The moss Physcomitrella patens gives insights into RNA interference in plants
As part of the joint project “RNAi and regulatory RNA in the genome-wide analysis of gene functions of green organisms”, Prof. Dr. Ralf Reski and Dr. Wolfgang Frank from the Department of Plant Biotechnology at the University of Freiburg are investigating the regulatory function of RNA fragments in the moss Physcomitrella patens. They are working in cooperation with Prof. Dr. Wolfgang Hess from Freiburg who is working with cyanobacteria and Prof. Dr. Detlef Weigel from Tübingen who is investigating the RNAi using Arabidopsis thaliana, the model organism of seed plants.
The way small regulatory RNAs act is examined using three plant model systems that represent different stages in the evolution of plants (Photo: Frank).
It has been known since the 1990s that it is not only protein-coding messenger ribonucleic acid (mRNA), transfer ribonucleic acid (tRNA) and ribosomal ribonucleic acid (rRNA) that are involved in the regulation of protein production, but also small RNA molecules, about 20 to 40 nucleotides long. These miRNA (micro RNA) sequences can either be located in certain regions of protein-coding genes or be part of genes that do not carry information required for the synthesis of proteins.
These genes are transcribed into precursors and subsequently processed further. The precursors have a particular structure. During the transcription of the gene, complementary base pairs of miRNA precursors bind to each other and form a double-stranded hairpin structure from which defined miRNA segments are excised. These RNA fragments are complementary to certain regions of an mRNA whose regulation they govern. The miRNA binds to these defined locations. This binding leads to the cleavage of mRNA, which is typical for plants, or to the inhibition of translation, something which is typical for animals. The specific function of miRNA therefore is the downregulation of the amount of proteins.
Promising RNomics approach
As part of the RNAi research project, Professor Frank is working on a number of different questions. “We know that the mechanism of RNA interference developed early during evolution. We also know that miRNA has a very conserved structure. This is also true for the miRNA targets,” explains Frank. Frank and his team are investigating the natural RNAi pathways that are present in Physcomitrella patens and what the different moss miRNAs look like. The scientists are comparing the identified moss miRNA pool with homologous miRNAs of Arabidopsis thaliana or rice. They hope that the comparison of sequences will lead them to the identification of compatible mRNA targets.
Frank uses an ‘RNomics approach’ for identifying a miRNA pool. This is a comprehensive RNA analysis using genomics and proteomics tools. The scientists will first fractionate the RNA, then produce clones and sequence them. “Bioinformatic tools have helped us to establish a non-redundant set of moss microRNA,” said Frank. The researchers still have to confirm that the selected sequences occur in the organism itself and hope to detect all moss miRNAs. That is why they also use bioinformatic tools and the fact that miRNA consists of very conserved sequences for something else. They are searching the genome of Physcomitrella patens, which is almost completely known, for homologies with other known plant miRNA sequences stored in a database. The computer will then calculate a potential structure for the areas on the right and left side of the predicted miRNA region and checks whether the sequence is able to form a hairpin structure that is typical for miRNA precursors. “We have found many miRNAs that are homologous to miRNAs in seed plants. However, we have also discovered miRNAs that are typical for moss,” said Frank.
Looking for miRNA targets
Frank and his team are also interested in the following question: which mRNAs are the targets of Physcomitrella patens miRNA. “Our experiments with different miRNAs show that the target RNAs code for important proteins. They are either important for the development and the signal transduction in plants or play a major role in the plants’ primary metabolism,” said Frank.
The researchers will conduct further studies in order to find out what happens if they remove a miRNA gene from the moss genome, alter the miRNA binding site in the target RNA or integrate homologous Arabidopsis thaliana genes. The moss plant has the extraordinary ability to precisely integrate DNA molecules into the genome through a mechanism known as homologous recombination. This process helps to integrate specific alterations in the moss genes. In addition, the predominant generation of the moss is haploid, so that any alteration in the genome has an immediate effect. This is why the biologists can stably and easily integrate Arabidopsis genes into the moss genome. “We are mainly interested whether such alterations lead to changes in the moss phenotype. This will tell us whether only miRNAs are conserved or whether also the protein function encoded by the particular mRNA has been conserved over thousands of years,” said the biologist.
Developmental disturbances and other drastic consequences
Comparison of a Physcomitrella patens wild-type plant with a transgenic Physcomitrella patens strain lacking a gene that is involved in the mechanism of RNA interference. The loss of gene function leads to drastic developmental disturbances in the transgenic plant strain. (Photo: Frank)
In other experiments, the researchers have mutated the binding site of the mRNA for ‘their’ miRNA. This deactivates the regulatory pathway and prevents mRNA from being cleaved. From this, the scientists can draw conclusions on the function of miRNA: “We can observe high mRNA levels and the plants show distinct developmental disorders. For example, the plant hormone auxin has a considerable effect on the development of the moss,” said Frank. The researchers also hope to find out what happens if miRNA is switched off. In order to do this, the team blocked several genes that code for precursor sequences of the miRNA and have thus switched off the regulation of all mRNAs under the control of this specific miRNA. The researchers found that the target sequence coded for a transcription factor and the plants grew faster.
“We also want to know how all this functions,” said Frank, emphasising the need to look at specific proteins that regulate the RNAi mechanism. “These proteins, which control particular RNAi pathways of plants, have homologous counterparts in mosses. We are particularly interested in the functional analysis of the moss proteins in order to deepen our understanding of the evolution of RNA interference,” said Frank.
If one of these proteins is switched off, the scientists observe drastic consequences. “The moss no longer produces leaves and does not grow further than the filament stage,” said the biologist.
The joint project is funded by the Landesstiftung Baden-Württemberg foundation. Coordinated by Dr. Martin Grauer, the foundation supports several research projects looking exclusively into RNAi.
kb – 24 August 2006
© BIOPRO Baden-Württemberg GmbH
Dr. Wolfgang Frank
Institute of Biology II
Phone: +49 (0)761/203-2820
Fax: +49 (0)761/202-6945