Approaches to define open and closed regions on RNA and their applications in studying the effects of interleukin-10 on lymphoma cells
Description
Understanding the structure of mRNA is pivotal for performing studies on its biogenesis and functions. This statement can be illustrated by a study to evaluate the applicability of an Exon-Junction Hypothesis in conferring mRNA specificity in a reverse transcription-polymerase chain reaction (RT-PCR) assay. This hypothesis was tested on several mRNAs of human cytokines, including interleukin-1$\beta$, interleukin-2, interferon $\gamma$, interleukin-4, and interleukin-13. Approximately three-quarters of the exon-junction primers were found to be mRNA-specific. However, it became apparent that the yields of RT-PCR were affected by the secondary structure of mRNA A series of conceptual and experimental approaches were developed to help elucidate the overall architecture of mRNA. The total nucleotide sequence of mRNA was folded based on an algorithm of energy minimization. The folded RNA structure was interpreted into discrete segments of Closed Regions and Open Regions. A procedure known as Segment Analysis was developed to help visualize the Closed Regions and Open Regions on a folded RNA. The double stranded structure of Closed Region was composed of neighboring sequences which by themselves can be refolded into a structure identical to that on the overall folded RNA. The double stranded stems of open region was specified by long range interactions. The nucleotide sequence of an open region will not be refolded into a structure identical to those on the overall folded RNA For experimental verification of the predicted Closed Regions and Open Regions, a scanning approach and a regional approach were developed. For the scanning approach, a mRNA molecule was systematically scanned in an unbiased manner by a series of RT-PCR assays. Using hIL-1$\beta$ as a model system, an inverse proportional relationship was found between the specific yields of the RT-PCR assays and the free energy levels of the Close Regions covered by the amplified RNA segments For the regional approach, RT-PCR assays were designed to target directly either a Closed Region or an Open Region. Using hIL-10 mRNA as test system, a strong inverse proportional relationship was again observed between the specific yields of RT-PCR and the free energy levels of Closed Regions and Open Regions The knowledge gained on mRNA structure was then applied on developing specific and sensitive RT-PCR assays for a study of effects of interleukin-10 on Burkitt's lymphoma cells. The cytokines IL-4, IL-10, IL-13, and their receptors were found to express in both EBV-positive and EBV-negative Burkitt's lymphoma cells. In addition, recombinant human IL-10 enhanced growth of Raji cells; whereas neutralizing monoclonal antibody against human and viral IL-10 inhibited cell growth. These results supported an autocrine role for hIL-10. In addition, several candidate responsive genes of IL-10 have been identified in lymphoma cells by comparing the transcript profiles of the tumor cells The most important aspect of this thesis research was the development of approaches to study the structure of mRNA by a combination of structural prediction based on minimal energy and experimental verification by RT-PCR. These insights into mRNA secondary structure are not only useful for molecular biology research on RNA splicing, stability, RNA-protein interactions, and translation, but are also indispensable in exploring clinically oriented investigations such as gene diagnosis and genetic therapy. (Abstract shortened by UMI.)