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Degree

Doctor of Philosophy, Case Western Reserve University, Molecular Biology and Microbiology, 1994.

Abstract

Using cell-free extracts derived from synchronously developing Ascaris embryos, we have studied the SL RNA and U6 snRNA sequence requirements for trans- and/or cis-splicing. Both chemical modification interference and mutational analyses revealed two essential elements within the SL RNA's snRNA-like domain, a short stretch on the 3′ side of stem II and the single stranded Sm binding site region between stems II and III. These two elements are necessary and sufficient to convert a piece of Ascaris U1 snRNA into a functional "SL RNA". Using psoralen cross-linking, we demonstrated that the SL RNA interacts with U6 snRNA by base pairing. Mapping of the U6/SL RNA cross-linked species placed the interaction within the 3′ end sequence of U6 and the Sm binding site region of SL RNA. The base pairing overlaps one of the two essential SL RNA sequence elements. Based on these observations, a working model for nematode pre-mRNA trans-splicing has been proposed. To compare and contrast the U6 snRNA sequence requirements for trans- and cis-splicing, we have developed a functional reconstitution system in which the U6 snRNA can be assayed. We performed a systematic mutagenesis where consecutive four base block substitutions were introduced throughout the U6 snRNA sequence. The ability of each mutant to support either trans- or cis-splicing has been tested in the reconstitution system. We have found that the U6 snRNA sequence requirements for trans- and cis-splicing are similar but not identical. Surprisingly, we have also observed branch formation between trans- or cis-splice substrate and U6 snRNA. More remarkably, one mutant with a four base change in a universally conserved region 5′ liberated "exon" of the U6 joined to the normal 3′ exon of the substrates. These results have several implications regarding the role of U6 snRNA in 5′ splice site recognition and the conformation of the U6 snRNA in functional spliceosome

Subject Headings

Biology, Molecular

Keywords

RNA; splicing

Advisor

Timothy W. Nilsen

Pages

206p.

Document number: case1061564747
Permalink: http://rave.ohiolink.edu/etdc/view?acc_num=case1061564747

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