Project description:Transcriptional response of Bacillus subtilis to ramoplanin in wild-type CU1065. Bacillus subtilis CU1065, WT (-RAM) vs. (+RAM) and liaR (yvqC) deletion (-RAM) vs. (+RAM). The experiment was conducted in triplicate using three independent total RNA preparations. Combined reference RNA was labeled with Cy3 and ramoplanin treated/untreated samples were labeled with Cy5.
Project description:mRNA cap addition occurs early during RNA pol II transcription, facilitating pre-mRNA processing and translation. We report that the mammalian mRNA cap methyltransferase, RNMT-RAM, promotes RNA pol II transcription, independently of mRNA capping and translation. In cells, sub-lethal suppression of RNMT-RAM reduces RNA pol II occupancy, net mRNA synthesis and pre-mRNA levels. Conversely, expression of RNMT-RAM increases transcription independently of cap methyltransferase activity. In isolated nuclei, recombinant RNMT-RAM stimulates transcriptional output; this requires the RAM RNA-binding domain. RNMT-RAM interacts with nascent transcripts along their entire length and with transcription associated factors including RNA pol II subunits, SPT4, SPT6 and PAFc. Suppression of RNMT-RAM inhibits transcriptional markers including histone H2B K120 ubiquitination, H3 K4 and K36 methylation, RNA pol II S5 and S2 phosphorylation and PAFc recruitment. These findings suggest that multiple interactions between RNMT-RAM, RNA pol II factors and RNA along the transcription unit stimulate transcription.
Project description:To explore the bacterial community profile of the gut of the African palm weevil and to identify the abundance and diversity of lignin degradation-associated bacteria in each gut segment.
Project description:We investigated the role of RNMT and its cofactor RAM in T cells using conditional knockout mouse models. We demonstrate that RNMT and RAM have broadly overlapping roles in CD4 T cell activation.
Project description:An optimal size of postembryonic root apical meristem (RAM) is achieved by a balance between cell division and differentiation. Despite extensive research, molecular mechanisms underlying the spatiotemporal coordination of cell division and differentiation remain largely unknown. Here, we report that ORESARA 15 (ORE15), an Arabidopsis PLATZ transcription factor preferentially expressed in the RAM, determines RAM size. The primary root length, RAM size, cell division rate, and stem cell niche activity were reduced in an ore15 loss-of-function mutant but enhanced in an activation-tagged line overexpressing ORE15 compared with the wild type. We further demonstrate that ORE15 regulates auxin transport and distribution, at least partly, by modulating the expression and localization of the auxin importer AUXIN RESISTANT 1 (AUX1). ORE15 forms mutually positive and negative feedback loops with auxin and cytokinin signaling, respectively. Collectively, our findings imply that ORE15 controls the RAM size by mediating the antagonistic interaction between auxin and cytokinin.
Project description:Long non-coding RNAs are important regulators of diverse biological prosesses. Here, we report on functional identification and characterization of a novel long intergenic noncoding RNA with MyoD-regulated and skeletal muscle-restricted expression that promotes the activation of the myogenic program, and is therefore termed Linc-RAM (Linc-RNA Activator of Myogenesis). Linc-RAM is transcribed from an intergenic region of myogenic cells and its expression is upregulated during myogenesis. Notably, in vivo functional studies show that Linc-RAM knockout mice display impaired muscle regeneration due to differentiation defect of satellite cells. Mechanistically, Linc-RAM regulates expression of myogenic genes by directly binding MyoD, which in turn promotes the assembly of the MyoD-Baf60c-Brg1 complex on the regulatory elements of target genes. Collectively, our findings reveal the functional role and molecular mechanism of a lineage-specific Linc-RAM as a regulatory lncRNA required for tissues-specific chromatin remodeling and gene expression.
Project description:The mRNA cap recruits factors essential for transcript processing and translation initiation. We report that regulated mRNA cap methylation is a feature of embryonic stem cell (ESC) differentiation. Expression of the mRNA cap methyltransferase activating subunit, RAM, is elevated in ESCs resulting in high levels of mRNA cap methylation and expression of Oct4 and Sox2 and other pluripotency-associated factors. During neural differentiation, RAM is suppressed which is required for loss of Oct4 and Sox2 and correct expression of neural markers. Cells were treated with control or RAM siRNA and cytosolic RNA or polysome RNA (actively translated) was sequenced.
Project description:Recently, we have identified a point mutation V338Y in mammalian nuclear-encoded mitochondrial protein MrpS5 that confers error-prone mitochondrial protein synthesis. In-vivo, mutation V338Y was associated with a distinct phenotype including impaired mitochondrial function, enhanced susceptibility to noise-induced hearing damage, anxiety-related behavioral alterations, and age-associated metabolic changes in muscle. To challenge the link between V338Y-mediated misreading and in-vivo phenotype, we introduced mutation G315R into the MRPS5 gene; MRPS5 G315R is homologous to the established bacterial ram mutation uS5 G104R. In contrast to the phylogenetically closely related bacterial translation, the corresponding MRPS5 G315R mutation did not affect the accuracy of mitochondrial protein synthesis. In addition, in the absence of mitochondrial misreading, homozygous mutant MRPS5G315R/G315R mice did not show phenotype distinct from wild-type animals.
Project description:Influenza A virus (FLUAV) poses a significant threat to both humans and animals. While vaccination serves as the primary defense against influenza, the effectiveness of currently approved vaccines is suboptimal. To address this issue, we have developed modified live virus (MLV) vaccines against influenza using genome rearrangement techniques targeting the internal gene segments of FLUAV. The rearranged M2 (RAM) strategy involves cloning the M2 ORF downstream of the PB1 ORF in segment 2 and incorporating multiple early stop codons within the M2 ORF in segment 7. Additionally, the IgA-inducing protein (IGIP) coding region was inserted into the HA segment to further attenuate the virus and enhance protective mucosal responses. RAM-IGIP viruses exhibit similar growth rates to wild type (WT) viruses in vitro and remain stable during multiple passages in cells and embryonated eggs. The safety, immunogenicity, and protective efficacy of the RAM-IGIP MLV vaccine against the prototypical 2009 pandemic H1N1 strain A/California/04/2009 (H1N1) (Ca/04) were evaluated in Balb/c mice and compared to a prototypic cold-adapted live attenuated virus vaccine. The results demonstrate that the RAM-IGIP virus exhibits attenuated virulence in vivo. Mice vaccinated with RAM-IGIP and subsequently challenged with an aggressive lethal dose of the Ca/04 strain exhibited complete protection. Analysis of the humoral immune response revealed that the inclusion of IGIP enhanced the production of neutralizing antibodies and augmented the antibody-dependent cellular cytotoxicity response. Similarly, the RAM-IGIP potentiated the mucosal immune response against various FLUAV subtypes. Moreover, increased antibodies against NP and NA responses were observed. These findings support the development of MLVs utilizing genome rearrangement strategies in conjunction with the incorporation of immunomodulators.