RESUMO
Successful plant reproduction depends on the adequate development of flower organs controlled by cell proliferation and other processes. The SCI1 gene regulates cell proliferation and affects the final size of the female reproductive organ. To unravel the molecular mechanism exerted by SCI1 in cell proliferation control, we searched for its interaction partners through semi-in vivo pulldown experiments, uncovering a cyclin-dependent kinase, NtCDKG;2. Bimolecular fluorescence complementation (BiFC) and co-localization experiments showed that SCI1 interacts with NtCDKG;2 and its cognate NtCyclin L in nucleoli and splicing speckles. The screening of a yeast two-hybrid (Y2H) cDNA library using SCI1 as bait revealed a novel DEAD-box RNA helicase (NtRH35). The interaction between the NtCDKG;2-NtCyclin L complex, and NtRH35 was also shown. Subcellular localization experiments showed that SCI1, NtRH35, and the NtCDKG;2-NtCyclin L complex associate with each other within splicing speckles. The Y2H screening of NtCDKG;2 and NtRH35 identified the conserved spliceosome components U2a', NKAP, and CACTIN. This work presents SCI1 and its interactors NtCDKG;2-NtCyclin L complex, and NtRH35 as new spliceosome-associated proteins. Our findings reveal a network of interactions and suggest that SCI1 may regulate cell proliferation through the splicing process. This study provides new valuable insights into the intricate molecular pathways governing plant development.
RESUMO
Apoptosis is the best-known programmed cell death that maintains tissue homeostasis in eukaryotic cells. The morphological characteristics include nuclear and cytoplasmic contraction and cytoplasmic blebbing, its biochemical hallmarks include caspase protease activity and DNA fragmentation. In rat ovaries, cell death is a normal process that occurs throughout the organism's life. Granulosa cells, the more abundant cell type forming the ovarian follicles, are eliminated via different routes of cell death. Most granulosa cells are eliminated through apoptotic cell death. In this work, we analyzed the behavior of nuclear components throughout the apoptotic process and determined how they are regionalized and conserved during follicular atresia in rat ovaries. Apoptosis was detected based on caspase-3 activity and DNA fragmentation using the TUNEL technique. We identified the transcription markers H3ac and RNA Pol II, and splicing factor SC35 by immunodetection. The nucleolar components were analyzed via light microscopy and transmission electron microscopy through immunodetection of the proteins nucleolin and nucleophosmin-1. The nuclear ultrastructure was analyzed using standard contrast and preferential ribonucleoprotein contrast. Our results demonstrate that during the progression of apoptosis, chromatin is remodeled to constitute apoptotic bodies; transcription and spliceosome elements are reorganized along with the nucleolar components. Additionally, the splicing and transcription factors are segregated into specific territories inside the apoptotic bodies, suggesting that transcriptional elements are reorganized during the apoptotic process. Our results indicate that apoptotic bodies not only are compacted, and chromatin degraded but all the nuclear components are progressively reorganized during cell elimination; moreover, the transcriptional components are preserved.
Assuntos
Apoptose , Atresia Folicular , Animais , Apoptose/genética , Cromatina/genética , Feminino , Atresia Folicular/metabolismo , Marcação In Situ das Extremidades Cortadas , Fatores de Processamento de RNA , RatosRESUMO
Ubiquitous eukaryotic non-coding circular RNAs are involved in numerous co- and post-transcriptional regulatory mechanisms. Recently, we reported full-length intronic circular RNAs (flicRNAs) in Entamoeba histolytica, with 3'ss-5'ss ligation points and 5'ss GU-rich elements essential for their biogenesis and their suggested role in transcription regulation. Here, we explored how flicRNAs impact gene expression regulation. Using CLIP assays, followed by qRT-PCR, we identified that the RabX13 control flicRNA and virulence-associated flicRNAs were bound to the HA-tagged RNA Pol II C-terminus domain in E. histolytica transformants. The U2 snRNA was also present in such complexes, indicating that they belonged to transcription initiation/elongation complexes. Correspondingly, inhibition of the second step of splicing using boric acid reduced flicRNA formation and modified the expression of their parental genes and non-related genes. flicRNAs were also recovered from chromatin immunoprecipitation eluates, indicating that the flicRNA-Pol II complex was formed in the promoter of their cognate genes. Finally, two flicRNAs were found to be cytosolic, whose functions remain to be uncovered. Here, we provide novel evidence of the role of flicRNAs in gene expression regulation in cis, apparently in a widespread fashion, as an element bound to the RNA polymerase II transcription initiation complex, in E. histolytica.
RESUMO
The advent of novel high-throughput sequencing techniques has revealed that eukaryotic genomes are massively transcribed although only a small fraction of RNAs exhibits protein-coding capacity. In the last years, long noncoding RNAs (lncRNAs) have emerged as regulators of eukaryotic gene expression in a wide range of molecular mechanisms. Plant lncRNAs can be transcribed by alternative RNA polymerases, acting directly as long transcripts or can be processed into active small RNAs. Several lncRNAs have been recently shown to interact with chromatin, DNA or nuclear proteins to condition the epigenetic environment of target genes or modulate the activity of transcriptional complexes. In this review, we will summarize the recent discoveries about the actions of plant lncRNAs in the regulation of gene expression at the transcriptional level.
Assuntos
Plantas/genética , RNA Longo não Codificante/genética , Transcrição Gênica/genética , Sequenciamento de Nucleotídeos em Larga Escala , Plantas/metabolismo , RNA Longo não Codificante/metabolismoRESUMO
Polycomb Repressive Complex 2 (PRC2) mediates transcriptional silencing by catalyzing histone H3 lysine 27 trimethylation (H3K27me3), but its role in the maturation of postmitotic mammalian neurons remains largely unknown. We report that the PRC2 paralogs Ezh1 and Ezh2 are differentially expressed during hippocampal development. We show that depletion of Ezh2 leads to increased expression of PSD-95, a critical plasticity gene, and that reduced PSD-95 gene transcription is correlated with enrichment of Ezh2 at the PSD-95 gene promoter; however, the H3K27me3 epigenetic mark is not present at the PSD-95 gene promoter, likely due to the antagonizing effects of the H3S28P and H3K27Ac marks and the activity of the H3K27 demethylases JMJD3 and UTX. In contrast, increased PSD-95 gene transcription is accompanied by the presence of Ezh1 and elongation-engaged RNA Polymerase II complexes at the PSD-95 gene promoter, while knock-down of Ezh1 reduces PSD-95 transcription. These results indicate that Ezh1 and Ezh2 have antagonistic roles in regulating PSD-95 transcription.