18 research outputs found
A practical approach to the understanding and teaching of RNA silencing in plants
Gene silencing, also called RNA interference (RNAi) is a specific
mechanism of RNA degradation involved in gene regulation, development
and defense in eukaryotic organisms. It became an important subject in
the teaching programs of molecular biology, genetics and biotechnology
courses in the last years. The aim of this work is to provide simple
and inexpensive assays to understand and teach gene silencing using
plants as model systems. The use of transient and permanent transgenic
plants for expressing reporter genes, like those derived from jellyfish
green fluorescent protein (gfp) encoding gene, provides a nice,
colorful and conclusive image of gene silencing. Three experimental
approaches to evidence RNA silencing are depicted. In the first
approach gene silencing is demonstrated after transient expression of
reporter genes in non-transgenic plants. In the second, silencing is
triggered against a reporter gene stably integrated into a transgenic
plant. The third approach involves the triggering of RNA silencing
against endogenous genes using viral vectors. In addition we illustrate
systemic gene silencing showing how the silencing signal is spread over
a plant and finally it is also demonstrated the suppression of gene
silencing. The first group of experiments is recommended to be tough on
undergraduate courses, the following two sections are recommended for
graduate courses. Hopefully, it will help students to understand this
important phenomenon and to unravel the importance of gene silencing as
a key gene regulation mechanism and as a molecular and biotechnological
tool
Virus infection elevates transcriptional activity of miR164a promoter in plants
Background: Micro RNAs (miRs) constitute a large group of endogenous small RNAs that have crucial roles in many important plant functions. Virus infection and transgenic expression of viral proteins alter accumulation and activity of miRs and so far, most of the published evidence involves post-transcriptional regulations.
Results: Using transgenic plants expressing a reporter gene under the promoter region of a characterized miR (P-miR164a), we monitored the reporter gene expression in different tissues and during Arabidopsis development. Strong expression was detected in both vascular tissues and hydathodes. P-miR164a activity was developmentally regulated in plants with a maximum expression at stages 1.12 to 5.1 (according to Boyes, 2001) along the transition from vegetative to reproductive growth. Upon quantification of P-miR164a-derived GUS activity after Tobacco mosaic virus Cg or Oilseed rape mosaic virus (ORMV) infection and after hormone treatments, we demonstrated that ORMV and gibberellic acid elevated P-miR164a activity. Accordingly, total mature miR164, precursor of miR164a and CUC1 mRNA (a miR164 target) levels increased after virus infection and interestingly the most severe virus (ORMV) produced the strongest promoter induction.
Conclusion: This work shows for the first time that the alteration of miR pathways produced by viral infections possesses a transcriptional component. In addition, the degree of miR alteration correlates with virus severity since a more severe virus produces a stronger P-miR164a induction.Instituto de BiotecnologĂaFil: Bazzini, Ariel Alejandro. Instituto Nacional de TecnologĂa Agropecuaria (INTA). Instituto de BiotecnologĂa; Argentina. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas; ArgentinaFil: Almasia, Natalia Ines. Instituto Nacional de TecnologĂa Agropecuaria (INTA). Instituto de BiotecnologĂa; Argentina. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas; ArgentinaFil: Manacorda, Carlos Augusto. Instituto Nacional de TecnologĂa Agropecuaria (INTA). Instituto de BiotecnologĂa; ArgentinaFil: Mongelli, Vanesa Claudia. Instituto Nacional de TecnologĂa Agropecuaria (INTA). Instituto de BiotecnologĂa; Argentina. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas; ArgentinaFil: Conti, Gabriela. Instituto Nacional de TecnologĂa Agropecuaria (INTA). Instituto de BiotecnologĂa; ArgentinaFil: Maroniche, Guillermo AndrĂ©s. Instituto Nacional de TecnologĂa Agropecuaria (INTA). Instituto de BiotecnologĂa; Argentina. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas; ArgentinaFil: Rodriguez, Maria Cecilia. Instituto Nacional de TecnologĂa Agropecuaria (INTA). Instituto de BiotecnologĂa; Argentina. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas; ArgentinaFil: Distefano, Ana Julia. Instituto Nacional de TecnologĂa Agropecuaria (INTA). Instituto de BiotecnologĂa; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas; ArgentinaFil: Hopp, Horacio Esteban. Instituto Nacional de TecnologĂa Agropecuaria (INTA). Instituto de BiotecnologĂa; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Del Vas, Mariana. Instituto Nacional de TecnologĂa Agropecuaria (INTA). Instituto de BiotecnologĂa; Argentina. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas; ArgentinaFil: Asurmendi, Sebastian. Instituto Nacional de TecnologĂa Agropecuaria (INTA). Instituto de BiotecnologĂa; Argentina. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas; Argentin
linc-mipep and linc-wrb encode micropeptides that regulate chromatin accessibility in vertebrate-specific neural cells
Thousands of long intergenic non-coding RNAs (lincRNAs) are transcribed throughout the vertebrate genome. A subset of lincRNAs enriched in developing brains have recently been found to contain cryptic open-reading frames and are speculated to encode micropeptides. However, systematic identification and functional assessment of these transcripts have been hindered by technical challenges caused by their small size. Here, we show that two putative lincRNAs (linc-mipep, also called lnc-rps25, and linc-wrb) encode micropeptides with homology to the vertebrate-specific chromatin architectural protein, Hmgn1, and demonstrate that they are required for development of vertebrate-specific brain cell types. Specifically, we show that NMDA receptor-mediated pathways are dysregulated in zebrafish lacking these micropeptides and that their loss preferentially alters the gene regulatory networks that establish cerebellar cells and oligodendrocytes - evolutionarily newer cell types that develop postnatally in humans. These findings reveal a key missing link in the evolution of vertebrate brain cell development and illustrate a genetic basis for how some neural cell types are more susceptible to chromatin disruptions, with implications for neurodevelopmental disorders and disease
