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Related Concept Videos

Ribosome Profiling02:24

Ribosome Profiling

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Ribosome profiling or ribo-sequencing is a deep sequencing technique that produces a snapshot of active translation in a cell. It selectively sequences the mRNAs protected by ribosomes to get an insight into a cell’s translation landscape at any given point in time.
Applications of ribosome profiling
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RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases. 
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Proteomics01:33

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A proteome is the entire set of proteins that a cell type produces. We can study proteomes using the knowledge of genomes because genes code for mRNAs, and the mRNAs encode proteins. Although mRNA analysis is a step in the right direction, not all mRNAs are translated into proteins.
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Reporter genes are a type of protein-coding gene that are often tagged to a gene of interest. Once inside a target cell, reporter genes usually produce visually identifiable characteristics like fluorescence and luminescence when expressed along with the gene of interest. Thus, reporter genes “report” the presence or absence of genes of interest in an organism, determine the gene expression pattern, or track the physical location of a DNA segment or protein in the cell.
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Related Experiment Video

Updated: Nov 1, 2025

TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of Drosophila Embryos
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Methods to Study Translated Pseudogenes: Recombinant Expression and Complementation, Targeted Proteomics, and RNA

Anne Parle-McDermott1,2, Niamh Bookey3,4, Paula Meleady4

  • 1Molecular Genetics Laboratory, School of Biotechnology, Dublin City University, Dublin, Ireland. anne.parle-mcdermott@dcu.ie.

Methods in Molecular Biology (Clifton, N.J.)
|June 24, 2021
PubMed
Summary
This summary is machine-generated.

Proving pseudogene translation into functional proteins is challenging. Researchers use PCR-based expression and proteomics to assess pseudogenic protein activity, localization, and function.

Keywords:
CloningDHFRExpression proteomicsPseudogeneRNA profiling

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Pseudogenes are DNA sequences similar to genes but generally considered non-functional.
  • Determining if pseudogenes are transcribed and translated into functional proteins presents significant technical hurdles, primarily related to specificity.

Purpose of the Study:

  • To outline methods for verifying the functional expression of pseudogenic proteins.
  • To enable the assessment of enzymatic activity, subcellular localization, and complementation capacity of pseudogenic proteins.

Main Methods:

  • Utilizing Polymerase Chain Reaction (PCR) to generate clones for exogenous expression of pseudogenic proteins (native or tagged).
  • Employing targeted proteomics analysis to detect endogenous pseudogenic proteins.
  • Assessing the full spectrum of endogenous RNA isoforms.

Main Results:

  • Exogenous expression facilitates purification, detection, and functional complementation assays in bacterial and mammalian systems.
  • Enzymatic activity, subcellular localization, and complementation with parental homologs can be evaluated.
  • Proteomics and RNA isoform analysis offer alternative routes to investigate pseudogenic protein functionality.

Conclusions:

  • The described PCR-based expression system provides a robust approach to validate pseudogenic protein function.
  • Targeted proteomics and comprehensive RNA analysis are crucial for a complete understanding of pseudogene potential.
  • These methods advance the study of pseudogene roles in cellular processes.