Investigating how mRNA Capping Enzyme regulates the c-Myc oncogene

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The c-Myc gene encodes a protein which is a key player in regulation of gene expression, whose malfunction and mutation is associated with many types of cancer. Currently, no therapies targeting c-Myc are available, thus this gene is a topic of intense research. Recently it was shown that the mRNA Capping Enzyme regulates c-Myc protein expression, but the mechanism is still largely unknown. Through biochemical and molecular biology experiments, we can deepen our understanding of the c-Myc oncogene regulation and in turn, bring us a step closer towards understanding the mechanisms of cancer, and potentially lead to new therapies. This paper will outline a potential mechanism by which Capping Enzyme regulates c-Myc gene expression which was discovered through a Nuffield Research Project.


The central dogma of biology states that genes encoded in the DNA get transcribed into mRNA transcripts, which are translated into proteins (Figure 1). Gene expression is a tightly regulated process. One of the key steps is the addition of a cap structure (mRNA Cap) to the 5’ end of the mRNA by mRNA Capping Enzymes. This cap serves as a marker for further transitional steps and prevents mRNA degradation by nucleases 1)Sianadh Dunn and Victoria H. Cowling, “Myc and mRNA capping”.

Untitled:maria:c-myc gene, mrna and protein diagram.tiff

(Figure 1; c-Myc transcription and translation)

Genetic mutations can spontaneously occur in genes within the cell, which change the gene expression and can cause cells to proliferate uncontrollably. Cancer and tumour formation is the result of at least two mutations in cancer-associated genes occurring within a cell, because cells have ‘tumour-suppressor’ mechanisms for dealing with a single mutation. For example, the first mutation could activate a gene that drives cancer and the second mutation could be one that prevents a tumour-suppressor mechanism, causing this cell to become cancerous and, after several cell divisions, form a tumour. It is crucial to understand how these cancer-causing genes (such as c-Myc when malfunctioning) are regulated. Only when we thoroughly research this we can produce a range of drugs and therapies that could specifically target these mechanisms (known as targeted cancer therapies). In theory these drugs would be much more efficient and less toxic than traditional cancer drugs or chemotherapies.


The c-Myc oncogene is an important gene in the human body, which controls over 15% of our genome 2)c-Myc gene”. The c-Myc gene expresses a c-Myc protein, which conducts many important cell functions such as growth, division and gene expression. In healthy cells, the c-Myc gene maintains a normal proliferation rate. However, it is thought that mutation or deregulation of the c-Myc gene occurs in 50% of all cancers, and often contributes to tumour initiation and progression. The c-Myc protein is challenging to therapeutically target in cancer patients since it plays an important regulatory role in the normal cells, so it is unfavourable to simply ‘block it’ 3)“Cancer Research – Targeting Myc”. Moreover, the c-Myc protein lacks an enzymatic active site which can be readily inhibited by small-molecule drugs. Therefore, current research is focused on the number of proteins regulating the gene (e.g. Capping Enzyme and CRD-BP).

These proteins may be more accessible to target and could lead to new methods of treating cancer. Targeted therapies may have an advantage over traditional chemotherapies, which cause a lot of damage to healthy cells. In a project, I investigated the various ‘stages’ of c-Myc expression such as c-Myc protein and mRNA transcription. It was shown that the mRNA capping enzyme regulates the expression of c-Myc 4)Olivia Lombardi et al, “c-Myc deregulation induces mRNA capping enzyme dependency”, but it is currently unknown if the mechanism is conducted directly or indirectly, i.e. mediated by other proteins mRNA Capping Enzyme regulates.


