RNAi Applications

RNAi Applications and Vigilin in RNA Metabolism

RNA Interference (RNAi)

The sequence of the human genome is effectively complete. In the emerging postgenomics era, one of the fundamental challenges is to determine the function of the approximately 30,000 genes in the human genome. A new technique called RNA interference, or RNAi, provides a quick and relatively easy way to analyze the function of any gene whose sequence is known. The basic idea of RNAi is that a double stranded RNA complementary to a segment of the mRNA is synthesized, introduced into the cell, and a cell system then degrades and destroys the mRNA complementary to the double stranded RNA. Any newly synthesized copies of that mRNA will be degraded soon after they are made. Since the mRNA has been destroyed and there is none of that specific mRNA available to be translated, there is no mRNA to serve as a template for synthesizing that protein. Once the pre-existing protein that was made from that mRNA before its destruction is degraded by the cell, the protein will be degraded over time and disappear from the cell. This process is sometimes referred to as gene silencing. The disappearance of a particular protein often results in an altered property of the cell (altered phenotype) or function of a metabolic pathway and allows that gene products function to be determined.

RNAi is a process of post-transcriptional silencing of the expression of a specific gene that exploits an ancient defense system for degrading the RNA of invading RNA viruses and other double-stranded RNAs. Variants of RNAi are seen in multicellular eukaryotes, from plants to humans. While early research applications of RNAi using long double-stranded RNAs were successful in Drosophila and nematodes, in human cells and other vertebrate cells, a second defense system is present. The presence of long double-stranded RNA activates the vertebrate interferon system resulting in the global degradation of virtually all mRNAs and the death of the cell. Studies of the pathway by which long dsRNAs are degraded in cells and trigger both the RNAi and interferon responses demonstrated that in an early step in this process the long dsRNAs are degraded to ~21 nucleotide double stranded fragments. The key finding was the demonstration that using a short 21 nucleotide double-stranded RNA did not trigger the interferon response and resulted in the specific degradation of the homologous mRNA. RNAi and the tiny siRNAs RNAs that mediate RNA interference and the micro RNAs that mediate a related process of translational repression have generated enormous interest and were recently named "Breakthrough of the Year" by Science (Dec. 20th 2002 issue).

Schematic of the Pathway of RNAi. (Modified from Dykxhoorn, D.M., Novina, C.D. and Sharp, P.A. 2003. Killing the Messenger: Short RNAs that Silence Gene Expression. Nature Revs. Mol. Cell. Biol. 4: 457-467.) Once an mRNA is cleaved by RNAi, since the ends of the mRNA are no longer protected by a 5' cap and a 3' poly(A) tail, the free ends of the two mRNA fragments are rapidly degraded by cell RNases.

Our long-standing interest in pathways of vertebrate mRNA degradation led us to early and extensive use of RNAi in our research and to efforts to both expand its applications and in so doing further delineate its mechanisms of action. A major problem of RNAi was that the effect wears off after a few days and the mRNA is no longer degaded. We developed a simple technique for long-term RNAi. This work and recent studies in other laboratories demonstrate that short interfering siRNAs are long-lived and act catalytically. Current work focuses on the development of other new applications of RNAi including regulated delivery systems.

RNAi Knockdown of Human Estrogen Receptor α. The upper panel is a Western blot performed using monoclonal antibody against hERα at the indicated times after transfection of MCF-7, human breast cancer cells, with either a control non-specific siRNA (pGL3, a luciferase siRNA), or with the hERα-specific siRNA using our long-term RNAi procedure. The bands were visualized and quantitated using ECL plus reagent and a PhosphorImager. Since ER is not required for the survival of MCF-7 cells, long-term RNAi is feasible. The lower panel shows quantitation of the ER level over time normailized to an actin internal standard. As expected maximum knockdown is 3 days post-transfection, with a very slow increase in ER levels thereafter. (D. Yu and D. Shapiro, unpublished observations)

Vigilin in RNA Metabolism

Our finding that vigilin likely plays a role in the estrogen-mediated stabilization of vitellogenin of vitellogenin mRNA led us to further studies of this unique protein. Vigilin is highly conserved in eukaryotes, ranging from yeast, to fruit flies, humans. Remarkably, vigilin contains 15 k-homology (KH) RNA binding domains. Many proteins mediating essential cell functions are members of families of closely related proteins. Because there are several family members with overlapping functions, mutations in a single family member are less likely to result in a lethal phenotype. However, vigilin (also called SCP160 in yeast and DDP1 in Drosophila) is unique. Our analysis of the human, mouse, yeast, nematode and Drosophila genomes doe not reveal closely related family members, or any other proteins containing more than 10 KH domains. We believe that the presence of so many RNA binding domains gives vigilin a great deal of functional flexibility. Also, our analysis of vigilin-RNA ineractions demonstrates that because there are so many KH domains in vigilin, there are no single point mutations that have much impact on vigilin's ability to bind RNAs (Kanamori, H., Dodson, R.E. and Shapiro, D.J. (1998) In Vitro Genetic Analysis of the RNA Binding Site of Vigilin, a Multi-KH Domain Protein. Mol. Cell. Biol. 18: 3991-4003).

