Final Report Summary - MIRNAEVOL (MicroRNAs, genomic evolution and the emergence of anatomical complexity)
The role of the teleost-specific (3R) whole genome duplication event (WGDE) on the genomic evolution of microRNA families.
Introduction:
microRNAs (miRNAs) play a fundamental role in the gene regulatory networks of both plants and animals. They regulate gene expression through a series of positive and negative feedback loops, both temporally and topologically, thus helping to control how an organism develops, grows, behaves, reproduces, and even dies.
As well as controlling developmental processes miRNAs play a key role in an organism's physiological response to environmental perturbations, such as: changes in salinity; nutrient levels; increases in water temperature; while playing a role in phenotypic plasticity. miRNAs have also been associated with major evolutionary transitions and it has been suggested that they may have led to enhanced rates of speciation within some teleost clades. Furthermore they are known to play a key role in many cancers and congenital diseases.
However, the mechanisms by which novel families of miRNAs evolve are poorly understood; previous work has suggested that they may evolve from introns, de novo synthesis from transcriptional units, transposable elements, or structured transcripts such as tRNA and snoRNA molecules. It has been hypothesised that WGDEs may explain high levels of miRNA family innovation. The role of the vertebrate-specific WGDEs (1R) has previously been investigated and found to play a minimal role in the origin of novel miRNA families. However, is 1R indicative of the more general phenomenon of WGDEs? To test this hypothesis the 3R WGDE will be investigated. Currently eleven miRNA families are believed to have evolved on the branch leading to teleosts, however, by sequencing the miRNA complement of four additional actinopterygians, it will be possible to observe if all eleven families evolved in conjunction with 3R or whether the process of evolution was more gradual.
Methods:
Small RNA libraries were constructed from the non-teleost actinopterygians polypterus and bowfin, and the teleosts osteoglossum and stickleback using 1ug of total RNA from the entire organism. This was used as the starting template for an Illumina TruSeq small RNA preparation kit. In this process 5' and 3' adaptors were added, reverse transcribed, and barcoded allowing multiplexing then amplified using polymerase chain reaction (PCR). Each barcoded taxon was then run on one third of a lane on a GAIIx sequencer, generating read counts of > 30 m for the four taxa.
The read data were then converted from FASTQ to FASTA format, the adaptors clipped, short reads (< 18 bp) and sequences containing unknown (N) bases were removed using Galaxy. Resulting reads were then initially analysed using miRDeep2. These were then corroborated by hand following established criteria to identify miRNAs rather than other RNA fragments such as siRNAs. Orthologous miRNAs were identified using an iterative search strategy in which all known miRNA sequences were downloaded from miRBase and a local basic local alignment search tool (BLAST) database was constructed using BlastStation-local. After each analysis newly identified miRs were included in the database for use in the next analysis.
Results & discussion:
Although previous workers have invoked genome duplication events as a driving force behind the evolution of novel miRNA families, this is only supported by incomplete taxon sampling. Classically only two fish taxa are included in genomic analyses of vertebrates: one being zebrafish and the other a derived member of the Percomorpha such as stickleback, medaka, or pufferfish. Such limited taxon sampling can create strong macrorvolutionary artefacts. Such artefacts are similar to those caused by the inclusion of only extant taxa in analyses of morphological innovation and the diversification of species, whereby prolonged periods of evolution are compressed into a single internal branch of a tree.
Given the large number of potential taxa which could be used to break the internal branch leading to clupeocephala, as well as the branch leading to the derived Percomorpha, it not surprising that a few carefully chosen taxa, which bracket 3R, were able to nullify the existing hypothesis.
In conclusion, this work clearly shows that 3R did nothing to enhance the rate of evolution of novel miRNA families and again, correlation due to poor taxon sampling does not equate to causality.
Introduction:
microRNAs (miRNAs) play a fundamental role in the gene regulatory networks of both plants and animals. They regulate gene expression through a series of positive and negative feedback loops, both temporally and topologically, thus helping to control how an organism develops, grows, behaves, reproduces, and even dies.
As well as controlling developmental processes miRNAs play a key role in an organism's physiological response to environmental perturbations, such as: changes in salinity; nutrient levels; increases in water temperature; while playing a role in phenotypic plasticity. miRNAs have also been associated with major evolutionary transitions and it has been suggested that they may have led to enhanced rates of speciation within some teleost clades. Furthermore they are known to play a key role in many cancers and congenital diseases.
However, the mechanisms by which novel families of miRNAs evolve are poorly understood; previous work has suggested that they may evolve from introns, de novo synthesis from transcriptional units, transposable elements, or structured transcripts such as tRNA and snoRNA molecules. It has been hypothesised that WGDEs may explain high levels of miRNA family innovation. The role of the vertebrate-specific WGDEs (1R) has previously been investigated and found to play a minimal role in the origin of novel miRNA families. However, is 1R indicative of the more general phenomenon of WGDEs? To test this hypothesis the 3R WGDE will be investigated. Currently eleven miRNA families are believed to have evolved on the branch leading to teleosts, however, by sequencing the miRNA complement of four additional actinopterygians, it will be possible to observe if all eleven families evolved in conjunction with 3R or whether the process of evolution was more gradual.
Methods:
Small RNA libraries were constructed from the non-teleost actinopterygians polypterus and bowfin, and the teleosts osteoglossum and stickleback using 1ug of total RNA from the entire organism. This was used as the starting template for an Illumina TruSeq small RNA preparation kit. In this process 5' and 3' adaptors were added, reverse transcribed, and barcoded allowing multiplexing then amplified using polymerase chain reaction (PCR). Each barcoded taxon was then run on one third of a lane on a GAIIx sequencer, generating read counts of > 30 m for the four taxa.
The read data were then converted from FASTQ to FASTA format, the adaptors clipped, short reads (< 18 bp) and sequences containing unknown (N) bases were removed using Galaxy. Resulting reads were then initially analysed using miRDeep2. These were then corroborated by hand following established criteria to identify miRNAs rather than other RNA fragments such as siRNAs. Orthologous miRNAs were identified using an iterative search strategy in which all known miRNA sequences were downloaded from miRBase and a local basic local alignment search tool (BLAST) database was constructed using BlastStation-local. After each analysis newly identified miRs were included in the database for use in the next analysis.
Results & discussion:
Although previous workers have invoked genome duplication events as a driving force behind the evolution of novel miRNA families, this is only supported by incomplete taxon sampling. Classically only two fish taxa are included in genomic analyses of vertebrates: one being zebrafish and the other a derived member of the Percomorpha such as stickleback, medaka, or pufferfish. Such limited taxon sampling can create strong macrorvolutionary artefacts. Such artefacts are similar to those caused by the inclusion of only extant taxa in analyses of morphological innovation and the diversification of species, whereby prolonged periods of evolution are compressed into a single internal branch of a tree.
Given the large number of potential taxa which could be used to break the internal branch leading to clupeocephala, as well as the branch leading to the derived Percomorpha, it not surprising that a few carefully chosen taxa, which bracket 3R, were able to nullify the existing hypothesis.
In conclusion, this work clearly shows that 3R did nothing to enhance the rate of evolution of novel miRNA families and again, correlation due to poor taxon sampling does not equate to causality.
