Arabidopsis thaliana: the species in which plant miRNAs were discovered

Mysterious Plant miRNAs: What About Them?

Welcome to the last article in this series! Last, but by no means least, we will look at the importance of plant miRNAs and how they differ from their animal counterparts.

When/How Were Plant miRNAs Discovered?

Plant miRNAs were first described in 2002, a decade after the seminal miRNA study in the nematode C. elegans (1). During this decade, the evidence was stacking up for miRNAs being abundant throughout eukaryotes, and Reinhart and co-workers went about looking for them in plants. Using a specially designed cloning strategy to find DICER cleavage products, the group found 16 candidate miRNAs in the Arabidopsis thaliana genome, based on their similarity to animal miRNAs (2).

What Did These miRNAs Look Like and How Did They Compare to Animal miRNAs?

  • Using a DICER target cloning strategy, 200 RNA molecules ranging from 20-nt to 24-nt in length with 5′-phosphate and 3′-hydroxyl groups were identified.
  • 16 of these sequences had adequate similarity to animal miRNAs to be considered plant miRNAs, and these were named miR156 to miR171.
  • Five of these 16 miRNAs were single copy, while the remaining 11 were represented at multiple loci, most likely due to duplication within the A.  thaliana genome (3 for a review covering genome duplication in Arabidopsis).
  • In line with miRNA inclusion criteria, the vast majority miRNAs were found outside of annotated genomic regions, thus not identified previously as genes.
  • Similarly to animal miRNAs, most of the plant miRNAs sequences began with a U.
  • Each of the plant miRNA loci were located in a genomic context where they could pair with a nearby genomic region to form a dsRNA hairpin structure resembling those required for DICER processing of animal miRNAs (2).

What Do We Now Know About miRNA Biogenesis and Function?

Our understanding of the biogenesis of miRNAs has improved over the years. Nowadays, it is well accepted that plant miRNAs are approximately 21 nucleotides long and are processed from stem-loop regions of long primary transcripts by a DICER-like enzyme prior to loading into silencing complexes. Once loaded into these complexes, they generally direct cleavage of complementary mRNAs, similarly to animal miRNAs.

While both plant and animal miRNAs base-pair with a complementary mRNA target, perfect sequence complementarity to an miRNA is observed only for some plant mRNAs (2).

A big difference between plant and animal miRNA biogenesis lies in their nuclear processing and export. In contrast to animal miRNAs, which are cleaved by two different enzymes, once inside and once outside the nucleus, both cleavages of plant miRNAs are performed by one enzyme, a Dicer homolog, called Dicer-like1 (DL1). Since DL1 is only expressed in the nucleus of plant cells, it is highly likely that both cleavages occur inside the nucleus.

Before plant miRNA duplexes can be transported from the nucleus, their 3′ overhangs are methylated by Hua-Enhancer1 (HEN1), a RNA methyltransferase protein. The methylated duplex can then be transported from the nucleus into the cytoplasm by the Hasty (HST) protein, an Exportin 5 homolog, where they disassemble and the mature miRNA is incorporated into the silencing complex/RISC (4). From this point on, plant miRNAs silence their targets in a similar manner to animal miRNAs.

What Do Plant miRNAs Do?

The discovery of miRNAs in the plant genome supports the claim that miRNAs arose early during eukaryotic evolution and underpins their importance in influencing gene expression since the beginning of multicellular life as we know it.

Plant miRNAs have now been implicated almost all aspects of plant biology. Studies are rapidly emerging describing diverse roles for miRNAs in flowering, plant defence (immunity), drought response, abiotic stress tolerance, leaf senescence, nutrient homeostasis and many more.

Despite their widespread roles, plant miRNAs are best known for their roles in all major stages of plant development. They typically play major roles at the core of gene regulatory networks, with important targets such as transcription factors involved in developmental processes (5).

Resources for Plant miRNAs:

  • miRPlant: If you’re a biologist with limited bioinformatics skills, then you’re in luck! This is a user-friendly interface where you can identify novel plant miRNAs from RNA sequencing data. Data is presented graphically where you can see the hairpin structure of novel predicted miRNAs (6).
  • miRDeep-P: This downloadable software is based on ultra-deep sampling of small RNA libraries by next generation sequencing. miRDeep-P can be used both to identify novel unannotated miRNAs in plant species and to assign expression status to individual miRNA genes. This software relies on a number of third party tools and is not for the bioinformatics dummy! (7).
  • MicroPC: This online plant miRNA resource has been built upon the results of large-scale expressed sequence tag (EST) analysis. This platform might be useful for you if you want to compare your miRNA sequencing data with known and stored miRNA sequences. Over 4000 miRNA candidates and almost 3000 miRNA targets spanning over 100 plant species are represented in MicroPC. This resource also facilitates miRNA and target prediction from user-entered nucleotide sequences (8).

That was it on plant miRNAs for now and marks the end of this miRNA series. Do you work with plant miRNAs? Have you found any additional resources to be particularly useful? We’d love to hear from you!

References

  1. R. C. Lee, R. L. Feinbaum, V. Ambros, The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75, 843-854 (1993).
  2. B. J. Reinhart, E. G. Weinstein, M. W. Rhoades, B. Bartel, D. P. Bartel, MicroRNAs in plants. Genes Dev 16, 1616-1626 (2002).
  3. M. T. Rutter, K. V. Cross, P. A. Van Woert, Birth, death and subfunctionalization in the Arabidopsis genome. Trends Plant Sci 17, 204-212 (2012).
  4. C. Lelandais-Briere et al., Small RNA diversity in plants and its impact in development. Curr Genomics 11, 14-23 (2010).
  5. M. W. Jones-Rhoades, D. P. Bartel, B. Bartel, MicroRNAS and their regulatory roles in plants. Annu Rev Plant Biol 57, 19-53 (2006).
  6. J. An, J. Lai, A. Sajjanhar, M. L. Lehman, C. C. Nelson, miRPlant: an integrated tool for identification of plant miRNA from RNA sequencing data. BMC Bioinformatics 15, 275 (2014).
  7. X. Yang, L. Li, miRDeep-P: a computational tool for analyzing the microRNA transcriptome in plants. Bioinformatics 27, 2614-2615 (2011).
  8. W. Mhuantong, D. Wichadakul, MicroPC (microPC): A comprehensive resource for predicting and comparing plant microRNAs. BMC Genomics 10, 366 (2009).

 

Image credit: Dawid Skalec

1 Comment

  1. Krishna Udaiwal on February 21, 2016 at 4:11 pm

    The greatest benefit, IMO, of miRs is that their functionality is so fundamentally simple, where theses 20-24 NT sequences which are anti-parallel to the target mRNA either bind permanently (siRNA) or lead to cleaving activity (miRNA), that makes them so important. Think about it in evolutionary term as well, where SNPs in miRNA could’ve directed entire gene changes irrespective of degenerative copies within a genome. As well, they induce gene silencing and not knockouts which may allow other potential complexities to the maturation cocktails in plants or animals than the knockout (all or nothing).

    I honestly am hoping this is starting to be taught in high-school biology, but that’s just my optimism.

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