Molecular Biology


    Protocols in Current Issue
    Mapping mRNA-18S rRNA Contacts Within Translation Initation Complex by Means of Reverse Transcriptase Termination Sites and RNAseq
    Authors:  Irene Díaz-López, René Toribio and Iván Ventoso, date: 08/20/2020, view: 1524, Q&A: 0
    [Abstract] The nucleotides involved in RNA-RNA interaction can be tagged by chemical- or UV-induced crosslinking, and further identified by classical or modern high throughput techniques. The contacts of mRNA with 18S rRNA that occur along the mRNA channel of 40S subunit have been mapped by site-specific UV crosslinking followed by reverse transcriptase ...
    Ribonucleoprotein Immunoprecipitation (RIP) Analysis
    Authors:  Jennifer L. Martindale, Myriam Gorospe and Maria L. Idda, date: 01/20/2020, view: 3904, Q&A: 0
    [Abstract] RNAs and RNA-binding proteins (RBPs) can interact dynamically in ribonucleoprotein (RNP) complexes that play important roles in controlling gene expression programs. One of the powerful ways to investigate changes in the association of RNAs with an RBP of interest is by immunoprecipitation (IP) analysis of native RNPs. RIP (RNP ...
    Protocol for Ribosome Profiling in Bacteria
    Authors:  Fuad Mohammad and Allen R. Buskirk, date: 12/20/2019, view: 4374, Q&A: 1
    [Abstract] Ribosome profiling provides information on the position of ribosomes on mRNA on a genomic scale. Although this information is often used to detect changes in gene expression under different conditions, it also has great potential for yielding insight into the mechanism and regulation of protein synthesis itself. First developed in yeast, ribosome ...
    Ectopic Gene Expression in Macrophages Using in vitro Transcribed mRNA
    Authors:  Pallavi Chandra and Jennifer A. Philips, date: 05/20/2018, view: 4717, Q&A: 0
    [Abstract] Macrophages are immune cells that contribute to host defense through various mechanisms including phagocytosis and antigen presentation. Their antimicrobial capacity is subverted by clinically important intracellular pathogens such as Mycobacterium tuberculosis. The study of host-pathogen interactions using these cells is therefore of ...
    Accurate, Streamlined Analysis of mRNA Translation by Sucrose Gradient Fractionation
    Authors:  Soufiane Aboulhouda, Rachael Di Santo, Gabriel Therizols and David Weinberg, date: 10/05/2017, view: 8133, Q&A: 0
    [Abstract] The efficiency with which proteins are produced from mRNA molecules can vary widely across transcripts, cell types, and cellular states. Methods that accurately assay the translational efficiency of mRNAs are critical to gaining a mechanistic understanding of post-transcriptional gene regulation. One way to measure translational efficiency is to ...
    Targeted Gene Mutation in Rice Using a CRISPR-Cas9 System
    Authors:  Kabin Xie, Bastian Minkenberg and Yinong Yang, date: 09/05/2014, view: 28567, Q&A: 0
    [Abstract] RNA-guided genome editing (RGE) using bacterial type II cluster regularly interspaced short palindromic repeats (CRISPR)–associated nuclease (Cas) has emerged as a simple and versatile tool for genome editing in many organisms including plant and crop species. In RGE based on the Streptococcus pyogenes CRISPR-Cas9 system, the Cas9 ...
    Individual-nucleotide-resolution UV Cross-linking and Immunoprecipitation (iCLIP) of UPF1
    Authors:  David Zünd and Oliver Mühlemann, date: 04/05/2014, view: 12294, Q&A: 0
    [Abstract] The fate of mRNA, in particular its stability, localization and rate of translation is regulated by RNA binding proteins assembling to messenger ribonucleoprotein (mRNP) complexes. To investigate the transcriptome-wide RNA binding sites of UPF1, the core factor of nonsense-mediated mRNA decay (NMD), we performed individual-nucleotide-resolution UV ...
    Cellular Translational Reporter Assay
    Authors:  Taishi Kimura and Kiyoshi Takeda, date: 03/20/2014, view: 8847, Q&A: 0
    [Abstract] The method described here allows measuring the effect of exogenously introduced modifications to in vitro-transcribed mRNA on the translation in cells. Using cells derived from knockout mice and control littermates, this method enables to compare the results in the presence or absence of specific gene products. In our lab, we used this ...
    Polysome Profiling Analysis
    Authors:  Masahiro Morita, Tommy Alain, Ivan Topisirovic and Nahum Sonenberg, date: 07/20/2013, view: 41823, Q&A: 2
    [Abstract] Polysome profiling is a method that allows monitoring of translation activity of mRNAs in cells and tissues. Once each polysome fractions are collected, the translation activity of each mRNA is analyzed using various molecular biology techniques such as Northern blotting, RT-PCR, microarray or deep-sequencing.
    Polysome Preparation, RNA Isolation and Analysis
    Authors:  Hailong Zhang and Muxiang Zhou, date: 11/05/2012, view: 25104, Q&A: 2
    [Abstract] During mRNA translation, 40S and 60S ribosomal subunits bind to target mRNA forming into an 80S complex (monosome). This ribosome moves along the mRNA during translational elongation to facilitate tRNA reading codon, where translation is activated and many monosomes can bind the same mRNA simutaneously, which forms polysomes. Polysomes can be ...

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