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Functional Significance of Structural Features and Intermediate Cleavages in the 5’ ETS of the Schizosaccharomyces pombe pre-rRNA

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Title: Functional Significance of Structural Features and Intermediate Cleavages in the 5’ ETS of the Schizosaccharomyces pombe pre-rRNA
Author: van Loggerenberg, Warren
Department: Department of Molecular and Cellular Biology
Program: Molecular and Cellular Biology
Advisor: Nazar, RossSeah, Stephen
Abstract: The pre-rRNA 5′ external transcribed spacer (5′ ETS), similar in size to that of the 18S rRNA, contains structural elements critical for rRNA maturation and is removed via a complex series of processing steps. This study further examined the functional significance of structural features and the intermediate cleavages (A’ and A0) in the 5’ ETS of Schizosaccharomyces pombe (S. pombe). Initial examination of processing intermediates in the 5' ETS based on RT-PCR indicated that the steps occurred in no specific order, suggesting alternate pathways for 5’ ETS processing and degradation. Mutational analyses at the A’ intermediate cleavage site indicated that this cleavage was not critical to rRNA maturation. Systematic mutations demonstrated that structural features in at least two of seven regions are important to protect the nascent pre-rRNA from degradation. RT- PCR mapping of the 5’ ETS region in cells with temperature sensitive nucleases refuted formation of the mature 5’ end of 18S rRNA by RNase III-like endonucleolytic cleavage across an extended hairpin. However, the results suggested a role in spacer degradation in that the endonucleolytic cleavages served as entry points for the nuclear 5’ exonuclease Dhp1p. Further mutational analyses around the 5’ ETS/18S rRNA junction indicated cleavage was not dependent on sequence and/or structure, but these sequences can dramatically affect rRNA yield. The results also suggested that the mature 5’ end of 18S rRNA is usually buried upon RNA processing, since changes that extended the position of A1 cleavage affected the 18S:25S rRNA ratios. Studies related to U3 snoRNA binding based on the expression of individual U3 snoRNA domains or mutations in a site complementary to the 5’ 18S rRNA pseudoknot were found also to affect the 25S:18S rRNA ratio significantly. Taken together, the present analyses raise the possibility that the 5’ end of the mature 18S rRNA actually may be the result of ribozyme cleavage while U3 snoRNA acts as a chaperone to stabilize the nascent rRNA structure. Although the intermediate steps were not critical to rRNA processing, the results indicated that failure to form a stable pre-ribosomal structure, as part of a ‘cellular checklist,’ results in the pre-rRNA being susceptible to degradation, and provides further evidence of quality control in ribosome biogenesis.
Date: 2018-01
Rights: Attribution-NonCommercial-NoDerivs 2.5 Canada
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Attribution-NonCommercial-NoDerivs 2.5 Canada Except where otherwise noted, this item's license is described as Attribution-NonCommercial-NoDerivs 2.5 Canada