenic regions play important roles in key biological processes.Analysis of published transcriptom datasets suggested some lincRNAs may be regulated by SERRATE,CBP20 and CBP80 in Arabidopsis. To further investigate the regulation, we use Arabidopsis lincRNA arrays v1 to detect lincRNA expression in se-2 and cbp20/80 double mutants.We found the expression levels of 940 lincRNAs (20%) out of the 4,634 lincRNAs with probes in array were significantly alerted in all mutants (P-value of eBays ANOVA < 0.05 & fold change of signal intensity > 2). This group included 525 up-regulated and 415 down-regulated lincRNAs.
Project description:MicroRNAs (miRNAs) play key regulatory roles in numerous developmental and physiological processes in animals and plants. The elaborate mechanism of miRNA biogenesis involves transcription and multiple processing steps. Here, we report the identification of a pair of evolutionarily conserved NOT2_3_5 domain containing proteins, NOT2a and NOT2b (previously known as At-NOT2 and VIP2, respectively), as components involved in Arabidopsis miRNA biogenesis. NOT2 was identified by its interaction with the Piwi/Ago/Zwille (PAZ) domain of Dicer-like 1 (DCL1), an interaction that is conserved between rice and Arabidopsis. Inactivation of both NOT2 genes in Arabidopsis caused severe defects in male gametophytes, and weak lines show pleiotropic defects reminiscent of miRNA pathway mutants. Impairment of NOT2s decreases the accumulation of pri-miRNAs and mature miRNAs and increases DCL1-containing nuclear speckle number in vivo. In addition, NOT2b protein interacts with Pol II and other miRNA processing factors including two cap-binding proteins, CBP80/ABH1, CBP20 and SERRATE (SE). Therefore, these results suggest that NOT2 proteins promote MIR transcription and facilitate efficient DCL1 recruitment in Arabidopsis. Examination of transcriptome of not2 and wild type by RNA-seq.
Project description:MicroRNAs (miRNAs) play key regulatory roles in numerous developmental and physiological processes in animals and plants. The elaborate mechanism of miRNA biogenesis involves transcription and multiple processing steps. Here, we report the identification of a pair of evolutionarily conserved NOT2_3_5 domain containing proteins, NOT2a and NOT2b (previously known as At-NOT2 and VIP2, respectively), as components involved in Arabidopsis miRNA biogenesis. NOT2 was identified by its interaction with the Piwi/Ago/Zwille (PAZ) domain of Dicer-like 1 (DCL1), an interaction that is conserved between rice and Arabidopsis. Inactivation of both NOT2 genes in Arabidopsis caused severe defects in male gametophytes, and weak lines show pleiotropic defects reminiscent of miRNA pathway mutants. Impairment of NOT2s decreases the accumulation of pri-miRNAs and mature miRNAs and increases DCL1-containing nuclear speckle number in vivo. In addition, NOT2b protein interacts with Pol II and other miRNA processing factors including two cap-binding proteins, CBP80/ABH1, CBP20 and SERRATE (SE). Therefore, these results suggest that NOT2 proteins promote MIR transcription and facilitate efficient DCL1 recruitment in Arabidopsis.
Project description:CBP20 (Cap-Binding Protein 20) encodes a small subunit of the cap-binding complex (CBC), which is involved in the conserved cell processes related to RNA metabolism in plants and, simultaneously, engaged in the signaling network of drought response, which is dependent on ABA. CBP20 (Cap-Binding Protein 20), which encodes a small subunit of the cap-binding complex (CBC). CBC is a heterodimer that is formed by two subunits – a small one that is encoded by CBP20 and a large subunit that is encoded by CBP80 (Cap-Binding Protein 80). Both the nucleotide and amino acid sequences of CBP20 are highly conserved across species from Saccharomyces to Homo sapiens. CBP20 is involved in very conserved cell processes that are related to RNA metabolism such as polyadenylation and splicing, miRNA biogenesis, and according to the most recent reports, to histone methylation (Kuhn et al. 2008; Kim et al. 2008; Gregory et al. 2008; Kmieciak et al. 2002; Laubinger et al. 2008; Li et al. 2016). Most striking, however, is its simultaneous engagement in ABA signaling during seed germination and drought response (Papp et al. 2004; Jäger et al. 2011). It was shown that an Arabidopsis cbp20 mutant exhibited a hypersensitivity to ABA during seed germination and a better performance under water deficit conditions than the WT (Papp et al. 2004; Jäger et al. 2011). Interestingly, the Arabidopsis knockout mutant in CBP80 (Cap-Binding Protein 80; ABA hypersensitive 1) exhibited a similar phenotype when exposed to ABA or drought stress (Hugouvieux et al. 2001; 2002; Daszkowska-Golec et al. 2013). In Solanum tuberosum, an amiR80.2-14 mutant (CBP80 silenced using artificial microRNAs) was also reported to be drought tolerant (Pieczyński et al. 2013). Water-deficiency conditions related gene expression analysis was performed in barley in two genotypes: the wild-type (WT) barley cultivar 'Sebastian’ and hvcbp20.ab mutant; in three time points: (1) optimal water conditions, (2) onset of drought stress and (3) after 10 days of prolonged drought stress in the second leaf; analyses were performed in three biological replicates. Here, we report the enhanced tolerance to drought stress of barley mutant in the HvCBP20 gene.Transcriptome analysis using the Agilent Barley Microarray integrated with observed phenotypic traits allowed to conclude that the hvcbp20.ab mutant exhibited better fitness to stress conditions by its much more efficient and earlier activation of stress-preventing mechanisms. The network hubs involved in the adjustment of hvcbp20.ab mutant to the drought conditions were proposed. These results enabled to make a significant progress in understanding the role of CBP20 in the drought stress response.