The role of small non-coding RNAs in host-baculovirus interaction and insect development

Jayachandran, Balachandran (2013). The role of small non-coding RNAs in host-baculovirus interaction and insect development PhD Thesis, School of Biological Sciences, The University of Queensland.

       
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Author Jayachandran, Balachandran
Thesis Title The role of small non-coding RNAs in host-baculovirus interaction and insect development
School, Centre or Institute School of Biological Sciences
Institution The University of Queensland
Publication date 2013
Thesis type PhD Thesis
Supervisor Sassan Asgari
Karyn Johnson
Total pages 65
Total colour pages 24
Total black and white pages 41
Language eng
Subjects 1001 Agricultural Biotechnology
0605 Microbiology
0703 Crop and Pasture Production
Formatted abstract
Intensive genomic and transcriptomic research in various organisms in recent decades have identified a new class of RNAs called non-coding RNAs (ncRNAs). ncRNAs were initially considered as “junk” RNA molecules since they had no role in protein synthesis.Rigorous studies in this field have revealed that they play crucial roles in gene regulation, genome stabilization and defense against invading pathogens. As part of the RNA interference (RNAi) response, small ncRNAs between 18-36 nucleotides can be classified into microRNAs (miRNAs), short interfering RNAs (siRNAs), and PIWI-interacting RNAs (piRNAs) according to their biogenesis and function.

In this study, we attempted per oz administration of synthetic miRNA mimics and inhibitors to larvae to study the function of miRNAs. For this, we identified a miRNA, har-miR-2002b,that is specifically expressed during larval stages of Helicoverpa armigera (cotton bollworm) and used it as an example. The supply of har-miR-2002b by feeding larvae on the miRNA mimic led to 40% larval mortality, reduced adult emergence, and 70% reduction in adult fecundity. A potential target for this miRNA was identified as the H.armigera trypsin-like serine protease (Ha-TLP). The oversupply of har-miR-2002b significantly reduced the Ha-TLP’s transcript and protein levels leading to less trypsin activity in the midgut. On the other hand, the application of har-miR-2002b inhibitor increased the Ha-TLP’s transcript levels both in vitro and in vivo. We also confirmed this negative interaction using a GFP reporter study. This is the first study which showed the potential of utilization of miRNA mimics and inhibitors in insect miRNA research by oral feeding and their potential use in pest control by targeting specific insect genes.

By utilizing the per oz administration of miRNA mimics/inhibitors developed during this project, we successfully established that Helicoverpa armigera single nucleopolyhedrovirus (HaSNPV) down-regulates the host gene H. armigera ecdysone receptor (Ha-EcR) but not the control gene Ha-TLP, via down-regulating miR-14 around 72 hours post-infection. In vivo and in vitro studies showed that in the presence of the synthetic miR-14 mimic the transcript levels of Ha-EcR significantly increased. This positive interaction was further confirmed by a GFP reporter assay. Further, application of the miRNA mimic led to reduced replication of the virus. These data suggest that by downregulating miR-14 HaSNPV reduces the Ha-EcR transcript levels for its own benefit.

In our next attempt to identify potential virus-encoded miRNAs upon HaSNPV infection in H. armigera, we discovered the generation of viral siRNAs (v-siRNAs), as a consequence of RNAi response by H. armigera. We determined that the RNAi response was mediated mainly by Dicer-2 but not Dicer-1. In addition, the v-siRNAs did not uniformly map to the viral genome but rather found in several hotspots. In Dicer-2 silenced HzFB cells, we found higher transcript levels of hotspot genes and as a consequence more viral replication. Further, we demonstrated that the transcripts of key viral structural and auxiliary genes were the main substrates for Dicer-2 to generate v-siRNAs. The results clearly provided the first demonstration of the presence of an active RNAi response against a DNA virus. However, the virus may take advantage of this response to regulate its replication to avoid premature death of its host. Overall, the results of this project provide additional information demonstrating the significant role of snRNAs in host-pathogen interaction.
Keyword MicroRNA
RNAi
Dicer-2
Small RNA
Helicoverpa armigera
Baculovirus
HaSNPV
Trypsin-like protease
Ecdysone receptor

 
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Created: Mon, 18 Nov 2013, 16:21:58 EST by Balachandran Jayachandran on behalf of Scholarly Communication and Digitisation Service