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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.
Abstract:
Polyadenylation of pre-mRNAs in the nucleus involves a specific endonucleolytic cleavage, followed by the addition of approximately 200 adenylic acid residues. We have assayed HeLa nuclear extracts for the activity that catalyzes the poly(A) addition reaction. The authenticity of the in vitro assay was indicated by the observation that the poly(A) tract added in vitro is approximately 200 nucleotides in length. We have fractionated nuclear extracts in order to define components involved in specific poly(A) addition. No single fraction from DEAE-Sephacel chromatography of a HeLa nuclear extract possessed the specific poly(A) addition activity. However, if the various fractions were recombined, activity was restored, indicating the presence of multiple components. Further fractionation revealed the presence of at least two factors necessary for the poly(A) addition reaction. The reconstituted system retains the characteristics and specificity seen in the crude extract. Additional purification of one of the factors strongly suggests it to be a previously characterized poly(A) polymerase which, when assayed in the absence of the other factor, can add AMP to an RNA terminus but without specificity. Thus, the other component of the reaction may provide specificity to the process. In contrast to the 3' cleavage reaction, the poly(A) addition machinery does not possess an essential RNA component, as assayed by micrococcal nuclease digestion, nor do anti-Sm sera inhibit the reaction. Thus, the total process of formation of a polyadenylated mRNA 3' end is complex and requires the concerted action of distinct nuclear components.
Insights
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.
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