Molecular Biology gene/ mRNA body

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Table of Contents

Introduction
Background
Preliminary Results
Future experiment proposals
Conclusion
Works Cited

Introduction

The expansion of poly-glutamines present in the Huntingtin proteins is solely responsible for the Huntington disease. The Huntington disease refers to a dominant autosomal neurodegenerative aberration. To understand the development of the Huntington disease, the function of normal Huntingtin proteins has to be elucidated. This is accomplished by purifying the Huntingtin proteins and Argonaute as associated proteins.

Studies have shown that Huntingtin proteins and Argo2 exist in P bodies and that a decline in Huntingtin led to a compromised RNA-mediated gene silencing. RNA-mediated gene silencing refers to the related processes that involve 21 to 25 nucleotide RNAs to repress the expression of specific target genes.

A change in the Huntingtin protein leads to a distinct neurodegeneration pattern. This paper, therefore, seeks to establish whether the Huntington disease protein is a contributory factor in RNA- mediated gene silencing. The paper is based on a research study titled Huntington’s disease protein contributes to RNA-mediated gene silencing through association with Argonaute and P bodies by Savas et al. (2008).

With regard to this, the paper seeks to test the hypothesis that Huntingtin protein causes a characteristic neurodegeneration pattern. This hypothesis is informed by a study on mouse striatal cells with mutant Htt that reveals the presence of fewer P bodies and a decreased gene silencing activity. This may suggest that transcriptional deregulation in Huntington disease is an outcome of the mutant Htt’s role post-transcriptional processes.

In order to test the hypothesis, Savas et al used a biochemical approach to purify proteins related to WT and mutant Htt and identified Ago2 as a co-purifying protein (Savas et al., p. 10820). Further co-localization tests showed the presence of Htt and Ago2 in processing P bodies, cytoplasmic foci that contain translationally repressed mRNAs with bound proteins (Savas et al., p. 10820).

The data suggested that normal Htt is a component of the P body and functions in the ost-transcriptional pathways (Savas et al., p. 10820). The results of the test indicate that Ago2 indeed co-purifies with Huntingtin. Specifically, Ago2 co-purifies with Htt 480 protein when over-expressed in cells.

The test further proved that Huntingtin is present in P bodies (Savas et al., p. 10822). It was also established that Huntingtin plays a role in small RNA- mediated processes (Savas et al., p. 10823). Finally, a comparison was made between normal Huntingtin and polyQ-expanded Huntingtin (Savas et al., p. 10824). The findings of this research may help in control and management of Huntington disease.

Background

The background of this research is based on RNA silencing, which refers to the process of sequence-specific regulation of gene expressions caused by double-stranded RNA (Fire et al, p. 806). It takes place in almost all eukaryotes. Some of the functions of the RNA silencing remain largely controversial while a few are well documented. RNA silencing processes mediate transcriptional and post-transcriptional gene silencing. Post-transcriptional gene silencing is characterized by mRNA degradation or translational repression.

Extensive complementarity between the double-stranded RNA and RNA is required for effective target mRNA degradation. However, imperfect complementarity may also produce effective translational repression, but without extensive target RNA degradation (Olsen & Ambros, p. 671). RNA silencing is a crucial process in both plants and animals as it allows for the regulation of development and the control of transposition events. It also plays an anti-viral role in plants and in insects such as Drosophila melanogaster (Voinnet, p. 206).

The processing of double-stranded RNA and siRNA requires the ribonuclease (RNase) 3 Dicer. After the RNA generation, one of the siRNA strands is incorporated into a complex with a constituent of the Argonaute protein family. The complex is then allowed to cleave or repress translation.

Scholars have attempted to describe the characterization of a new RNA polymerase that is responsible for the gene silencing. One such scholar is David Balaucombe who was able to identify the sd4 mutant, which was ineffective in the siRNA production and methylation of a retro-element, and which also indicated a partial loss of trans-gene silencing. Balaucombe’s studies revealed that plants encode subunits for a fourth polymerase in addition to the common DNA-dependent RNA polymerases 1, 2 and 3.

Mutational studies show that polymerases are able to silence certain transposons and repetitive DNA involving RNA-dependent RNA polymerase Rdr2 and RNase 3 Dcl3. This possibly explains why chromatin silencing depends on RNA transcription. In addition, Pol 4 may be resistant to DNA or chromatin modifications that impact on polymerases 1, 2 and 3 and, therefore, is able to maintain the silenced state.

Preliminary Results

Savas et al conducted their experiment to test the hypothesis that Huntingtin protein causes a characteristic neurodegeneration pattern. Preliminary results posted a number of findings. First and foremost, it was concluded that Ago2 co-purifies with Huntingtin.

This was informed by a discovery-based Htt purification plan, which was aimed at identifying the interactions and differences between proteins that co-purify with WT and mutant Htt.

In the experiment, HeLa cells were used to create stable cell lines that express Flag-tagged Htt N-terminal 590 aa with 25 or 97 glutamines (Flag-Htt 590-25Q or Flag-Htt 590-97Q) (Savas et al., p.10820). A cytoplasmic S100 fraction was prepared from these cells and subjected to immune-purification with Flag-M2 agarose followed by peptide elution (Savas et al., p.10820).

HeLa cells are purposely used because they provide large materials that help in identification of interacting proteins. During the test, SDS/PAGE was used to isolate the peptide eluted fraction, which was then stained with coomassie blue (Savas et al., p.10820). This produced the right molecular weight of Flag-Htt590 and a limited number of nonstoichiometric co-purifying proteins.

