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Structure-function analysis of the U14 small nuclear RNA of Saccharomyces cerevisiae
Abstract
The U14 RNA of Saccharomyces cerevisiae is an essential small nuclear RNA (snRNA) associated with the nucleolus. Repression of U14 RNA synthesis was shown to result in impaired production of 18S rRNA, manifest as a dramatic decrease in the ratio of mature 18S and 25S RNAs. This effect was evident within one generation after the onset of U14 gene repression and correlated well with depletion of U14 snRNA. Results from pulse-chase assays revealed the basis of the imbalance to be underaccumulation of 18S RNA and its 20S precursor. This effect appears to result from: (1) impairment of processing of the 35S rRNA transcript at sites that define the 20S species and (2) rapid turnover of an unusual 18S-containing intermediate. These results constitute the first demonstrated involvement of an essential snRNA in rRNA maturation. Functional mapping was directed at assessing the importance of sequence elements that are: (1) conserved among the yeast and smaller mouse U14 RNAs and (2) unique to the yeast species. First, the functional equivalency of the yeast and mouse U14 RNAs was examined in a test strain, in which wild-type U14 synthesis is induced by galactose and repressed by glucose. The experimental RNAs included mouse U14 and several yeast:mouse bi- and tri-partite hybrid RNAs, all transcribed from yeast U14 gene signals in a plasmid. Next, plasmid-encoded yeast U14 DNA was subjected to deletion and base substitution mutagenesis. Functional characterization showed that phylogenetically conserved sequences in the yeast and mouse U14 RNAs are interchangeable, provided that a terminal stem composed of 5$\sp\prime$ and 3$\sp\prime$ segments is preserved. This result argues that the yeast and mouse U14 RNAs are functional homologs and that the U14 RNAs are universally important. The result also identified two internal yeast specific elements which are required for function of U14 in yeast. An effort was also made to develop a genetic system for analyzing structural features of rDNA involved in 18S rRNA processing, with a view to establishing U14-dependent pre-rRNA maturation assays. To this end, a plasmid-borne rDNA operon was constructed, in which the 18S RNA coding sequence was "tagged" by an oligonucleotide insertion. The hybridization tag was shown to behave as a silent mutation with no apparent interference with rRNA processing, ribosome assembly or incorporation of tagged ribosomes into polysomes. The analysis of deletions constructed with the tagged operon revealed that 18S RNA production in Saccharomyces cerevisiae requires colinear expression of intact 25S RNA.
Subject Area
Molecular biology
Recommended Citation
Li, Haodong V, "Structure-function analysis of the U14 small nuclear RNA of Saccharomyces cerevisiae" (1991). Doctoral Dissertations Available from Proquest. AAI9207426.
https://scholarworks.umass.edu/dissertations/AAI9207426