Monthly Archives: September 2015

Plant miRNA evolution update

So here’s another figure I’ve prepared for the Plant Cell review article I am writing. This is an update on the patterns of miRNA conservation across land plants according solely to the information present in miRBase release 21.

For this analysis, I first placed each land plant miRNA family in miRBase 21 into one of eight broad plant groups (Eudicots-Rosids, Eudicots-Asterids, Basal Eudicots, Monocots, Basal Angiosperms, Gymnosperms, Lycophytes, and Bryophytes). I then defined a conserved miRNA family as one which had at least one high-confidence annotation, and which was annotated in two or more of these broad groups. By this definition, there are just 36 conserved families out of the more than 2,000 that are currently annotated.


This figure also highlights a major issue in the large-scale analysis of land plant miRNA conservation .. highly unequal sampling density within the different groups. Rosids and monocots have received the most attention, with large numbers of species represented, and high numbers of overall annotations (barcharts at the top). In contrast, basal eudicots, basal angiosperms, lycophytes, and bryophytes are much less well-sampled, with each group represented at present by just one species in miRBase 21. Inferring secondary losses in some of these lineages (basal eudicots, basal angiosperms, lycophytes) is NOT believable.

This chart also illustrates how important the high-quality miRNA annotation set for bryophytes is (the sole species represented is Physcomitrella patens). Based on the presence of high-confidence annotations in both Physcomitrella and one or more angiosperm group, we can confidently say that nine families (miR156, miR160, miR166, miR171, miR319, miR390, miR477, miR529, and miR535) were most likely present in the last common ancestor of all land plants. Another three families (miR167, miR395, and miR408) might also belong in the ultra-conserved set, but they are less certain because their Physcomitrella annotations are not high-confidence. Another two families (miR396 and miR482) clearly predate divergence of all seed plants. Other patterns are less certain because of the frequent presence of annotations that are not (yet) known to be high confidence. I think this again highlights the need for a systematic review of miRNA annotations, based on re-analysis of all available small RNA-seq data with a single, high-confidence MIRNA identification methodology. We are working toward this goal in my lab.

Plant microRNAs in miRBase 21

So I’ve been asked to contribute a review article to The Plant Cell on the general subject of microRNA / small RNA evolution in plants.  I set out to make an up to date figure on microRNA annotations across plants based solely on the annotations in miRBase 21. The results were surprising to me.

72 different land plant species are represented in miRBase 21, with a total of 2,247 different miRNA FAMILIES annotated. Note, that is families, not loci. Seemingly a huge diversity of different miRNA sequences.

But, the issue is that many of these annotations are likely to be false positives .. either other types of regulatory small RNAs, or even worse, just degraded garbage. The curators of miRBase, since release 20, have set out to try and make a ‘high-confidence’ list of microRNA loci, based on their internal parsing of public small RNA-seq data. (See their paper in NAR). In miRBase 21, from plants, there are just 176 high-confidence miRNA families (a high-confidence family is a family for which at least one locus has a high-confidence designation).  Furthermore, only 17 of the 72 plant species have ANY high-confidence annotations at all! The scatterplot below illustrates this, as we can see that most species have few annotated families and no high-confidence ones.


Scatterplot of numbers of high-confidence miRNA families from 72 plant species as a function of the total number of annotated families. Data are coded according to broad taxonomic groups. A few species of interest are labeled. Data were processed from miRBase 21.

Of course, there are some caveats here. The biggest is that the ‘high-confidence’ designation is based on whether or not the miRBase folks have analyzed the available high-throughput small RNA-seq data for a given species. In some cases, they may not have (though I haven’t dug into that specifically). The second caveat is that all species are NOT equally treated here. Some species (for instance, Oryza sativa, Arabidopsis thaliana) have high-quality reference genomes and have had lots of experimental attention over the years. Many others have neither of these traits, and so their annotations are necessarily more piecemeal. Overall I think this points out the need clearly for a more uniform approach to retrospective analysis of miRBase annotations.

I was also pleased to see that Physcomitrella patens has a very high percentage of high-confidence miRNA families .. since my lab annotated the vast majority of those families!

