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Polyadenylate sequences on Newcastle disease virus mRNA synthesized in vivo and in vitro
Abstract:
Polyadenylate [poly(A)] sequences are associated with the 35 and 50S Newcastle disease virus (NDV)-specific RNAs as well as all six to seven of the 18-22S NDV-specific messenger RNAs extracted from infected chicken embryo cells. The poly(A) associated with the 18-22S RNA has an average size of 120 to 130 nucleotides. The 18-22S RNA synthesized in vitro by NDV's virion-bound polymerase contains six to seven species of the same size and relative proportions as its intracellular counterpart. This in vitro synthesized 18-22S RNA also contains covalently linked poly(A) sequences which, although variable in size, are usually larger and more heterogeneous than those from the infected cell. In vitro RNA synthesis is supported not only by magnesium (at an optimal concentration of mM) but by manganese (at an optimal concentration of 0.5 to 1.0 mM) as well. However, the major product made in the presence of manganese, although sedimenting at 18 to 22S, differs somewhat from the product made in the presence of magnesium.
Insights
Newcastle disease virus (NDV) infection involves polyadenylate [poly(A)] sequences on viral RNAs. In vitro studies show NDV polymerase synthesizes 18-22S RNAs with poly(A) tails, revealing insights into viral RNA processing.
Area of Science:
- Virology
- Molecular Biology
- RNA Biology
Background:
- Polyadenylate [poly(A)] sequences are crucial post-transcriptional modifications in eukaryotic and viral RNAs.
- Newcastle disease virus (NDV) is an avian paramyxovirus with a segmented RNA genome.
Purpose of the Study:
- To investigate the presence and characteristics of poly(A) sequences in NDV-specific RNAs.
- To analyze the in vitro synthesis of NDV 18-22S messenger RNAs and their associated poly(A) tails.
Main Methods:
- Extraction and characterization of NDV-specific RNAs from infected chicken embryo cells.
- In vitro RNA synthesis using NDV virion-bound polymerase.
- Analysis of RNA size, poly(A) tail length, and synthesis conditions (Mg2+ vs. Mn2+).
Main Results:
- Poly(A) sequences were found on both 35/50S and 18-22S NDV RNAs.
- In vitro synthesized 18-22S RNAs mimicked intracellular counterparts in size and proportion.
- In vitro poly(A) tails were larger and more heterogeneous than intracellular ones.
- Manganese ions supported in vitro RNA synthesis but yielded a slightly different product compared to magnesium.
Conclusions:
- NDV virion-bound polymerase can synthesize 18-22S RNAs with covalently linked poly(A) tails.
- The in vitro system provides a model for studying NDV RNA processing and polyadenylation.
- Divalent cations (Mg2+ and Mn2+) influence the in vitro RNA synthesis products.