RNA interference is a crucial system in cell functionality. This pathway plays a large role in regulating genes by inducing gene silencing. One of the required elements in all RNAi pathways is the Dicer protein. The role of Dicer in RNAi is to cut dsRNA into small interference RNAs and stimulate loading into the RISC complex that later down the line facilitates gene silencing. This process normally functions as a defense against infections but in Giardia Lamblia, or Giardia intestinalis, parasites the RNAi machinery is used to prolong infection by evading host cell defenses. By a process called antigenic variation, Giardia parasites are able to switch expression of their surface antigens so they are no longer targeted by the host immune responses. This is intriguing because the Giardia Dicer protein is a vital piece of machinery in regulating antigenic variation. The GDicer’s role is to degrade dsRNA, from the siRNAs created new recombinant proteins (VSPs) direct the generation of antisense RNAs. This in turn leads to the production of new surface proteins that are expressed in order to elude identification by the host cell. This process highlights the versatility of the Dicer protein and how different variations of structures can function in various pathways. For example, Giardia Dicer actually lacks many structural domains found in other kinds of Dicer protein such as the Human Dicer. The missing domains include the amino terminal helicase and the carboxy-terminal domain (dsRBD). This makes the GDicer a much smaller molecule in comparison to others like the HDicer. The evidence of these missing domains also bring to question what the necessary components of Dicer are and influence more research into the specific domain functions of the molecule.
Figure A: Highlights the differences in structure by showing the primary sequences of Human Dicer and Giardia Dicer.
The Giardia Intestinalis Dicer protein is a small molecule represented in a "hatchet"-like shape composed of multiple alpha helices and beta sheets . The GDicer protein contains three major subunits that together are considered the universal core functional unit of dicer proteins. The three domains are the and . The PAZ Domain is the recognition site of the protein for dsRNA and the RNase III domains are responsible for cutting the backbones of the dsRNA. The two RNase subunits are connected to the PAZ Domain by a long alpha helix known as the . This alpha helix is surrounded by the and together these form the “handle” of the hatchet. The domains are also categorized into three different regions; the RNase Region made up of both RNase III domains and a , the connector helix and N-Terminus make up the Platform Region that is structurally composed of the an antiparallel Beta sheet and three alpha helices, and the third region is the PAZ Domain . The are structurally rigid but are connected by flexible hinges. These hinges allow the dicer protein to conformationally change in order to accommodate variations of dsRNA during binding.
During dsRNA binding the flexible hinges of the dicer protein allow many diverse dsRNA structures to bind. During binding there are two distinct kinks or hinges present in the protein structure that allow for the molecule to conformationally change in order to accommodate varieties of RNA structures. The first hinge is induced by the presence of a Proline - in the connector helix that sits on the apex of the kink. There is no direct point that causes the second hinge to form, it is a result of the conformational change that shifts along the protein. dsRNA binding begins in the PAZ Domain where the double stranded compound is first recognized. The PAZ Domain contains a large extended loop that recognizes a 3prime 2-nucleotide overhang present in the RNA sequences. This loop is called the and it helps form the RNA binding pocket. The RNase subunits sit adjacent to each other to form a that each contain a metal ion to form a two-metal ion mechanism of catalysis. This method of catalysis employs both RNase III subunits to have metal binding sites that are 4.2A and 5.5A apart. This relatively matches the width of the dsRNA major groove. Mn2+ is the metal ion found in the of each RNase III subunits . In each domain the binding site is found between four strictly conserved acidic . These are the cleavages sites for the RNA backbone.
Dicer is an enzyme involved in the process of RNA interference (RNAi) in both unicellular and multicellular eukaryotes. Since the Giardia Dicer lacks the helicase, DUF 283, and dsRBD sequence found in human Dicer, it can be used to deepen our understanding of processes in RNAi and conserved structures. dsRNA cleavage by Giardia Dicer is magnesium dependent while other divalent cations can support catalytic activity. Further, the mechanism of Dicer-catalyzed dsRNA processing is conserved. The presence of discrete dicing intermediates separated by 25 nucleotides indicates Giardia dicer processes dsRNA from the helical end which reflects what is observed in human Dicer. This suggests that Dicer works as a “molecular” ruler that is able to recognize dsRNA and cleave it at a specific distance from the helical end. As Dicer’s cleavage products play key roles in promoting cellular homeostasis through the fine-tuning of gene expression, dysregulation of Dicer activity can lead to disease and deepening our understanding of this protein can assist in obtaining a more complete understanding of RNAi.
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Prucca CG, Slavin I, Quiroga R, Elías EV, Rivero FD, Saura A, Carranza PG, Luján HD. Antigenic variation in Giardia lamblia is regulated by RNA interference. Nature. 2008 Dec 11;456(7223):750-4. doi: 10.1038/nature07585. PMID: 19079052.
Rogers, Kara. "RNA interference". Encyclopedia Britannica, 6 Oct. 2023, https://www.britannica.com/science/RNA-interference. Accessed 3 December 2023.
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