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Related Concept Videos

mRNA Stability and Gene Expression02:51

mRNA Stability and Gene Expression

The structure and stability of mRNA molecules regulates gene expression, as mRNAs are a key step in the pathway from gene to protein. In eukaryotes, the half-life of mRNA varies from a few minutes up to several days. mRNA stability is essential in growth and development. The absence of the proteins regulating its stability, such as tristetraprolin in mice, can cause systemic issues, including bone marrow overgrowth, inflammation, and autoimmunity.
Cis-acting Elements involved in mRNA stability
Pre-mRNA Processing: Modification of pre-mRNA Ends01:35

Pre-mRNA Processing: Modification of pre-mRNA Ends

In eukaryotic cells, transcripts made by RNA polymerase are modified and processed before exiting the nucleus. Unprocessed RNA is called precursor mRNA or pre-mRNA to distinguish it from mature mRNA.
Once about 20-40 ribonucleotides have been joined together by RNA polymerase, a group of enzymes adds a cap to the 5' end of the growing transcript. In this process, a 5' phosphate is replaced by modified guanosine that has a methyl group attached (7-methyl guanosine). This 5' cap helps the cell...
pre-mRNA Processing02:01

pre-mRNA Processing

In eukaryotic cells, transcripts made by RNA polymerase are modified and processed before exiting the nucleus. Unprocessed RNA is called precursor mRNA or pre-mRNA to distinguish it from mature mRNA.
Once about 20-40 ribonucleotides have been joined together by RNA polymerase, a group of enzymes adds a “cap” to the 5’ end of the growing transcript. In this process, a 5’ phosphate is replaced by modified guanosine that has a methyl group attached to it (7-Methyl guanosine). This 5’ cap helps the...
Pre-mRNA Processing02:01

Pre-mRNA Processing

In eukaryotic cells, transcripts made by RNA polymerase are modified and processed before exiting the nucleus. Unprocessed RNA is called precursor mRNA or pre-mRNA to distinguish it from mature mRNA.
Once about 20-40 ribonucleotides have been joined together by RNA polymerase, a group of enzymes adds a “cap” to the 5’ end of the growing transcript. In this process, a 5’ phosphate is replaced by modified guanosine that has a methyl group attached to it (7-Methyl guanosine). This 5’ cap helps the...
Nonsense-mediated mRNA Decay02:27

Nonsense-mediated mRNA Decay

The Upf proteins that carry out nonsense-mediated decay (NMD) are found in all eukaryotic organisms, including humans. Each protein has an individual role, but they need to work in collaboration. Upf1 is an ATP-dependent RNA helicase that unwinds the RNA helix. Because Upf1 can unwind any RNA, Upf2 and Upf3 are required to help Upf1 discriminate between nonsense and normal mRNAs.
Usually, Upf3 binds to an Exon Junction Complex (EJC) at mRNA splice sites. If a ribosome fully translates the mRNA,...
Nonsense-mediated mRNA Decay02:27

Nonsense-mediated mRNA Decay

The Upf proteins that carry out nonsense-mediated decay (NMD) are found in all eukaryotic organisms, including humans. Each protein has an individual role, but they need to work in collaboration. Upf1 is an ATP-dependent RNA helicase that unwinds the RNA helix. Because Upf1 can unwind any RNA, Upf2 and Upf3 are required to help Upf1 discriminate between nonsense and normal mRNAs.
Usually, Upf3 binds to an Exon Junction Complex (EJC) at mRNA splice sites. If a ribosome fully translates the mRNA,...

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Probing RNA Structure with Dimethyl Sulfate Mutational Profiling with Sequencing In Vitro and in Cells
10:34

Probing RNA Structure with Dimethyl Sulfate Mutational Profiling with Sequencing In Vitro and in Cells

Published on: December 9, 2022

Analysis of mRNA decapping.

Shin-Wu Liu1, Xinfu Jiao, Sarah Welch

  • 1Rutgers University, Department of Cell Biology and Neuroscience, Piscataway, New Jersey, USA.

Methods in Enzymology
|December 30, 2008
PubMed
Summary
This summary is machine-generated.

mRNA decay regulates gene expression through decapping enzymes like Dcp2 and DcpS. This study details methods to monitor their activities in human cells, aiding research into mRNA degradation pathways.

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Area of Science:

  • Molecular Biology
  • Gene Expression Regulation
  • Biochemistry

Background:

  • mRNA decay is crucial for controlling gene expression in eukaryotes.
  • Two main pathways, 5' to 3' and 3' to 5', degrade mRNA, both starting with poly(A) tail removal.
  • Decapping, the hydrolysis of the 5'-cap structure, is a key mRNA degradation step.

Purpose of the Study:

  • To describe methods for monitoring the decapping activities of human decapping enzymes.
  • To enable the study of Dcp2 and DcpS enzyme functions in mRNA decay.

Main Methods:

  • Bacterial expression of human decapping enzymes.
  • Monitoring decapping activities of both bacterially expressed and endogenous human enzymes.
  • Utilizing established biochemical assays to quantify enzyme function.

Main Results:

  • Established protocols for measuring Dcp2 and DcpS decapping activities.
  • Demonstrated the ability to assess enzyme function using both recombinant and native enzyme sources.
  • Provided a foundation for further investigation into the specific roles of Dcp2 and DcpS.

Conclusions:

  • The described methods allow for robust monitoring of Dcp2 and DcpS decapping activities.
  • These techniques are applicable to both purified and endogenous enzyme preparations.
  • Facilitates deeper understanding of mRNA decay mechanisms and gene expression regulation.