Breaking & Entering

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Using RNA Interference (RNAi) and Cell-permeable Peptides to Investigate Protein Function

It is well accepted that the major role of RNA is to transmit gene messages, via messenger RNA (mRNA), in order to produce protein. However, key cellular processes such as transcription, splicing, mRNA decay and translation are regulated by both protein factors and non-coding small RNAs. MicroRNAs (miRNA) represent one group of such small RNAs that regulates gene expression by interfering with the translation or triggering the decay of many key mRNAs. This miRNA-mediated regulation was first observed in plants in the early 1990s at the University of Arizona1, and was termed co-suppression. In 1998, Fire et al. defined this mechanism in C. elegans, naming it 'RNA interference'2.

It was observed that when a double-stranded RNA molecule was introduced into the cells of C. elegans, it interfered with gene expression. The discovery of this endogenous mechanism led to the development of the RNA interference (RNAi) technique that has revolutionized the field of molecular biology for the last six years. Moreover, some uses for RNAi outside of the laboratory have been found; it is also being tested as a drug to treat diseases such as cancer, viral infection and immunological related diseases, among others3. It has elicited so much interest and raised so many possibilities that the pioneers in the RNAi field have recently been awarded the Nobel Prize. RNAi has been found to be a cheaper, faster and a less labourious alternative of gene knockdown in order to study the function of a specific gene.

By knocking down a specific mRNA by RNAi, it is possible to observe an almost complete depletion of the corresponding protein, leading to a loss of function for the gene-product, when compared to the wild-type. RNAi mainly acts by blocking gene expression at the post transcriptional level (mRNA) when short, double-stranded RNA molecules (ex: small interfering RNA [siRNA], short hairpin RNA [shRNA]) are introduced in the cells.

These short RNAs act by using an endogenous cellular system, which involves the RISC complex to recognize the target mRNA, leading to its rapid decay4. However, since these short RNAs are only approximately 21 base pairs long, it was found that off-target, non-specific effects could also occur5. To address this issue, rescue experiments have been used to prove specificity and eliminate artifacts due to off-target effect. If depleting a protein may be considered the first step in studying its function, it would be safe to say that returning the level of that protein to what it once was is the second step. Such an approach is known as a 'rescue', since the phenotype resulting from the lack of a functional protein has been 'corrected' by having the protein level returned.

Various methods have been developed to perform rescues, but there exists a barrier which each method has had to tackle. In fact, siRNA administration has also had to contend with this obstacle. All cells are delineated by a lipid bilayer, known as the cell membrane. This membrane serves as a selective barrier for what may enter the cell. Nucleic acid material (including siRNA duplexes) for example, cannot simply traverse the membrane. Instead, a process known as transfection is often used, whereby the cells have genetic material added into them. One of the most popular ways of doing this involves adding a compound that forms positively charged lipid vesicles, also known as liposomes. Since these are positively charged and DNA is negative, they associate together, and are thought to be taken into the cell through endocytosis.

Alternative methods of transfection include using the compound CaPO4, which can increase permeability between the lipids forming the membrane, or cells may simply be shocked (known as electroporation), but these methods can produce a lower transfection efficiency, cause more cell damage, are less reproducible, or may simply be more inconveniencing. Transfection can also be used in rescuing cells, whereby a cDNA expressing the gene that is being inhibited by the siRNA duplexes is added back to the cells. To protect this cDNA from the siRNA duplexes, silent mutations (which cause variations in the DNA code, but not in the protein product) can be performed in the siRNA-target sequence6. Although this approach allows the expression of the depleted protein in siRNA-treated cells, it faces the limitation of transfection efficency. Likewise, creating a silent mutation gene variant can be time consuming due to the different steps required to optimize this approach.

To address the transfection efficiency issue, several methods using RNA or DNA viruses have been developed. During infection, viruses can be engineered to insert the desired small, silent mutated DNA, similarly to the method just described above, except that the viruses require no additional compounds for transfection with much higher transfection efficiency than regular cDNA7.

Alternatively, DNA can be encapsulated in its own neutral liposome (unlike transfection as described earlier, where it simply associates with the vesicle), and this capsule can fuse into the membrane of the cell, eventually allowing the DNA to enter. While these methods do not involve the strain of chemical treatment on the cells, they are much more time-consuming when it comes to preparing the materials. Additionally, administering DNA by these methods results in a different expression level of the gene than would normally be observed, making it difficult to compare the rescue protein level to the endogenous level of the target protein.

More recently, a non-gene based approach was developed to rescue the expression of siRNA-mediated depleted proteins. Instead of administering DNA, which must then be transcribed and translated to produce protein, the protein itself can be given to cells. Again due to the selective entry cell membranes permit, modifications have been required. A discovery in 1988 revolutionized this field when researchers noted that an HIV protein (the Tat Transactivator) was capable of passing through the cell membrane 8, 9. It was found that certain proteins contain a motif that allow them to physically interact with the exterior of the cell membrane, leading to their rapid intake inside the cell. In 1991 other ‘cell-permeable peptides’ (CPPs) were found10, directing more attention towards these compounds. It was shown that by conjugating the permeabilization region of these CPP proteins to another protein, the chimera could likewise freely enter the cell11.

