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https://hdl.handle.net/2142/23629
Description
Title
Molecular analysis of peasnRNA variants
Author(s)
Hanley, Brian Andrew
Issue Date
1991
Doctoral Committee Chair(s)
Schuler, Mary A.
Department of Study
Biology, Molecular
Chemistry, Biochemistry
Discipline
Biology, Molecular
Chemistry, Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Chemistry, Biochemistry
Language
eng
Abstract
Five small nuclear RNAs (snRNAs) (U1, U2, U4, U5, U6) found in the nucleus of eukaryotic cells mediate the excision of introns from pre-mRNAs. The structure and expression of the snRNA components have been well documented in animal and yeast systems but little information has existed on the structure and expression of the splicesomal snRNAs involved in plant intron excision. To further define the snRNA components involved in intron excision, a clone library has been constructed from anti-m$\sb3$G immunoprecipitated snRNAs expressed in pea seedlings. cDNA clones representing U1, U2, U4, and U5 snRNAs expressed in seedling tissue have been isolated and sequenced. Comparison of the pea snRNA variants with other organisms suggest that functionally important primary sequences are conserved phylogenetically even though the overall sequences have diverged substantially. Structural variations occur in regions required for U1-specific protein binding suggesting that alternate U1 snRNP particles may exist in vivo. Sequence variations in the U2 and U4 snRNAs are limited to the 3$\sp\prime$ terminal regions of the molecules. In light of this sequence analysis, it is clear that the dicot snRNA variants do not differ in sequences implicated in RNA:RNA interactions with pre-mRNA. Instead sequence differences occur in regions implicated in the binding of small ribonucleoproteins and may result in the formation of unique snRNPs during development.
In an effort to elucidate the function of the numerous pea snRNA variants, the expression of U1-U6 snRNA variants in pea seedling development was examined. Comparison of the snRNAs in pea seeds and seedlings has revealed that four (U1, U2, U4, U5) of the five snRNAs required for pre-mRNA splicing have differentially- and developmentally-regulated forms detectable on northerns. Selected subsets of plant snRNAs accumulate at particular stages in plant development. These variants are potentially required for the splicing of developmentally regulated transcripts, which, in some cases possess extremely heterogeneous splice sites.
To analyze the regulatory elements in U2 snRNA gene, a six nucleotide linker has been inserted into the U2 snRNA between stem-loops III and IV. Experiments have demonstrated that heterologous U2 snRNAs can be expressed in plant nuclei for trans-complementation studies. (Abstract shortened with permission of author.)
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