Related Experiment Video
Updated: Jul 18, 2026
09:16
Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro
Published on: May 3, 2014
Achieving specific RNA cleavage activity by an inactive splicing endonuclease subunit through engineered
1Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306, USA.
Journal of Molecular Biology
|December 19, 2006
Summary
Inactive RNA endonuclease subunits become active through oligomerization. Assembly mode dictates RNA specificity, revealing new insights into enzyme regulation and function.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Protein-protein interactions regulate enzyme activity and substrate specificity.
- RNA endonucleases are crucial for RNA processing and regulation.
- Controlling RNA endonuclease activity is vital for specific RNA targeting.
Purpose of the Study:
- To investigate how an inactive RNA splicing endonuclease subunit can be activated.
- To determine the correlation between oligomerization and RNA specificity.
- To engineer a self-assembling catalytic subunit with RNA cleavage activity.
Main Methods:
- Oligomerization studies of RNA endonuclease subunits.
- RNA cleavage assays to assess activity and specificity.
- Site-directed mutagenesis guided by 3D structural data.
- Analysis of higher-order oligomer formation.
Main Results:
- An inactive RNA endonuclease subunit is activated solely by oligomerization.
- The assembly mode of the subunit influences its RNA specificity.
- Engineered catalytic subunits exhibit specific RNA cleavage activity and form functional tetramers.
- A higher-order oligomer species displays unique RNA cleavage specificity.
Conclusions:
- Oligomerization is a key mechanism for activating and modulating RNA endonuclease function.
- The assembly state of RNA endonucleases dictates their RNA cleavage specificity.
- Engineering self-assembling subunits offers a route to novel enzymatic activities and specificities.
Related Concept Videos
01:32RNA Splicing
Splicing is the process by which eukaryotic RNA is edited before its translation into protein. The RNA strand transcribed from eukaryotic DNA is called the primary transcript. The primary transcripts that become mRNAs are called precursor messenger RNAs (pre-mRNAs). Eukaryotic pre-mRNA contains alternating sequences of exons and introns. Exons are nucleotide sequences that code for proteins, whereas introns are the non-coding regions. In RNA splicing, introns are removed and exons are bonded...
01:32RNA Splicing
Splicing is the process by which eukaryotic RNA is edited before its translation into protein. The RNA strand transcribed from eukaryotic DNA is called the primary transcript. The primary transcripts that become mRNAs are called precursor messenger RNAs (pre-mRNAs). Eukaryotic pre-mRNA contains alternating sequences of exons and introns. Exons are nucleotide sequences that code for proteins, whereas introns are the non-coding regions. In RNA splicing, introns are removed and exons are bonded...
02:18Alternative RNA Splicing
Alternative RNA splicing is the regulated splicing of exons and introns to produce different mature mRNAs from a single pre-mRNA. Unlike in constitutive splicing where a single gene produces a single type of mRNA, alternative splicing allows an organism to produce multiple proteins from a single gene and plays an important role in protein diversity.
There are five types of alternative RNA splicing that vary in the ways the pre-mRNA segments are removed or retained in the mature mRNA. The first...
There are five types of alternative RNA splicing that vary in the ways the pre-mRNA segments are removed or retained in the mature mRNA. The first...
02:47Ribozymes
The term ribozyme is used for RNA that can act as an enzyme. Ribozymes are mainly found in selected viruses, bacteria, plant organelles, and lower eukaryotes. Ribozymes were first discovered in 1982 when Tom Cech’s laboratory observed Group I introns acting as enzymes. This was shortly followed by the discovery of another ribozyme, Ribonulcease P, by Sid Altman’s laboratory. Both Cech and Altman received the Nobel Prize in chemistry in 1989 for their work on ribozymes.
Ribozymes can be...
Ribozymes can be...
01:11Restriction Enzymes
Restriction enzymes are bacterial enzymes used to cut DNA in a sequence-specific manner. To cleave DNA, they bind to specific palindromic sequences called restriction sites. Such palindromic DNA sequences or inverted repeats are commonly found in regions of functional significance, such as the origin of replication, gene operator sites, and regions containing transcription termination signals.
The host bacteria protect their own genomic DNA from these enzymes by methylating these sites. Some...
The host bacteria protect their own genomic DNA from these enzymes by methylating these sites. Some...
01:32Pre-mRNA Processing: RNA Splicing
Splicing is the process by which eukaryotic RNA is edited before its translation into protein. The RNA strand transcribed from eukaryotic DNA is called the primary transcript. The primary transcripts that become mRNAs are called precursor messenger RNAs (pre-mRNAs). Eukaryotic pre-mRNA contains alternating sequences of exons and introns. Exons are nucleotide sequences that code for proteins, whereas introns are the non-coding regions. In RNA splicing, introns are removed and exons are bonded...