Related Experiment Video
Updated: Aug 2, 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
Multiple factors are required for poly(A) addition to a mRNA 3' end
M A McDevitt1, G M Gilmartin, W H Reeves
1Howard Hughes Medical Institute, Laboratory of Molecular Cell Biology, Rockefeller University, New York, New York 10021.
Genes & Development
|May 1, 1988
Summary
This study identifies at least two essential factors for polyadenylation, the process of adding a poly(A) tail to mRNA. One factor is poly(A) polymerase, while another component appears to confer specificity to this crucial RNA processing step.
Area of Science:
- Molecular Biology
- Gene Expression Regulation
- RNA Processing
Background:
- Polyadenylation is a critical step in mRNA maturation, involving endonucleolytic cleavage and the addition of a poly(A) tail.
- This process occurs in the nucleus and is essential for mRNA stability, export, and translation.
Purpose of the Study:
- To identify and characterize the molecular components responsible for specific poly(A) addition in HeLa nuclear extracts.
- To elucidate the mechanism and factors involved in the in vitro polyadenylation reaction.
Main Methods:
- Fractionation of HeLa nuclear extracts using DEAE-Sephacel chromatography.
- Assay of poly(A) addition activity in fractionated and reconstituted nuclear extracts.
- Characterization of factors using micrococcal nuclease digestion and anti-Sm sera.
Main Results:
- Poly(A) addition activity was dependent on multiple components within the nuclear extract, requiring recombination of fractions for restoration.
- At least two distinct factors were identified as necessary for specific poly(A) addition.
- One factor was identified as poly(A) polymerase, capable of non-specific AMP addition, while the second factor appears to confer specificity.
Conclusions:
- The polyadenylation process requires the coordinated action of multiple nuclear components, including poly(A) polymerase and a specificity-conferring factor.
- Unlike the cleavage reaction, the poly(A) addition machinery does not appear to require an essential RNA component.
- Understanding these factors is key to comprehending the complexity of mRNA 3' end formation.
Related Concept Videos
02:01pre-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...
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...
02:01Pre-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...
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...
02:41Chromatin Structure Regulates pre-mRNA Processing
In eukaryotic cells, nascent mRNA transcripts need to undergo many post-transcriptional modifications to reach the cell cytoplasm and translate into functional proteins. For a long time, transcription and pre-mRNA processing were considered two independent events that occur sequentially in the cell. However, it has now been well established that transcription and pre-mRNA processing are two simultaneous processes that are precisely regulated inside the cell.
The chromatin structure, especially...
The chromatin structure, especially...
02:36Transfer RNA Synthesis
One of the unique features of tRNA is the presence of modified bases. In some tRNAs, modified bases account for nearly 20% of the total bases in the molecule. Altogether, these unusual bases protect the tRNA from enzymatic degradation by RNases.
Each of these chemical modifications is carried by a specific enzyme, post-transcription. All of these enzymes have unique base and site-specificity. Methylation, the most common chemical modification, is carried by at least nine different enzymes, with...
Each of these chemical modifications is carried by a specific enzyme, post-transcription. All of these enzymes have unique base and site-specificity. Methylation, the most common chemical modification, is carried by at least nine different enzymes, with...
02:33Initiation of Translation
Initiating translation is complex because it involves multiple molecules. Initiator tRNA, ribosomal subunits, and eukaryotic initiation factors (eIFs) are all required to assemble on the initiation codon of mRNA. This process consists of several steps that are mediated by different eIFs.
First, the initiator tRNA must be selected from the pool of elongator tRNAs by eukaryotic initiation factor 2 (eIF2). The initiator tRNA (Met-tRNAi) has conserved sequence elements including modified bases at...
First, the initiator tRNA must be selected from the pool of elongator tRNAs by eukaryotic initiation factor 2 (eIF2). The initiator tRNA (Met-tRNAi) has conserved sequence elements including modified bases at...
01:35Pre-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...
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...