Date

January 2003

UMMS Affiliation

Graduate School of Biomedical Sciences, Pharmacology and Molecular Biology

Document Type

Dissertation, Doctoral

Subjects

Gene Expression Regulation, Viral; Gene Products, tat; HIV-1; Positive Transcriptional Elongation Factor B; RNA Interference; RNA, Small Interfering; Academic Dissertations; Dissertations, UMMS

Disciplines

Life Sciences | Medicine and Health Sciences

Abstract

Gene expression of human immunodeficiency virus type-1 (HIV-1), which causes Acquired Immunodeficiency Syndrome (AIDS), is regulated at the transcriptional level, where negative factors can block elongation that is overcome by HIV Tat protein and P-TEFb. P-TEFb, a positive elongation transcription factor with two subunits, CDK9 and Cyclin T1 (CycT1), catalyzes Tat-dependent phosphorylation of Ser-5 in the Pol II C-terminal domain (CTD), allowing production of longer mRNAs. Ser-5 phosphorylation enables the CTD to recruit mammalian mRNA capping enzyme (Mce1) and stimulate its guanylyltransferase activity. This dissertation demonstrates that stable binding of Mce1 and cap methyltransferase to template-engaged Pol II depends on CTD phosphorylation, but not on nascent RNA. Capping and methylation doesn't occur until nascent pre-mRNA become 19-22 nucleotides long. A second and novel pathway for recruiting and activating Mce1 involved direct physical interaction between the CTD, Tat and Mce1. Tat stimulated the guanylyltransferase and triphosphatase activities of Mce1, thereby enhancing the otherwise low efficiency of cotranscriptional capping of HIV mRNA. These findings imply that multiple mechanisms exist for coupling transcription elongation and mRNA processing at a checkpoint critical to HIV gene expression.

To elucidate P-TEFb's function in human (HeLa) cells, RNA interference (RNAi) was used to degrade mRNA for hCycT1 or CDK9. Down-regulation of P-TEFb expression by RNAi can be achieved without causing major toxic or lethal effects and can control Tat transactivation and HIV replication in host cells. High-density oligonucleotide arrays were used to determine the effect of P-TEFb knockdown on global gene expression. Of 44,928 human genes analyzed, 25 were down-regulated and known or likely to be involved in cell proliferation and differentiation. These results provide new insight into P-TEFb function, its potent role in early embryonic development and strong evidence that P-TEFb is a new target for developing AIDS and cancer therapies.

To fulfill the promise of RNAi for treating infectious and human genetic diseases, structural and functional mechanisms underlying RNAi in human cells were studied. The status of the 5' hydroxyl terminus of the antisense strand of short interfering RNA (siRNA) duplexes determined RNAi activity, while a 3' terminus block was tolerated in vivo. A perfect A-form helix in siRNA was not required for RNAi, but was required for antisense-target RNA duplexes. Strikingly, crosslinking siRNA duplexes with psoralen did not completely block RNAi, indicating that complete unwinding of the siRNA helix is not necessary for RNAi in vivo. These results suggest that RNA amplification by RNA-dependent RNA polymerase is not essential for RNAi in human cells.

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