The gel lanes were then partitioned and subjected to in-gel tryptic digestion, which was followed by ESI-MS/MS based peptide sequencing (Savas et al., p.10820). Many of the distinct co-purifying polypeptides corresponded with the Argonaute proteins. This confirmed that the Ago family of proteins is responsible for small RNA gene-mediated silencing (Peters and Meister, p. 611).

Proteins associated with Ago2 from HEK293T were purified separately and 40 unique peptides corresponding to the endogenous Htt was identified (Savas et al., p.10822). This interaction was established by the co-precipitation and immune-blotting. Tests further revealed that Huntingtin is present in P bodies.

This was investigated by the localization of transfected Htt and Ago2 by confocal microscopy (Savas et al., p.10822). This was done by transfecting HeLa cells with Myc-Htt590-25Q and Flag-Ago2 and then examined by indirect immuno-flourescence (Savas et al., p.10822). It was observed that the cells expressing these proteins had distinct foci, indicating strong co-localization of the two proteins (Savas et al., p.10822).

Preliminary results also link Huntington to small RNA-mediated processes (Savas et al., p.10823). This finding was investigated by using a Luc reporter to determine the effect of Htt knockdown on the silencing of the mRNA transcripts (Savas et al., p.10823).

This was accomplished by transfecting HeLa cells with GW182, Htt, or Ago2 siRNA and further co-transfecting them with SV40 promoter-Luc reporter plasmid and siRNA to Luc (Savas et al., p.10823). The results of this experiment indicated that cells transfected with Ago2 siRNA posted a five-time increase in reporter activity (Savas et al., p.10823). This confirms that Huntingtin plays a role in small RNA-mediated processes.

Future experiment proposals

A possible future experiment could involve the isolation of Flag-tagged WT (25Q) and polyQ-expanded (97Q) Htt protein complexes from HeLa cell cytoplasmic S100 fraction. Eluates from immuno-purrified Flag Htt 590 should be resolved by 10% SDS/PAGE and visualized by coomassie blue staining (Savas et al., p.10823). Verification of Ago2 as an Htt590 interactor should be done before probing the presence of antibodies in the Flag immuno-precipitates from the cell lysates.

Cytoplasmic extract from HEK293T cells stably expressing Flag-Ago2 or Flag-vector (Mock) should be immuno-precipitated with α-Flag M2 antibody and probed with α-Htt (HDB4E10, Abcam) or α-Flag antibody. HeLa cells should then be co-transfected with Myc-Htt590–25Q, -97Q or deletion mutant (ΔQ or ΔP) and Flag-Ago2.

Ago2 should be immuno-precipitated with α-Flag antibody and the presence of Htt be determined by immuno-blotting with α-Myc antibody. For the experiment to be more accurate, it important to verify the components of the reactions at each stage. Care should also be taken while extracting and culturing of specimens to be used in the experiments.

The RNA-mediated processes have continued to intrigue the science world. However, in spite of the rapid technological advances, studies and experiments on these processes remain largely rudimentary. Consequently, they present several weaknesses, which future attempts should address. Future experiments should take into account the quality of siRNA used in sequencing, invasive transfection reagents and differences in cell lines and culture conditions (Frantz, p. 763).

Future experiments should also build on the RNAi-dependent chromatin-based silencing pathway that was reported in fission yeast Schizosaccharomyces pombe. This may lead to better methods and guidelines for siRNA sequence selection, which are essential in combating the current setbacks caused by the trial-and-error methods.

In addition, there is need to develop more efficient system of delivery and regulation of tissue-specific expressions of siRNA. Lastly, it is important to develop additional RNAi protocols for genome-wide screens. This will assist in the accurate identification of genes involved in specific biological processes.

This experiment is expected to yield results that further prove that Huntingtin disease protein contributes to RNA-mediated gene silencing through association with Argonaute and P bodies. This is because Huntingtin is present in P bodies and this association is highlighted in these experiments.

Conclusion

The various tests conducted for the purpose of this research have gone a long way in establishing Huntingtin and the RNA-mediated gene silencing through association with Ago2 and P bodies. The tests were able to show that Ago2 co-purifies with Huntingtin, that Huntingtin is Present in P bodies and was able to compare normal and polyQ-expanded Huntingtin.

The findings of this report will hopefully pave the way for new ways of tackling certain gene-mediated complexes. A part from further equipping the world of medicine, it could lead to better plant and animal production as it would be possible to engineer genes to achieve certain qualities in organisms. More research is needed in this area in order to achieve long term success.

Works Cited

Fire, Andrew et al. “Potent and Specific Genetic Interference by Double-Stranded RNA in Caenorhabditis Elegans”. Nature 391 (1998): 806-811. Print.

Frantz, Simon. “Studies Reveal Potential Pitfalls of RNAi.” Nat. Rev. Drug Discovery 2 (2003): 763-764. Web.

Olsen, Philip H. and Ambros, Victor. “The Lin-4regulatory RNA Controls Developmental Timing in Caenorhabditis Elegans by Blocking LIN-14 Protein Synthesis After the Initiation of Translation.” Dev. Biol. 216.2 (1999): 671-680. Web.

Peters, Lasse and Meister, Gunter. “Argonaute Proteins: Mediators of RNA Silencing.” Mol Cell 26 (2007): 611–623. Web.

Savas, Jeffrey N. et al. “Huntington’s Disease Protein Contributes to RNA-mediated Gene Silencing Through Association with Argonaute and P Bodies.” PNAS 105.31 (2008): 10820-10825. Web.

Voinnet, Oliver. “Induction and Suppression of RNA Silencing: Insights from Viral Infections.” Nat. Rev. Genet. 6 (2005): 206-220. Print.

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