functional characterisation of miRNAs
Dr. Eileen Knorr
The analysis of complex phenomena such as immunity and stress responses requires sophisticated methods if vertebrates are used as models, reflecting the overlapping interactions between many redundantly-acting proteins. However, most developmental and immunological signaling pathways are highly conserved between insects and vertebrates, therefore insects offer an opportunity to study these processes in simper models.
The red flour beetle Tribolium castaneum (see developmental stages to the left) has emerged as a genetically-tractable model organism which is particularly suitable in the fields of insect evolutionary and developmental biology. The T. castaneum genome has been fully sequenced, and other advantages include its short generation interval, high fecundity, the ability to rear large numbers of insects in captivity, and the availability of molecular tools such as transposon-mediated germ-line transformation.
This method allows the creation of enhanced green fluorescent protein (EGFP) marker lines to facilitate phenotypic analysis. T. castaneum is also an established model for potent and systemic RNA interference (RNAi), a specific post-transcriptional gene silencing mechanism which is triggered by double stranded RNA (dsRNA). RNAi has become a powerful tool for the functional analysis of specific genes, since the resulting phenocopy is associated with the loss-of-function phentype of the gene of interest. RNAi can be elicited in any T. castaneum tissue by the injection of dsRNA into the body cavity. Furthermore, injection of dsRNA into pupal or adult stages of females induces parental RNAi in the offspring without harmful injections. This method has been used successfully to study a variety of physiological processes, including pattern formation during development, hormone signaling and cuticle formation.
1. Insect immune response
Insects are the most successful group of animals in terms of species diversity, with more than one million species spanning nearly all terrestrial habitats. One of the reasons for this tremendous success is their potent and highly-sophisticated immune system, even though it is restricted to innate immune responses.
We focus on the identification of novel immunity-related genes in T. castaneum. Bioinformatic analysis of the T. castaneum genome (and genomic or EST databases from other species) allows the identification and characterization of known genes, but other approaches are required to identify novel genes. We use suppression subtractive hybridization (SSH), which is particularly suitable for the discovery of new genes, and unsuspected functions for known genes. We then carry out RNAi experiments allowing the functional characterization of the sequences we have identified.
MicroRNAs (miRNAs) are non-coding RNAs approximately 22 nucleotides in length that have been found in diverse species including many animals and plants. Their major function is the post-transcriptional regulation of gene expression by base-pairing with the 3ʹ untranslated regions of target messenger RNAs. A single miRNA can regulate hundreds of different target genes and more than 30 per cent of the genes in animals are thought to be regulated in this manner. Therefore, miRNAs play an important role in many biological processes. Thousands of miRNAs have been identified since the discovery of the first examples 20 years ago, but most are computational predictions without experimental confirmation.
Our project therefore focuses on the expression and functional characterization of miRNAs from the model beetle Tribolium castaneum, and the use of microarray analysis to study miRNA expression under different forms of stress, including starvation, infection and heat shock. This approach has, for the first time, identified T. castaneum miRNAs with differential and sex-specific expression profiles in response to environmental stress. In the future, we will focus on the functional characterization of single miRNAs and the identification of physiological target genes.
|group members:||Dr. Eileen Knorr (Arbeitsgruppenleiterin)