The identification of hidden signals in human mRNA sequences
In the regulatory genomics laboratory the group decipher genomes. They aim to discover signals that control where and when genes are turned on or off. Much of this information is found in the section before the coding regions for proteins (promoters), but more recently the search has been extended to other sections of the genes.
Many genes provide the instructions to make proteins - they 'code' for proteins. These proteins have specific roles in cells. Massive worldwide efforts, including the human genome project, have been able to discover most of the protein coding genes in the human genome. There are over 25,000 human protein coding genes, encoding proteins as diverse as haemoglobin (oxygen carrier), insulin (hormone), or trypsin (digestive enzyme).
Surprisingly, protein coding regions only make up only about 1% of the three billion base genome. Most of rest of the human genome was once considered to be 'junk' DNA.
However, ongoing animal genome sequencing projects have shown a much larger amount, about 3-5% of the genome is similar between vertebrates. Thus different species for example human, chicken and dogs have conserved regions. This is in addition to what is needed to code for the proteins. These parts of the genome have also been conserved during the evolution of vertebrates.
These conserved parts are expected to contain most of the information required to program cells to perform different functions. For example to control, or 'turn on' genes in the right cells or times, haemoglobin in blood cells, trypsin in the digestive system, or regulate the synthesis of monoamine oxidase in the brain. This information directs cells to develop into different types, to divide or die, to perform their proper function. If the process goes wrong cells may grow aberrantly, as cancer cells.
Another part of the regulation is done through regions in the molecules that encode proteins, messenger RNAs (mRNAs). Each protein coding gene produces mRNAs that are translated into the appropriate proteins. However, about 40% of this mRNA sequence is not translated (untranslated) known as the UTR. Some of these regions contain regulatory information. Well known human examples include directions for the regulation of iron balance and inflammation.
Mutations in these regulatory elements have been associated with disease, as are the better known mutations in the protein encoding regions. Mutations in these regulatory elements have been shown to contribute to some types of cancer, heart disease, arthritis and diabetes.
The Brown group have developed a new integrated approach to decipher this regulatory information. Analysis has utilised high throughput genomic data and the Brown group's bioinformatic techniques. This identified regions subject to 'purifying selection' . Some are also similar to known functional elements and we predict that these are key regulators of gene expression. The outcome of these studies has been to greatly extend the range of known functional RNA elements in the human genome. Some of this data is publically available in the Transterm and UTRPathDB databases. A more detailed review of software for mRNA analysis is also available as part of the Transterm database help.
These projects involve a combination of computer (bioinformatic) and experimental tools to test predictions. Bioinformatics was recently highlighted as a key field of expertise for the life sciences in the future. It arose at the interface of the fields of biology, mathematics and computer science to permit interpretation of large quantities of biological data, particularly DNA sequences. The recent availability of large amounts of sequence data, particularly complete sequences, has revolutionised the study of many organisms. This work is part of the Otago University Area of Research Excellence entitled 'Bioinformatics and Computational Biology'.
This study takes the approach of inferring the biology of translation from the DNA blueprint, then testing the ideas experimentally in tissue culture cells. New assays have been developed which utilise the power of modern microscopic and cell biological techniques. The elements discovered include stability/instabilty elements (e.g. AU rich elements) and those for mRNA localisation in mammalian cells.
A database of translational control elements, mining large amounts of sequence data- Transterm
Genomic information is stored in large repositories located around the world. Data is continuously poured into these databases, then copied or mirrored to individual countries or institutions. This data is collated and mined at the University of Otago in the TransTerm project.
Transterm was initially a database of sequence contexts about the stop and start codons of many species found in GenBank. But it has been extended Transterm now also contains codon usage data for these same species and summary statistics for the sequences analysed. A database of translational control elements is now an ongoing feature of our TransTerm database. A brief review of mRNA Motifs in mRNA is available via the Transterm help.
Translational control in hepatitis B virus protein synthesis
Viruses frequently use unusual regulatory mechanisms to make essential proteins. Commonly these involve translational control or 'recoding' mechanisms. These are potential sites for anti-viral agents. Most of these recoding mechanisms are poorly understood, but these events are usually directed by sequences and structures in the viral RNAs. These signals may be short adjacent RNA sequences or more complex structures. The synthesis of HBV polymerase (P) protein involves a complex process that differs from host cell protein synthesis, and is a potential target for antiviral agents. The P protein translation initiation site (AUG) is preceded by multiple potential initiation sites, to make any P protein these must somehow be bypassed. P protein is essential for HBV maintenance and multiplication. A new hypothesis for P protein synthesis has been postulated and tested. The study involves testing the expression of reporter constructs containing part of the viral genome in animal cells. This study will identify the key components of this process, and suggests new avenues for treatment of HBV targeted at this mechanism.
Cis Regulatory RNA elements in human mRNAs- A new method to identify the sites of RNA-protein interactions in living cells.
Protein expression depends significantly on the stability, translation efficiency and localization of mRNA. These qualities are largely dictated by the RNA-binding proteins associated with an mRNA. We have developed a new method to visualize and localize RNA-protein interactions in living mammalian cells. This methods uses trimolecular fluorescence complementation assay (TriFC) to locate RNA-protein interactions where they occur in living cells using confocal microscopy.
Using this method, we found that the fragile X mental retardation protein (FMRP) isoform 18 and the human zipcode-binding protein 1 ortholog IMP1, an RNA transport factor, were present on common mRNAs. These interactions occurred predominantly in the cytoplasm, in granular structures. In addition, FMRP and IMP1 interacted independently of RNA. Tethering of FMRP to an mRNA caused IMP1 to be recruited to the same mRNA and resulted in granule formation. The intimate association of FMRP and IMP1 suggests a link between mRNA transport and translational repression in mammalian cells.
In search of conserved mRNA localization and anchoring mechanisms
We have a new international collaborative program with Professor Anne Spang (Biozentrum, Basel, Switzerland) and Chris Brown (Otago University, New Zealand) to identify RNA localization zipcodes in yeast and mammalian cells and to test their conservation of function.
Although protein localization mechanisms have been intensively investigated over the last few decades, the localization of RNAs, which are at least as diverse as proteins, has been much less widely studied. Yet RNA localization is crucial for normal development, and defects in the process may underlie numerous diseases, particularly neuropathologies (more...)
Current Group (1/2012)
Dr Xiaowei Sylvia Chen (Research Fellow) PhD (2007) and postdoctoral studies at Massey University (publications)
Dr Dan Garama (Assistant Research Fellow) PhD (2011)
Stewart Stevens (PhD candidate)
Ambarish Biswas (PhD candidate)
Josh Gagnon (Software Developer)
Andrew Sarman (MSc student)
Tania Bracey-Brown (Assistant Research Fellow)
Related information from the University of Otago