Functional significance of the simian virus 40 late mRNA polyadenylation signal RNA secondary structure / Holly Hans Henry.

Format:

Book

Manuscript

Microformat

Thesis/Dissertation

Author/Creator:

Henry, Holly Hans.

Contributor:

Alwine, James C., advisor.

University of Pennsylvania.

Language:

English

Subjects (All):

Penn dissertations--Cell and molecular biology.

Cell and molecular biology--Penn dissertations.

Cell and Molecular Biology.

Academic Dissertations as Topic.

Medical Subjects:

Cell and Molecular Biology.

Academic Dissertations as Topic.

Local Subjects:

Penn dissertations--Cell and molecular biology.

Cell and molecular biology--Penn dissertations.

Physical Description:

xii, 138 pages : illustrations (some color) ; 29 cm

Production:

2000.

Summary:

The SV40 late polyadenylation signal (SVLPAS) is very efficient and characterized by its structure which is more complex than most known mammalian polyadenylation (PA) signals. Specifically, it contains efficiency elements both upstream and downstream of the AAUAAA. Also, the downstream region contains three defined elements (two U-rich and one G-rich) instead of the single U- or GU-rich element found in most PA signals. Many reports have indicated that secondary structure in RNA may play a significant role in RNA processing and other functions. Given the complexity of the SVLPAS, we have used nuclease structure analysis techniques to determine the secondary structure of the SVLPAS. We find that the SVLPAS has a functionally significant secondary structure. The region upstream of the AAUAAA is predominantly sensitive to single strand specific nucleases. In contrast, the region downstream of the AAUAAA contains a prominent region within the U-rich element closest to the AAUAAA which is sensitive to the double strand specific nuclease RNase V1. A characteristic pattern of four contiguous RNase V1-sensitive regions, denoted structural region (SR) 1, 2, 3 and 4, is detected. Linker scanning mutants were constructed in the downstream region and analyzed both for structure and function using in vitro cleavage analyses. These data show that the downstream structural regions, particularly SR3, are functionally significant. We discuss the possibility of downstream secondary structure as general functional feature of polyadenylation signals.

The importance of the SVLPAS RNA secondary structure are also discussed in regards to U1A protein binding. Previously our lab has illustrated the U1A protein as factor involved in 3' end processing of the SVLPAS. These data suggested the importance of the SVLPAS upstream efficiency elements (USEs) in protein binding. We have further characterize this binding and will show the importance of sequences both upstream and downstream of the SVLPAS AAUAAA for protein binding. Through mutational analysis we show the correlation between the presence of the SVLPAS RNA secondary structure and U1A binding suggesting a role for structure in binding. The use of optical biosensor technology allowed us to further characterize this interaction and show one of the first descriptions of this technology to study RNA/protein interactions. We show a mutation in the downstream region which affects RNA secondary structure and 3' end processing, also decreases U1A protein binding by lowering the association rate of protein with the RNA. Thus these data allow us to begin to understand the mechanisms involved in U1A protein binding to the SVLPAS.

Notes:

Supervisor: James C. Alwine.

Thesis (Ph.D. in Cell and Molecular Biology) -- University of Pennsylvania, 2000.

Includes bibliographical references.

Local Notes:

University Microfilms order no.: 99-65489.

OCLC:

244971141