One of the most efficient cell-permeable peptides is the Drosophila transcription factor Antennapedia (AP) or more precisely, the third helix of the AP homeodomain12. A proposed explanation for its high cellular uptake efficiency when compared to the majority of CPPs is that since the sequence used is derived from a protein that transfers between cells, it is possible that during evolution its permeability characteristics have improved13. The mechanism by which the AP-peptide is taken into the cell has not yet clearly been determined. For years, it was believed that the AP homeodomain translocates through the cell membrane of all cell types without the need of active intracellular transporters. It was suggested that the AP permeable peptide interacts with the plasma membrane via electrostatic interactions and is internalized by micropinocytosis or by the formation of inverted micelles13. Recent studies support the converse, however, demonstrating the requirement of an energy dependant pathway for translocation14, 15. This discrepancy can in fact be viewed as a disadvantage of the use of AP peptides, because it is always preferable to understand how the tools one is using actually work. Likewise, the production of proteins in large quantities can be quite costly, and so this must also be considered when choosing to use CPPs for rescue. However, the in vivo effects of AP-mediated delivery of conjugated polypeptides or proteins justifies the use of cell-permeable as a method for protein delivery16.

If the goal is to delineate the function of a given protein, the use of RNAi knockdown and AP-conjugated proteins for rescue go hand-in-hand. In fact, it is now common to use rescue in order to validate that the loss of phenotype observed with RNAi treatment is specific for the target gene. An example of this may be given from the research done in the laboratory. HuR is an mRNA binding protein that was hypothesized to play a role in the formation of muscle fibers17. To test this hypothesis both RNAi and cell-permeable-mediated rescue approaches were combined. Using these methods it was demonstrated that HuR is required for the formation of skeletal muscle fibers17. Depleting the expression of HuR protein in the embryonic mice muscle cells C2C12 led to the complete inhibition of muscle fiber formation, commonly called myogenesis17.

To confirm that this inhibition was in fact due specifically to the loss of HuR, AP-HuR-GST and GST-AP (a control) fusion proteins, a 60 kDa and a 26 kDa protein, respectively, were added to these cells prior to induction of differentiation. Then, when myogenesis was initiated, it was possible to observe the rescue of C2C12 differentiation in the cells that had been treated with AP-HuR-GST, whereas those treated with GST-AP were unable to differentiate17. These results clearly demonstrated that HuR was specifically responsible for the rescue, and that its siRNA-mediated silencing was specific. This approach has been used to rescue the expression of other proteins by us as well as other groups.

Figure 1. The use of RNA interference (siRNA) and a cell-permeable peptide (AP) to demonstrate the specific role of HuR in myotube formation. (A) The inhibitory effect of silencing HuR endogenous expression in myoblast cells via siRNA prevents the formation of muscle fibers17. (B) The addition of AP-HuR-GST to the siRNA-HuR-treated embryonic muscle cells (C2C12) reestablishes muscle fiber formation17. This is one example of how RNA interference and cell-permeable peptides can be used together to investigate protein function.

Even though the great majority of proteins fused to the AP homeodomain are effectively translocated into cells (>95% transfection efficiency), rescue experiments using AP peptides are still underrated. Moreover, not only can CPPs be used for rescue experiments, but they also serve other research purposes. For example, in the case of cell lines that are difficult to transfect such as differentiated neurons or muscle fibers, the AP peptides could be used as a carrier to any protein with >90% efficiency. Furthermore, in vivo trials are attempting to target a specific cell type with CPPs, while preventing non-specific entry into other cell types. These are just some examples of how cell-permeable peptides are utilized, and in years to come it will not be surprising to see further uses develop for CPPs. In the field of molecular biology research, innumerable tools are available to discover more about the biology systems studied, and by combining modern techniques, the field can advance at an even faster rate.

Acknowledgements
This work was supported by an FRSQ fellowship and a CIHR operating grant (Mop-57680) to I.G. I.G. is a recipient of TierII Canada
Research Chair.

References
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Christopher von Roretz and Pascal Beauchamp are Master's students in the laboratory of Dr. Imed Gallouzi, at McGill University. Von Roretz's research focuses on studying the relationship between the mRNA-binding protein, HuR, and apoptosis, while Beauchamp studies the role of HuR on muscle differentiation. Imed-Eddine Gallouzi, PhD, is an Assistant Professor in the Department of Biochemistry at McGill University, Montreal. Gallouzi's laboratory specializes in the study of HuR and the various roles it plays in biological systems. HI.E.G is also a TierII Canada Research Chair.