It has been found that when Capping Enzyme is knocked down, c-Myc protein levels are subsequently decreased. But how does this happen? These were the following hypotheses:

  • Capping Enzyme regulates c-Myc mRNA stability, protein stability or both.
  • Capping Enzyme is regulating other proteins/genes that can in turn be acting on c-Myc (eg CRD-BP). CRD-BP (Coding Region Determinant – Binding Protein) is a protein bound to CRD on the c-Myc mRNA at the 3’ end, and regulates c-Myc mRNA stability (by either binding to the mRNA or not). As Capping Enzyme has the potential to regulate a number of genes, CRD-BP may be one of them and thus c-Myc may be regulated this way. (See Figure 2)

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(Figure 2)

(Figure 2; potential c-Myc regulation through CRD-BP)


To investigate the mechanism by which c-Myc is regulated two experiments were performed.

Experiment 1

A Capping Enzyme knockdown was performed to investigate its effect on c-Myc mRNA levels. Capping Enzyme, c-Myc and a control gene were knocked down (this means the gene will stop being expressed) in HeLa cells using siRNA transfection methods with the help of a RNAiMax lipid-based transfection reagent, and incubated for 72 hours before performing RT-qPCR and analysing genes of interest – c-Myc, Capping Enzyme, CRD-BP and GAP-DH (a house-keeping gene used as a control). siRNAs are non-coding RNAs which bind to mRNA molecules (with fully or partly complementary sequences) and reduce their stability, thus controlling the levels of these mRNAs present in the cells 5)“Regulation after Transcription”. Whereas, transfections are the process of introducing foreign genetic material into a cell. In transfections, liposomes are used (lipid bilayers that form particles around the siRNA). The lipid structure is similar to that of a cell membrane, and fuses into the cell releasing its contents. The siRNAs then interfere with the specific target sequence, and ‘block’ a gene in the mRNA. This stops the gene’s expression whose effect can be observed on other genes or components in the cell. RT-qPCR is a technique used to amplify nucleic acids such as RNA to observe their relative quantity to a control in the cell[1]. mRNA expression levels obtained were normalised to GAP-DH. Results shown in Figure 3 were obtained. Three technical replicates were performed and the average expression is shown. The table was further processed into Figure 3 where the relative expression of genes was shown when genes of interest were knocked-down. The error bars represent the variation between technical replicates as determined by standard error of the mean (SEM), n=3.

Experiment 2

In this experiment, the effect of Capping Enzyme knockdown on c-Myc protein stability was investigated. A Capping Enzyme knockdown, alongside a control, were performed on HeLa cells using a siRNA transfection method, and the cells were incubated for 72 hours before treating them with a protease inhibitor (this stops all protein, including c-Myc, degradation in a cell) and its controlfor two hours. The protein concentration from each sample was measured using a Bradford Protein Assay (used to quantify protein concentrations in cells), and SDS-PAGE (method which separates proteins based on their size, and allows their relative quantification and detection) was conducted to obtain the relative concentrations of c-Myc protein and Actin, a control protein, in the cells investigated. The intensity of the bands was quantified and Figure 5 was obtained. The results were further processed into Figure 6 shown.


Experiment 1

mRNA of interest expression level relative to GAP-DH
Gene Sample Ctrl Expression SEM
CE ctrl siRNA * 1 0.064
CE CE siRNA 0.2689 0.0359
CE c-Myc siRNA 1.20573 0.06863
C-MYC ctrl siRNA * 1 0.06393
C-MYC CE siRNA 0.42709 0.02163
C-MYC c-Myc siRNA 0.63726 0.04566
CRD-BP ctrl siRNA * 1 0.02213
CRD-BP CE siRNA 0.71637 0.02232
CRD-BP c-Myc siRNA 0.82146 0.03174

(Figure 3)

(Figure 4)

How to interpret Figure 5: The genes of interest are shown along the x-axis, and their expression is shown relative to a control – y-axis. The colours represent the siRNA knock-downs. Eg, if Capping Enzyme was knocked-down, the c-Myc gene expression decreased (compare the red bar to the blue control bar above the C-MYC Gene).