Our studies of vigilin have focused on how a protein with 15 RNA binding domains interacts with nucleic acids and on using RNAi to analyze the proposed functions of vigilin.

Vigilin-Nucleic Acid Interactions

We used a novel type of in vitro genetic selection to identify key features of a high affinity RNA binding site for vigilin. Surprisingly, high affinity binding of vigilin to RNA requires an approximately 75 nucleotide, largely unstructured RNA containing multiple (A)nCU and UC(A)n motifs. Using information from the in vitro selection, we predicted and subsequently identified a high afifnity vigilin binding site at the 3'-end of human dystrophin mRNA (Kanamori, H., Dodson, R.E. and Shapiro, D.J. (1998) In Vitro Genetic Analysis of the RNA Binding Site of Vigilin, a Multi-KH Domain Protein. Mol. Cell. Biol. 18: 3991-4003). (Dystrophin is a muscle protein. Defective production of dystrophin causes muscular dystrophy.) The 3' ends of some other mRNAs did not bind vigilin. Based on these studies we concluded that vigilin binds to RNAs with a high degree of specificity. Subsequent binding studies established that vigilin interactions with nucleic acids are likely based on processive binding. In this model, random thermal motion creates a contact area between a high affinity binding site on the RNA and a sub-set of vigilin's KH domains. This unstable complex can either dissociate, or progress to form a more stable complex. After the RNA makes a productive contact with vigilin, it transiently tethers the two molecules together. Due to the close proximity of vigilin and the RNA, random thermal motion of the unstructured RNA brings it into close proximity to additional vigilin KH domains and thereby established multiple contacts between additional vigilin KH domains and less favorable lower affinity RNA binding sites. Most, or all, of vigilin's 15 KH domains and the RNA make multiple low affinity contacts, which together stabilize the vigiln-RNA interaction and result in high affinity binding of vigilin to RNA (Dodson and Shapiro, 2002).

Although our studies and those of some other researchers suggested a role for vigilin in RNA metabolism, other researchers in yeast and Drosophila and mammalian cells suggested that vigilin functions to regulate chromosome partioning at mitosis, or to regulate the overall efficiency of translation. To test these models for vigilin action, we carried out RNA interference. RNAi mediated knockdown of vigilin is rapid and triggers the apoptotic death of both dividing and non-dividing human cells. Since vigilin is essential for viability of cells that are not undergoing mitosis, it must have an essential function independent of any potential role in chromosome portioning at mitosis. Experiments carried out after substantial vigilin knockdown, but before the cells have begun to die, showed that vigilin knockdown does not alter the overall rate of protein synthesis, indicating that vigilin is not a regulator of the overall translation rate. Finally, knockdown of vigilin triggers caspase-dependent apoptosis (Goolsby and Shapiro, 2003). These data support the view that vigilin's essential functions are neither chromosome partioning nor control of overall translation, and are consistent with vigilin playing a critical role in mRNA metabolism. Current work aims to identify functions of vigilin in human cells.

Vigilin is Essential for the Viability of Non-dividing Human Cells. Non-dividing HeLa cells were transfected with either a control pGLL3 (luciferase non-specific siRNA) or with the vigilin-specific Vig2 siRNA and photographed two days later. Most of the cells transfected with the vigilin-specific siRNA have died. Some of the cells on the plate transfected with Vig2 siRNA are dying. There are a few healthy cells on the plate and these are likely to be untransfected HeLa cells (From Goolsby and Shapiro 2003)

Some Recent Publications

Dodson, R.E. and Shapiro D.J. (2002) Regulation of Pathways of mRNA Destabilization and Stabilization. In K. Moldave Ed. Progress in Nucleic Acids Res. and Molec. Biol. 72: 129-164

Goolsby, K.M. and Shapiro, D.J. (2003) RNAi-mediated Depletion of the 15 KH Domain Protein, Vigilin, Induces Death of Dividing and Non-dividing Human Cells but Does Not initially Inhibit Protein Synthesis. Nucleic Acids Res. In Press.

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