During the course of this research, many of the widely accepted tenets surrounding the mechanisms of plant miRNA mediated gene regulation have been reappraised. Notable amongst these is the view that plant miRNA almost exclusively regulate their targets through transcript cleavage. This prevailing view meant that miRNA/target interaction could be relatively easily confirmed through modified 5ꞌ RACE assays. It is generally accepted today that translational attenuation by plant miRNAs is widespread. However, the mechanism whereby translational repression occurs in plants is yet to be found and no technique has yet to be developed that can positively show direct binding of miRNA to its target mRNA. When targets are regulated without cleavage, detection of
miRNA/target interaction by 5ꞌ RACE is not possible and retention of uncleaved and untranslated transcripts in the cell may also distort evidence of target down-regulation through RT-PCR. Therefore, in the absence of a cleavage product, confirmation of down-regulation of a target is restricted to proteomic studies which are relatively time consuming and expensive. Ideally, detection of translational attenuation should be coupled to deep sequencing studies in the same way as Taqman probes and 5ꞌ RACE, perhaps from examination of miRNA/target pairing after AGO immune-precipitation. Although the depth of sequencing now available enables a highly stringent predictive process, bioinformatic analysis is only ever predictive. Therefore, direct evidence of miRNA/target pairing, although not necessarily demonstrating a biologically significant relationship, would add a level of predictive confidence not currently available. This situation was encountered with functional analysis of miR166g-5p. Over-expression of
150
miR166g-5p resulted in a clear phenotype and it was shown that it acts independently of miR166g-3p. However, despite the cleavage products of nine predicted targets being tested for none were positively identified. This means that the mode of action of miR166g-5p cannot be directly placed into the nodulation pathway. Hairy root transformation produces chimeric transgenic roots. While this is a useful system for relatively fast phenotypic screening, full functional analysis of miRNA ideally requires stable transformation and the generation of double or triple mutants. Some of the miRNA and targets presented here may prove to be suitable for functional analysis in Arabidopsis where superior tools, such as an extensive range of mutants already exist. Where this is not possible and in the absence of improved molecular tools, thorough miRNA/target functional analysis in M. truncatula will be a long term endeavour. However, the research presented here and in the previous the microarray work by Holmes et al has produced a wide ranging and solid platform for future studies.
6.1.8
Conclusions
This thesis set out to elucidate the miRNA environment of the M. truncatula root meristem. Deep sequencing and bioinformatic analysis revealed multiple highly conserved miRNAs with strong and/or differential expression patterns within and between meristematic and non-meristematic tissues. Many of these have described functions in other species where they are chiefly involved in developmental and stress related functions. Conserved miRNAs not previously found in M .truncatula were identified. From expression patterns, target prediction and homologue function it was found that many of these are strong candidates for regulators of root growth. The main strength of this thesis is the depth of sequencing employed. This enabled a high degree of fidelity in miRNA prediction as sequential filtering removed all but a small fraction from the data set. This fidelity was demonstrated through the successful functional analysis of two previously unstudied miRNA, miRN304 and miR166g-5p. It was demonstrated that over-expression of the each of these hairpin precursors was sufficient to produce measurable phenotypes. The exact mode of action of each of these, however, remains unclear. miRNA biogenesis and target regulation is highly dynamic and relies on multicomponent processing by various members of the DICER-LIKE and
151
either by cleavage or translational attenuation fully understood. For these reasons the placing of any given miRNA, with confidence, into a genetic pathway is a challenging and time consuming task. We are confident however, that further study of miRN304 and miR166g-5p will show both to have important roles in M. truncatula root
organogenesis. Furthermore this study has identified several strong candidates for future studies of miRNA involved in root architecture, and that these studies will be essential in completing the genetic framework required for meristem formation and function.
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7
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