study of nonsense mediated mrna decay

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Project 1:

Nonsense-mediated mRNA decay (NMD) is a quality-control mechanism that selectively degrades aberrant mRNAs harboring nonsense codons (premature termination codons; PTC). If translated, these mRNAs can produce truncated proteins with deleterious activities. NMD is a highly conserved pathway that exists in all eukaryotes examined to date. Nearly, a third of human genetic diseases are caused by mutant genes with nonsense or frame shift mutations resulting in PTC. Recently, NMD was also shown to target retroviruses as well as mRNA expressed from subset wild type cellular genes.

Although a lot of research has been done to study the mechanism of NMD question remains how the machinery involved distinguishes between premature and normal stop codons. The current consensus is that a second downstream signal dictates whether a stop codon is premature or not. While this second signal appears to vary in different species and possibly in different transcripts, a common feature is that it must be downstream of the stop codon to elicit NMD. Early studies revealed that the second signal in mammalian cells is somehow delivered by an intron downstream of the stop codon. This explained why normal transcripts with stop codons located in last exon are not subject to NMD. Recent studies suggest that rather than being a single linear pathway, mammalian NMD has several branches.

NMD poses a significant challenge to the stability of retroviral. Retrovirus is dependent on host transcription and translation machinery and must overcome mRNA quality control measures to ensure their genome is translated in an efficient and timely manner. It could be achieved by hijacking cellular pathways for encoding elements stabilize its own genome. Recently, a RNA stability element which facilitates NMD evasion was identified immediately downstream of the gag termination codon in Rous sarcoma virus. It is reasonable to believe that HIV-1 has devised strategies to circumvent the problem of NMD.

Figure 1: Schematic representation of the mammalian Green Fluorescent Protein (GFP) based NMD reporter assay. The GFP target gene construct expressed with or without a premature termination codon mutation.

I propose to develop Green Fluorescent Protein (GFP) based assays to study mechanism of NMD evasion by HIV-1. GFP transcript will be expressed using a strong CMV promoter will be cloned in frame with NMD substrate RNA sequence. Once the hybrid transcript is expressed in mammalian cells it will be subject to NMD and reduced translation into GFP (figure 1). NMD of this transcript will be monitored by PCR, RNAse protection assay and visualizing cells using fluorescence spectroscopy for expression of GFP. Overexpression and knockdown factors involved in NMD (for e.g. UPF1) will be used to confirm reliability and reproducibility of the assay. This assay will serve as a useful tool to investigate both trans acting factors as well as cis acting HIV element involved in hijacking of NMD pathway.

Several key components required for NMD have been identified; but all the factors that modulate NMD have not been completely elucidated. Elucidating the factors involved will lay a foundation for understanding the NMD transcriptional networks in cells. The GFP reporter assay described above is adaptable to high throughput format and will be subsequently utilized to find new factors involved in NMD pathway. Initially, based on survey of literature, I will perform a manual siRNA screen for factors (for e.g. RhoxF2) which may have a potential role in NMD pathway. This would be first utilized to do a medium throughput screen to identify new factors involved in this pathway. The unified NMD model will make many testable predictions and will hopefully provide a useful framework for future mechanistic investigations. This in turn may lead to therapies for diseases involving the NMD RNA surveillance pathway.

Project 2:

Chikungunya virus (CHIKV), a member of the Alphavirus genus, is a serious potential threat to human health in many areas. It has caused millions of cases of serious, but not life-threatening, illness characterized by fever, rash, and a painful arthralgia. Information about CHIKV is growing rapidly but much remains to be discovered. Strategies utilized by CHIKV for replication, its genetic evolution, and its interactions with the host and vector are poorly understood. CHIKV is a small (about 60-70 nm-diameter), spherical, enveloped, positive-strand RNA virus. Its genome is of approximately 12 kb which encodes two ORF of poly-proteins. These poly-proteins are further processed into structural (C, E3, E2, 6K, E1) and non structural proteins (nsP1, nsP2, nsP3, nsP4) by cellular and viral proteases. There are no approved antiviral treatments currently available for CHIKV. Currently, CHIKV infection is treated symptomatically, usually with non-steroidal anti-inflammatory drugs or steroids, bed rest, and fluids.

Among the virus-encoded enzymes, nsP2 constitutes an attractive target for the development of antiviral drugs. It is a multifunctional protein of approximately 90 kDa with a helicase motif in the N-terminal portion of the protein while the papain-like protease activity resides in the C-terminal portion. The nsP2 protease is an essential enzyme whose proteolytic activity is critical for virus replication. I propose to generate a fluorescent reporter protein as a target for nsP2 protease and develop a cell based FRET assay to screen inhibitors of (Figure 2).

The FRET reporter will have nsP2 protease target site as linker between fusion of FRET Donor and Acceptor fluorescent protein. Upon expression in mammalian

Figure 2: Schematic representation of the FRET based reporter assay. The FRET donor and acceptor fusion protein will be linked with nsP2 protease target sequence. In presence of nsP2 linker will be cleaved and separate the donor and acceptor leading to loss of FRET signal from acceptor protein.

cells this protein is expected to show good FRET signal which can be easily quantified using a plate reader. Co-expression of nsP2 enzyme, will lead to cleavage of FRET reporter protein at linker site. This will separate the fluorescence donor and acceptor and will lead to loss of FRET signal. When an appropriate inhibitor is added in the system, the FRET signal will be restored. Also, viability of cells and toxicity of tested drugs can be easily followed by monitoring the fluorescence emission of acceptor.

Once this system is developed several active pharmaceutical inhibitors will be screened for activity. My priority would be to first screen known clinical available inhibitors of papain family of protease (for e.g. Cathepsins) which can be readily used for therapy. Further, the utility of reporter system can be expanded to study other of inhibitors for CHIKV which may affect different steps in viral life cycle and can be monitored by following fluorescence signal.

Similar system can also be adopted to screen inhibitors for other viral proteases (for e.g. Dengue virus, Hepatitis C virus).