Research

***Graduate student positions available for 2018!***

The regulation of gene expression at the translational level is fundamental for normal cellular homeostasis and survival. Because control of translation is relatively quick and efficient, cells can regulate gene expression quickly in response to environmental stresses. During some cellular stresses such as viral infection, endoplasmic stress (ER) stress and apoptosis, the cell adapts by inhibiting overall protein synthesis. However, it is apparent that a subset of mRNAs are translated under these conditions. These mRNAs, in general, encode for proteins that are important for the cell to adapt to the environmental stress. The identification of these specialized mRNAs is crucial in our understanding of fundamental gene expression mechanisms and how cells respond to stress and viral infection.  We are addressing several main questions:

A) Which mRNAs are translated during cell stress and virus infection?  By identifying these specialized mRNAs, we will gain insights into the network of genes that respond to and adapt to cell stress and virus infection. We are especially interested in understanding the cell responses in cells that contribute to diabetes, cancer and virus infection.

B) How are these mRNAs translated when overall protein synthesis is shutoff? These mRNAs must contain cis-acting RNA elements that can recruit the ribosome. We are using biochemical and molecular appproaches to elucidate how these mRNAs are translated.

C) How do cells respond to virus infections? When viruses infect cells, the host cells respond by inducing an innate antiviral immune response. Conversely, viruses have evolved strategies to counteract and evade these host antiviral responses.  We are interested in identifying the signaling pathways and mechanisms that contribute to host antiviral immunity and infection.

Projects

1) One of the major interests in the lab is to understand how viruses interact with the host cell. Specifically, we are interested in how viruses hijack the host translational machinery for proper expression of viral proteins at the expense of host gene expression. A current focus is on an unusual non-canonical mechanism of translation found in an internal ribosome entry site (IRES) of a the dicistroviridae family.  Members of this family include the Cricket paralysis virus (CrPV) and the honey bee viruses such as Israeli acute paralysis virus (IAPV) and Kashmir Bee virus. IRESs are typically long, structured RNA elements, which can directly recruit ribosomes and normally requires a subset of translation initiation factors. Remarkably, unlike translation of the majority of mRNAs, this type of IRES contains domains that functionally mimic a tRNA to initiate translation independent of an AUG start codon, initiator Met-tRNA, and initiation factors. Thus, the IRES has evolved specific RNA elements that recruit and hijack the ribosome. Currently, we are using biochemical approaches to identify and delineate the RNA elements that manipulate specific functions of the ribosome. The study of the dicistrovirus IRESs has served as a model for understanding other IRESs found in some viruses such as hepatitis C virus, HIV and poliovirus and in some cellular IRES such as the oncogene, c-myc, and the angiogenesis factor, VEGF.

We recently have discovered that a subset of dicistroviruses contain a hidden open reading frame, called ORFx, just downstream of the IRES. We demonstrated that the IRES can drive translation in 0 and +1 reading frames, thus revealing a novel viral strategy to increase coding capacity. Interestingly, specific mutations in the IRES can uncouple 0 and +1 frame translation, thus suggesting the IRES adopts distinct conformations that direct reading frame selection. We are using biochemical approaches to gain insights into translational reading frame selection by the IRES and how this may be regulated during virus infection. We are also addressing the role of ORFx, which we hypothesize has a role in counteracting antiviral immunity or a specific step of the viral life cycle.

The secondary structure of the CrPV IRES. Conserved nucleotide positions are shown in uppercase, and nonconserved nucleotides are in lowercase. Numbering refers the nucleotide position within the CrPV RNA genome. Helical regions are indicated by a black dash between nucleotides. The CCU triplet and the GCU alanine codon, which encodes for the first amino acid, occupy the P- and A-sites of the ribosome, respectively. Underlined nucleotides represent the two amino acid residues in the viral capsid protein. PK denotes a pseudoknot structure and SL denotes a stem-loop.

2) Viruses have evolved numerous strategies to efficiently and preferentially amplify their genome in the host cell. Furthermore, the virus has to evade the host antiviral response while at the same time competes for specific host cellular machinery (i.e. the ribosome). By revealing the mechanisms of the battle between the virus and host, we will have a better understanding of the host response to viral infection and may also uncover novel targets for antiviral therapies. Our lab is using the cricket paralysis virus infection as a model system to reveal fundamental virus host interactions. Using a Drosophila as a model host system, we are presently employing RNA-seq and ribosome profiling approaches to identify the host antiviral response. Furthermore, we are also examining the mechanism by which host protein synthesis is shutoff during CrPV infection in order to understand how the virus recruits ribosomes from host mRNAs.

3) Positive sense RNA viruses such as poliovirus, Hepatitis C virus and coxsackievirus translate their genomes to express a long polyprotein, which is then processed by a virus-encoded protease to produce the individual mature viral proteins. The viral protease also target host proteins to affect specific cell functions and to dampen the host antiviral responses.  However, the complete repertoire of host protein targets of viral proteases is not known. In collaboration with Chris Overall and Honglin Luo, we are using a novel proteomics approach to enrich for the target proteins that are cleaved by viral proteases. Current projects are focused on identifying the repertoire of host targets of proteases from poliovirus and coxsackievirus.

4) Because the CrPV IRES has the unusual ability to recruit ribosomes directly without the aid of initiation factors, we hypothesize that the IRES can be exploited to drive translation of genes in diseased cells.  We are exploring the therapeutic approach of delivering IRES-containing mRNAs using liposome-nanoparticles to solid tumours in collaboration with Pieter Cullis’s lab.

5) The endoplasmic reticulum (ER) is a specialized organelle in the cell where proteins destined to be secreted are folded and glycosylated. The balance of folded proteins in the ER has to be tightly regulated for proper cellular function. For instance, the chronic accumulation of unfolded proteins in the ER (called ER stress) is associated with the progression of diabetes and with diseases where specific missense mutations in genes cause the improper folding of proteins in the ER. The cell senses and responds to ER stress by activating a stress response called the unfolded protein response (UPR). One arm of the UPR is a rapid and dramatic inhibition of protein synthesis in order to decrease the accumulation of unfolded proteins in the ER. Paradoxically, the inhibition of protein synthesis triggers the preferential translation of specialized mRNAs, some of which are vital for the cell to adapt to the ER stress. In collaboration with Jim Johnson, we aim to identify the subset of these mRNAs through microarray-based and next-generation sequencing approaches and to elucidate the noncanonical mechanisms by which these mRNAs are translated during ER stress.

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