Analysis – Experiment 1

In Capping Enzyme knockdown (red bars), it is observed that Capping Enzyme controls c-Myc expression (i.e. mRNA stability). This is seen as when Capping Enzyme is knocked down, the expression of c-Myc decreases, which confirms previous studies [1]. It is also seen that CRD-BP expression levels are decreased when Capping Enzyme is knocked down – this suggests a possible mechanism by which Capping Enzyme controls c-Myc expression. This has not been previously observed and may be a novel way in which the c-Myc oncogene is regulated.

Experiment 2

Actin (control protein) Protein Concentration c-Myc Protein Concentration
Control 15492.317 Control 11149.752
Control CE 14384.853 Control CE 12138.995
Protease Inhibitor Ctrl 17598.095 Protease Inhibitor Ctrl 35383.413
Protease Inhibitor CE 16013.267 Protease Inhibitor CE 23959.836

(Figure 5)

Figure 6: Ctrl and CE are two separate treatments of siRNA knockdowns. Actin and c-Myc are the two protein expressions observed

(Figure 6)

Analysis – Experiment 2

When Capping Enzyme is knocked down and cells are treated with the protease inhibitor, c-Myc protein levels still decreased. From previous studies we know that when Capping Enzyme is knocked down, c-Myc protein levels decrease [4]. If Capping Enzyme was regulating c-Myc protein stability/degradation, c-Myc protein levels would have not been affected by Capping Enzyme knockdown in the presence of the inhibitor as this treatment stops all protein degradation. However, since c-Myc protein levels do decrease, this is an indication that Capping Enzyme does not regulate c-Myc protein stability/degradation. Thus another mechanism is working in place (and this could potentially be through the regulation of CRD-BP by Capping Enzyme, affecting c-Myc mRNA stability indirectly.

Conclusion / Outlook

Through various molecular biology and biochemistry techniques, I have determined that:

  1. Capping Enzyme is responsible for c-Myc expression regulation (i.e. mRNA stability)
  2. Capping Enzyme does not regulate c-Myc protein stability.

I have also suggested that Capping Enzyme might indirectly regulate c-Myc protein levels through the mRNA binding protein, CRD-BP.

These results have contributed to our knowledge of Capping Enzyme regulation of c-Myc and its stability. Performing biological replicates of these experiments would confirm these results. They could potentially lead to novel therapies for cancer targeting. This field of work can be extended along several directions – the mechanism between CRD-BP and c-Myc could be investigated, and seeing if targeting CRD-BP can kill cancer cells which are driven by the c-Myc gene.


  •  CRD – Coding Region Determinant is a region to which the CRD-BP (Coding Region Determinant – Binding Protein) binds
  •  Gene – sequence of DNA that codes for a protein
  • Nuclease – an enzyme in cells that degrades nucleic acids
  • Oncogene – a cancer causing gene
  • Proliferation – cell growth and division
  •  Protease – an enzyme which breaks down protein in the cell
  • Transcription – The process by which a complementary mRNA strand is made from the DNA in the nucleus.
  • Translation – The process by which the mRNA gets translated into protein in the ribosomes located in the cytoplasm.


I would like to thank the Nuffield Foundation and University of Dundee for giving me an opportunity to carry out my research project. I would also like to thank my scientific supervisor Dr Victoria Cowling, my two outstanding supervisors: Olga Suska and Olivia Lombardi for giving up their time and supporting me throughout my project (and for supplying the placement with lots of laughter and Wotsit celebrations), and all the other members of the lab (Fran, Aneesa, Alison, Jo and Dhaval) for making this project inspirational and amazing!


  1. Example: suppose the control was 100%, and an abundance of 60% for a protein was obtained – this means that the levels of it’s expression decreased in the cell

References   [ + ]

1. Sianadh Dunn and Victoria H. Cowling, “Myc and mRNA capping”
2. c-Myc gene”
3. “Cancer Research – Targeting Myc”
4. Olivia Lombardi et al, “c-Myc deregulation induces mRNA capping enzyme dependency”
5. “Regulation after Transcription”

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