Metabolic and miRNA Profiling of TMV Infected Plants Reveals Biphasic Temporal Changes
Plant viral infections induce changes including gene expression and metabolic components. Identification of metabolites and microRNAs (miRNAs) differing in abundance along infection may provide a broad view of the pathways involved in signaling and defense that orchestrate and execute the response in plant-pathogen interactions. We used a systemic approach by applying both liquid and gas chromatography coupled to mass spectrometry to determine the relative level of metabolites across the viral infection, together with a miRs profiling using a micro-array based procedure. Systemic changes in metabolites were characterized by a biphasic response after infection. The first phase, detected at one dpi, evidenced the action of a systemic signal since no virus was detected systemically. Several of the metabolites increased at this stage were hormone-related. miRs profiling after infection also revealed a biphasic alteration, showing miRs alteration at 5 dpi where no virus was detected systemically and a late phase correlating with virus accumulation. Correlation analyses revealed a massive increase in the density of correlation networks after infection indicating a complex reprogramming of the regulatory pathways, either in response to the plant defense mechanism or to the virus infection itself. Our data propose the involvement of a systemic signaling on early miRs alteration
miR-430 regulates zygotic mRNA during zebrafish embryogenesis
Abstract Background Early embryonic developmental programs are guided by the coordinated interplay between maternally inherited and zygotically manufactured RNAs and proteins. Although these processes happen concomitantly and affecting gene function during this period is bound to affect both pools of mRNAs, it has been challenging to study their expression dynamics separately. Results By employing SLAM-seq, a nascent mRNA labeling transcriptomic approach, in a developmental time series we observe that over half of the early zebrafish embryo transcriptome consists of maternal-zygotic genes, emphasizing their pivotal role in early embryogenesis. We provide an hourly resolution of de novo transcriptional activation events and follow nascent mRNA trajectories, finding that most de novo transcriptional events are stable throughout this period. Additionally, by blocking microRNA-430 function, a key post transcriptional regulator during zebrafish embryogenesis, we directly show that it destabilizes hundreds of de novo transcribed mRNAs from pure zygotic as well as maternal-zygotic genes. This unveils a novel miR-430 function during embryogenesis, fine-tuning zygotic gene expression. Conclusion These insights into zebrafish early embryo transcriptome dynamics emphasize the significance of post-transcriptional regulators in zygotic genome activation. The findings pave the way for future investigations into the coordinated interplay between transcriptional and post-transcriptional landscapes required for the establishment of animal cell identities and functions
A practical approach to the understanding and teaching of RNA silencing in plants
Gene silencing, also called RNA interference (RNAi) is a specific
mechanism of RNA degradation involved in gene regulation, development
and defense in eukaryotic organisms. It became an important subject in
the teaching programs of molecular biology, genetics and biotechnology
courses in the last years. The aim of this work is to provide simple
and inexpensive assays to understand and teach gene silencing using
plants as model systems. The use of transient and permanent transgenic
plants for expressing reporter genes, like those derived from jellyfish
green fluorescent protein (gfp) encoding gene, provides a nice,
colorful and conclusive image of gene silencing. Three experimental
approaches to evidence RNA silencing are depicted. In the first
approach gene silencing is demonstrated after transient expression of
reporter genes in non-transgenic plants. In the second, silencing is
triggered against a reporter gene stably integrated into a transgenic
plant. The third approach involves the triggering of RNA silencing
against endogenous genes using viral vectors. In addition we illustrate
systemic gene silencing showing how the silencing signal is spread over
a plant and finally it is also demonstrated the suppression of gene
silencing. The first group of experiments is recommended to be tough on
undergraduate courses, the following two sections are recommended for
graduate courses. Hopefully, it will help students to understand this
important phenomenon and to unravel the importance of gene silencing as
a key gene regulation mechanism and as a molecular and biotechnological
tool
Optimized CRISPR-RfxCas13d system for RNA targeting in zebrafish embryos
CRISPR-Cas systems have been used to induce DNA mutagenesis for gene function discovery. However, the development of tools to eliminate RNAs provides complementary and unique approaches to disrupt gene expression. Here, we present a workflow to perform specific, efficient, and cost-effective mRNA knockdown in zebrafish embryos using our in vivo optimized CRISPR-RfxCas13d (CasRx) system. Although the described protocol focuses on mRNA knockdown in zebrafish embryos, it can also be applied to other vertebrates.This work was supported by Ramon y Cajal (RyC-2017-23041), PGC2018-097260-B-I00 grant and MDM-2016-0687 program (Spanish Ministerio de Ciencia, InnovaciĂłn y Universidades, and European Union), Universidad Pablo de Olavide (UPO) Research and the Springboard programs from UPO and CABD, respectively (M.A.M.-M.). This study was supported by the Stowers Institute for Medical Research. A.A.B. was awarded a Pew Innovation Fund and the US National Institutes of Health (R01 GM136849). This work was performed as part of the research for the obtainment of a G.dS.P degree., Graduate School of the Stowers Institute for Medical Research. The CABD is an institution funded by Pablo de Olavide University, Consejo Superior de Investigaciones CientĂficas (CSIC), and Junta de AndalucĂa. L.H.-H. is a recipient of a predoctoral fellowship from Ministerio de Ciencia