— Mike Axtell

miRNAs in Ectocarpus are a distinct, but share similarities to plants/animals

microRNAs and the evolution of complex multicellularity: identification of a large, diverse complement of microRNAs in the brown alga Ectocarpus

James E. Tarver, Alexandre Cormier, Natalia Pinzon, Richard S. Taylor, Wilfrid Carre, Martina Strittmatter, Herve Seitz, Susana M. Coelho and J. Mark Cock

PMID: 26101255

This paper focuses on miRNA analyses in Ectocarpus (esp) discussing the evolutionary background and considerations of the origin of miRNA loci for given lineages of organism.  I found it interesting because it took systems with less established miRNA backgrounds and used a broad context for its mechanism and role.  As brown alga are thought to have independently evolved multicellularity, which gives particular insight for the possible role of miRNAs in this process.  Additionally, ectocarpus has a large suite of homologues known to be associated with miRNA function in plants and animals, making the origin and similarities of this mechanism interesting (Table 2).

miRNA sequencing

To assess questions about the miRNA makeup in ectocarpus, the authors performed sRNA-seq on male and female NILs, aligning reads with bowtie-2 and characterizing loci with mirDeep (animal and plant versions).  They followed a strict set of requirements for identification of miRNAs, namely that:

  1. Must include at least a 15 bp pair within a hairpin
  2. Both mir and mir* must be present
  3. Precise dicing
  4. 3p product must extend 2 bp beyond the 5p product

The outcome of this analysis resulted in 63 families with a total of 64 loci, most of which were new and even filtering out many loci from previous studies which failed to meet requisites.  The clearly result of this is that nearly all miRNAs found within Ectocarpus have no other family members. Even when looking in only the seed region of a loci, the authors found that even low identity (>75%) cutoffs retained the vast majority of loci in separate families.  

One of the hypotheses of the paper was to indicate that there would be expression specificity between male and female individuals, which was not supported via northern blot (Figure 1).  

In the prediction of targets for miRNAs, the authors implemented the tool TAPIR, looking for high-complementarity targets.  This process yielded 160 targets, available in Table S3.  Despite the lack of family expansion, apparently several of the miRNA are found to redundantly target the same genes.

Origin of miRNAs

The authors suggest that the main genomic origin for miRNA loci in ectocarpus is likely from intronic regions of transcribed genes, but only by deduction.  A large proportion of miRNAs were found to be located within protein-coding genes, in intronic regions and commonly stranded with the gene.  miRNAs found within genes were not found to co-express significantly, hurting the case that these would be expressed simultaneously…  I don’t know if this evidence is damning, as there could be any number of factors affecting the measured expression levels…  

When looking at the evolutionary origins of esp-MIRs, the authors found some interesting results.  According to the authors, miRNA loci-loss occurs very slowly, and usually in only exceptional cases.  Is this true?  A paper is cited that has a differing opinion, but is discounted as an “over-estimation”.  To examine mirs in closely related species, they looked at two closely related alga, as well as two more distant diatoms.  Despite the relationship, NO mir loci matches were found through blast search.  Would we expect this?  This means that this lineage has evolved its own set of miRNAs.  

As for multicellularity, the authors contend that the presence of miRNAs is associated with developmental complexity.  They support this argument by saying that there is a correlation between number of cell types/ developmental characters and the complexity of miRNA systems in an organism.  Just looking purely at the number of families, they show that higher order plants and animals have more while lower organisms have less.  I’m not sure how convincing this is, as its highly speculative and doesn’t talk much about an evolutionary mechanism.

Mechanistically, esp-mirs seem to have commonalities with plant and animal miRNAs, having similar fold-backs to land-plants and 21-mers as the most common mature.  The paper indicates that esp-AGO2 is 40% identical with HsAGO2, but they don’t speak to AtAGO2.  Important differences include that there is a vastly higher ratio of mir:mir* in terms of read detection (>400 fold).  

Other thoughts…

If this is the case, it seems likely that the authors may be missing significant portions of miRNAs, considering the required depth for identifying a star sequence.  Similar to our lab’s philosophy with shortstack (setting a very high bar for miR discovery), the authors seem to be concerned with false positives, striving for only including confirmed miRs, and even pleading for higher standards for mir identification in the field.  Considering this, it seems interesting to me that they do not speak to a requirement for high expression levels for a mir, something that I thought was tacitly required, though we don’t implement this standard either.


Printing on a Fabric Poster

Hi Everyone!

Mike suggested that I post some information on how and where to print fabric posters, like the one I made for ASPB this year.

There is a great website providing information on this whole topic, written by Jessica Polka through the American Society of Cell Biologists.  This article is extremely informative, and can easily walk you through the steps to printing your own.

Some thoughts of mine:

  • The cost for printing is comparable to, if not better than anywhere in town.  About $25 for their slow service which takes about 2 weeks and about $45 for a rush order (around 3 days to turn-around).
  • Quality is excellent!  As good as any poster you’ve seen.  If you don’t believe me, come look at mine.
  • The only drawback is some size restrictions…  The quoted price can print a poster that is 36″x 58″, which is fine, but to make it larger would cost more.