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Related Concept Videos

Ribosome Profiling02:24

Ribosome Profiling

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
Ribosome profiling has many applications, including in vivo monitoring of translation inside a particular organ or tissue type and quantifying new protein synthesis levels.
The technique helps...
Translational Regulation01:29

Translational Regulation

Translational regulation in prokaryotes ensures efficient protein synthesis by controlling ribosome access to mRNA. This regulation is mediated by secondary RNA structures, including translational riboswitches, RNA thermometers, and small RNAs (sRNAs), which respond to intracellular and environmental signals to modulate gene expression.Translational RiboswitchesRiboswitches in the leader region of mRNAs can regulate translation by altering the accessibility of the Shine-Dalgarno (SD) sequence,...
Leaky Scanning02:28

Leaky Scanning

During most eukaryotic translation processes, the small 40S ribosome subunit scans an mRNA from its 5' end until it encounters the first start AUG codon. The large 60S ribosomal subunit then joins the smaller one to initiate protein synthesis. The location of the translation initiation is largely determined by the nucleotides near the start codon as there may be multiple translation initiation sites present on the mRNA.  Marilyn Kozak discovered that the sequence RCCAUGG (where R stands for...
DNA Microarrays02:34

DNA Microarrays

Microarrays are high-throughput and relatively inexpensive assays that can be automated to analyze large quantities of data at a time. They are used in genome-wide studies to compare gene or protein expression under two varied conditions, such as healthy and diseased states. Microarrays consist of glass or silica slides on which probe molecules are covalently attached through surface functionalization. Most commonly, the slides are prepared through the chemisorption of silanes to silica...

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Quantitative Immunofluorescence to Measure Global Localized Translation
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Published on: August 22, 2017

Translational control analysis by translationally active RNA capture/microarray analysis (TrIP-Chip).

Kenji Kudo1, Yaguang Xi, Yuan Wang

  • 1Mitchell Cancer Institute, Mobile, AL 36688, USA.

Nucleic Acids Research
|February 4, 2010
PubMed
Summary
This summary is machine-generated.

Researchers developed a novel method to study gene translation in small cell samples. This technique reveals changes in actively translating messenger RNAs (mRNAs) using heat shock protein 70 (hsp70) chaperones, enabling sensitive analysis of translational control.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Post-transcriptional regulation plays a crucial role in gene expression.
  • Studying actively translating messenger RNAs (mRNAs) is essential for understanding gene regulation.
  • Current methods are often limited by the need for large cell quantities.

Purpose of the Study:

  • To develop a novel technique for studying translational control in small cell populations.
  • To isolate and analyze actively translating mRNAs associated with polysomes.
  • To enable high-throughput expression profiling of translationally regulated genes.

Main Methods:

  • Utilized molecular chaperones (hsp70s) that bind to nascent polypeptide chains on polysomes.
  • Developed affinity capture beads to isolate hsp70-polysome complexes.
  • Applied high-throughput expression profiling to captured mRNAs.
  • Validated the approach using in vitro translation systems and HCT-116 colon cancer cells.

Main Results:

  • Demonstrated feasibility with thymidylate synthase (TS) mRNA in vitro.
  • Showed increased polysome-associated mRNA levels for TS and p53 in HCT-116 cells after 5-fluorouracil treatment, despite unaltered steady-state mRNA levels.
  • Successfully identified translational rate differences from as few as 500 cells.

Conclusions:

  • The developed approach effectively isolates actively translating mRNAs from limited cell samples.
  • This method reveals translational regulation dynamics not apparent from steady-state mRNA analysis.
  • The technology facilitates the study of translational control in clinical specimens with small cell numbers.