Protocols in Current Issue
    Monitoring Real-time Temperature Dynamics of a Short RNA Hairpin Using Förster Resonance Energy Transfer and Circular Dichroism
    Authors:  Martin Balcerowicz, Marco Di Antonio and Betty Y. W. Chung, date: 03/20/2021, view: 1038, Q&A: 0

    RNA secondary structures are highly dynamic and subject to prompt changes in response to the environment. Temperature in particular has a strong impact on RNA structural conformation, and temperature-sensitive RNA hairpin structures have been exploited by multiple organisms to modify the rate of translation in response to temperature changes.

    Trypanosomatid, fluorescence-based in vitro U-insertion/U-deletion RNA-editing (FIDE)
    Authors:  Wolf-Matthias Leeder, Elisabeth Kruse and H. Ulrich Göringer, date: 03/05/2021, view: 439, Q&A: 0

    Gene expression within the mitochondria of African trypanosomes and other protozoan organisms relies on a nucleotide-specific RNA-editing reaction. In the process exclusively uridine (U)-nucleotides are site-specifically inserted into and deleted from sequence-deficient primary transcripts to convert them into translatable mRNAs. The reaction is

    Identification of Intrinsic RNA Binding Specificity of Purified Proteins by in vitro RNA Immunoprecipitation (vitRIP)
    Authors:  Marisa Müller, Tamas Schauer and Peter B. Becker, date: 03/05/2021, view: 956, Q&A: 0

    RNA-protein interactions are often mediated by dedicated canonical RNA binding domains. However, interactions through non-canonical domains with unknown specificity are increasingly observed, raising the question how RNA targets are recognized. Knowledge of the intrinsic RNA binding specificity contributes to the understanding of target

    Plant ARGONAUTE Protein Immunopurification for Pathogen Cross Kingdom Small RNA Analysis
    Authors:  Florian Dunker, Bernhard Lederer and Arne Weiberg, date: 02/05/2021, view: 700, Q&A: 0

    Over the last decade, it has been noticed that microbial pathogens and pests deliver small RNA (sRNA) effectors into their host plants to manipulate plant physiology and immunity for infection, known as cross kingdom RNA interference. In this process, fungal and oomycete parasite sRNAs hijack the plant ARGONAUTE (AGO)/RNA-induced silencing complex

    Ribosome Purification from an α-proteobacterium and rRNA Analysis by Northern Blot
    Authors:  Liang Yin and Caroline S. Harwood, date: 12/05/2020, view: 650, Q&A: 0
    [Abstract] Ribosomes are an integral part of cellular life. They are complex molecular machines consisting of multiple ribosomal proteins and RNAs. To study different aspects of ribosome composition, many methods have been developed over the decades. Here, we describe how to purify ribosomes from the α-proteobacterium Rhodopseudomonas palustris ...
    Fluorescent Polysome Profiling in Caenorhabditis elegans
    Authors:  Dan Shaffer and Jarod A Rollins, date: 09/05/2020, view: 1761, Q&A: 0
    [Abstract] An important but often overlooked aspect of gene regulation occurs at the level of protein translation. Many genes are regulated not only by transcription but by their propensity to be recruited to actively translating ribosomes (polysomes). Polysome profiling allows for the separation of unbound 40S and 60S subunits, 80S monosomes, and actively ...
    RNA Stability Measurements Using RT-qPCR in Arabidopsis Seedlings
    Authors:  Tianran Jia and Brandon H. Le, date: 07/20/2020, view: 1372, Q&A: 0
    [Abstract] Steady-state mRNA levels are determined by both the rates of transcription and degradation. Regulation of mRNA stability and/or degradation are key factors that can significantly affect mRNA levels and its biological functions. mRNA stability can be measured indirectly after transcription inhibition. This protocol described a rapid and sensitive ...
    Preparation of Yeast tRNA Sample for NMR Spectroscopy
    Authors:  Marjorie Catala, Alexandre Gato, Carine Tisné and Pierre Barraud, date: 06/20/2020, view: 1446, Q&A: 0
    [Abstract] Transfer RNAs (tRNAs) are heavily decorated with post-transcriptional modifications during their biosynthesis. To fulfil their functions within cells, tRNAs undergo a tightly controlled biogenesis process leading to the formation of mature tRNAs. In addition, functions of tRNAs are often modulated by their modifications. Although the biological ...
    Biochemical Pulldown of mRNAs and Long Noncoding RNAs from Cellular Lysates Coupled with Mass Spectrometry to Identify Protein Binding Partners
    Authors:  Anca F. Savulescu, Stoyan Stoychev, Sipho Mamputha and Musa M. Mhlanga, date: 06/05/2020, view: 2126, Q&A: 0
    [Abstract] RNA binding proteins (RBPs) interact with cellular mRNAs, controlling various steps throughout the lifetime of these transcripts, including transcription, cellular transport, subcellular localization, translation and degradation. In addition to binding mRNA transcripts, a growing number of RBPs are shown to bind long noncoding RNAs (lncRNAs), ...
    Confocal and Super-resolution Imaging of RNA in Live Bacteria Using a Fluorogenic Silicon Rhodamine-binding Aptamer
    Authors:  Regina Wirth, Peng Gao, G. Ulrich Nienhaus, Murat Sunbul and Andres Jäschke, date: 05/05/2020, view: 3734, Q&A: 0
    [Abstract] Genetically encoded light-up RNA aptamers have been shown to be promising tools for the visualization of RNAs in living cells, helping us to advance our understanding of the broad and complex life of RNA. Although a handful of light-up aptamers spanning the visible wavelength region have been developed, none of them have yet been reported to be ...

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