Research Summaries

IRES Initiated Translation

We are studying the unusual mechanism of translation initiation by internal ribosome entry in certain viral (i.e. Hepatitis C virus, picornaviruses and some insect viruses) and cellular mRNA molecules. In the conventional scanning mechanism of translation initiation, which operates on most mRNA molecules, 40S subunits are recruited at or near the 5' end of the mRNA. Subsequently, the 40S ribosomal subunits are predicted to scan the mRNA in a 5' to 3' direction until the first AUG codon is encountered as start site for protein synthesis. However, certain viral and cellular mRNAs, notably encoding proto-oncogenes and regulatory genes, contain long 5' noncoding regions with multiple AUG codons. Thus, the translation initiation rate in these mRNAs is predicted to be low according to the scanning model; alternatively, other translation initiation mechanisms may operate to ensure efficient translation. Indeed, some of such mRNAs with long leaders contain internal ribosome entry sites which can bind ribosomes directly. Much of our work has been focusing on the mechanism and prevalence of internal ribosome binding. Specifically, we are addressing the following questions: Which cellular and viral mRNAs can be translated by internal ribosome binding? What are the cellular gene products that mediate internal ribosome binding? Is internal initiation regulated in the cell? What is the molecular basis for designating a given AUG codon as start site codon?

Posttranscriptional Control of Gene Expression by microRNAs

RNAi and microRNAs (miRNA) modulate eukaryotic post-transcriptional gene expression. Both types of regulation involve small non-coding RNA molecules of approximately 22 nucleotides. The most extensively studied of the two modes is the RNAi pathway, in which small interfering RNAs (siRNAs) bind to mRNA targets with perfect complementarity, and stimulate their decay. In contrast, miRNAs (endogenous non-coding RNA molecules) regulate gene expression by RNA decay and translational inhibition. A miRNA ribonucleoprotein particle (miRNP), containing the ~21 nt miRNA strand, typically recognizes sequences within the 3’ UTR of target mRNAs. The degree of sequence complementarity between the miRNA and the 3’ UTR mRNA binding site is proposed to dictate the miRNA mode of action. Similar to the RNAi pathway perfect complementarity triggers miRNP-mediated cleavage of the target mRNA, while imperfect base-pair interactions are proposed to inhibit translation. To understand the specific control mechanisms miRNAs employ to control gene expression, we are interested in identifying and characterizing cellular miRNAs, their targets, and cellular factors required for miRNA-mediated translational repression and miRNA-directed RNA decay.

Roles for microRNA-122 in Hepatitis C Virus Gene Amplification

Approximately 3% of the world’s population is infected with Hepatitis C virus (HCV) and in the US, chronic HCV infection is the most common factor leading to liver transplantations. HCV is an enveloped RNA virus classified within the Flaviviridae family. The positive-sense, single-stranded HCV RNA genome encodes a single open reading frame (ORF). At the 5’ and 3’ ends of the genome are conserved untranslated regions (UTRs) which are essential for expression of the long ORF and genome replication. Our laboratory recently reported that the highly abundant liver-specific microRNA miR-122 modulates HCV gene expression. Sequestration of miR-122 with antisense oligonucleotides in a cell line harboring autonomously replicating HCV (also known as replicon cells) resulted in a dramatic reduction of HCV gene expression. This surprising positive regulation of HCV gene expression by a miRNA was confirmed by complementation studies in cells expressing HCV RNA with altered miR-122 recognition sites and exogenously added compensatory mutant miR-122. Additionally, unlike previous miRNA associations with the 3’ UTR of target mRNAs, the miR-122-HCV genome interaction occurred specifically within the 5’ UTR. An interaction between viruses and miRNAs is however not unprecedented as a number of viruses encode their own miRNAs to manipulate viral and cellular gene expression. This is, however the first report of a cellular miRNA promoting gene expression. We are currently examining the mechanism by which miR-122 modulates HCV RNA levels, as well as searching for cellular targets of miR-122 and their regulation by miR-122.

Virus-Host Interactions

With limited anti-viral approaches, associations with human cancers, and the continuing emergence of new and highly pathogenic viruses (e.g. Hendra and Nipah viruses, and the SARS [severe acute respiratory syndrome] virus), viruses continue to contribute greatly to the public health burden. In order to develop effective antiviral therapies we need to understand both the virus life cycle, and how the virus and host interact. As the virus strives to usurp the cell, and the host mounts its defensive response, virus-host interactions are not only intricate but also multifaceted. Our current research integrates the use microarrays, proteomics and bioinformatics to examine the effect of poliovirus and cricket paralysis virus on gene expression, protein production, and posttranslational modifications and processing events.

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