Author Archives: Nate Johnson

Mobile sRNAs can induce methylation on a genome-wide scale

The paper I chose to discuss was “Mobile small RNAs regulate genome-wide DNA methylation”, from the Ecker and Baulcombe groups. (PMID: 26787884, doi: 10.1073/pnas.1515072113). This goal of this study was to identify mobile sRNA loci and methylation loci, based on their interaction. To do this, the authors used shoot/root grafts of wild types: Col-0 and C24 and a mutant lacking siRNA formation: dcl234, as to elucidate the requirement of a mobile signal. Mobile sRNAs were identified where WT shoot could produce transcripts that were sequenced in dcl234 roots.

They identified 3 relevant classes of mobile-sRNAs and targets: direct interaction, indirect interaction and de novo methylation.  Direct interaction was shown where mobile-sRNAs were were enriched in a methylation site, whereas indirect had methylation due to mobile-sRNAs, but have no clear sRNA culprit. De novo loci are shown where methylation is induced by mobile-sRNAs in Col-0 root by a C24 shoot, but is not present with a Col-0 shoot. This study found widespread accounts of direct and indirect loci, as well as significant de novo methylated sited.

The huge quantity of loci identified as indirectly methylated was an interesting point of this paper. The authors have several thoughts for why this might occur, focusing on a possible secondary signal bridging the mobile signal and methylation or the possibility of aggressive threshold for mobile-sRNA significance inducing false negatives. Another point brought up is the possibility of requiring perfect matching with sRNA alignment resulting in missing valid secondary alignment of transcripts. This certainly seems possible to me, as allowing for a single mis-match opens a much more inclusive set of sRNA targets. These could be biologically relevant despite the mis-match.

Genomically, these direct and indirectly targeted loci are localized in transposable elements, while depleted in coding regions. This is true with both CHH and CHG methylation. Using mutant libraries from another study (Stroud et al. 2013 – Cell), the authors made a clear connection that mobile-sRNA methylation targets are dependent on DRM1/2. This makes a strong case for methylation through an AGO-dependent pathway.

This was an interesting paper, which gave strong evidence to support several of the claims. As an observational study, it gave support to previous work which indicated methylation but on a loci-specific scale. It is clear that this is taking place on a much broader level.

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.


Annotation of Soybean small RNAs reveals non-canonical phased siRNAs

Siwaret Arikit (a,b), Rui Xia (a,b), Atul Kakrana (b), Kun Huang (a,b), Jixian Zhai (a,b), Zhe Yan (c), Oswaldo Valdés-López (d), Silvas Prince (e), Theresa A. Musket (e), Henry T. Nguyen (e), Gary Stacey (c), and Blake C. Meyers (a,b)

a Department of Plant and Soil Sciences, University of Delaware, Newark, Delaware 19711

b Delaware Biotechnology Institute, University of Delaware, Newark, Delaware 19711

c Division of Plant Science, University of Missouri, Columbia, Missouri 65211

d Unidad de Morfologia y Función, FES Iztacala, Universidad Nacional Autónoma de México, Los Reyes Iztacala, Tlalnepantla 54090, Mexico

e National Center for Soybean Biotechnology and Division of Plant Sciences, University of Missouri, Columbia, Missouri 65211

PMID: 25465409

doi: 10.1105/tpc.114.131847


This article is an interesting take on the challenges associated with small RNA annotation from Blake Meyers.  Done on a large-scale basis in soybean, this project seeked to classify small RNA loci based on more modern interpretations, making use of both small RNA-seq libraries and degradome PARE sequencing.

First, the authors re-evaluated miRBase-20 genes based on several rules, namely trying to clarify genes that act canonically as miRNAs from ones that don’t.  This came in the form of several classes: (1) miRNAs that are weakly expressed but resemble siRNAs, (2) genes that are likely siRNAs, (3) genes that marginally meet the strict definition of miRNA and (4) well characterized and defined miRNAs.  530 plant miRNA aligned to the soybean genome, and fell under the following classifications: (1) 191 weakly expressed, (2) 203 siRNA-like, (3) 15 marginal miRNAs and (4) 121 highly expressed and canonical miRNAs.  This breakdown made some of the failings of miRBase pretty apparent, as so few of these genes could be clearly defined as miRNAs in soy.  Also, it seems clear that these genes make up a spectrum of classifications, as these classes had to be defined by some seemingly arbitrary cutoffs for strand and abundance ratios.  It is a challenge to define these classes.  The authors also used these cutoffs to filter and identify new candidate miRNAs, through which they found numerous canonical and novel genes.  The mapping procedure used in this study is a bit non-descript, as they just mention using Bowtie to map perfectly matched reads, and filtered out structural RNAs.  It looks like they allow multi-mapping reads with up to 20 alignments.  I would expect that if this procedure was refined using a method like butter, we might see less ambiguous and weakly expressed miRNAs.  Are these erroneous?

Another portion of this article I found interesting was their attempts to identify phasiRNA loci, where they identified 504 loci with a “stringent threshold” for their phasing P-value.  Almost all of the found loci overlapped protein coding genes.  The intriguing part about their PHAS loci identifications is that they found some non-canonical patterns of phasing from variants of TAS3 loci.  These included circumstances that required 3-hits from a miRNA to trigger phasi induction, as well as phasing in a downstream direction.  If we have PHAS loci like this in a dataset analyzed by shortstack, in my understanding it should be annotated without a problem… (is this correct?).

The most highly represented group of genes targeted by phasiRNA in soybean encode NB-LRR proteins, which have over 300 members characterized in legumes.  The authors cite several hypotheses for why this family is so plentiful as targets, hypothesizing that the phasiRNA act as regulators in the absence of a pathogen trigger, or that this is control over a rapidly expanding gene-family, citing studies by Shivaprasad et al., 2012 and Kallman et al., 2013, respectively.  Could it be both?  I will have to read some of their cited papers to get more context for phasiRNA gene-regulation.

This paper also has a huge amount of information on tissue specificity of phasi and micro-RNA genes, providing a more complete picture on this regulation in soy.  They saw wide diversity in tissue specific small RNA expression, finding several sub-groups of highly specific sRNA genes.  Overall, a very interesting article with a large amount of content, making it hard to show all of it here.

miRNA annotation in Capsella rubella (Camelineae) indicates rapid divergence

Rapid divergence and high diversity of miRNAs and miRNA targets in the Camelineae

Lisa M. Smith, Hernan A. Burbano, Xi Wang, Joffrey Fitz, George Wang, Yonca Ural-Blimke and Detlef Weigel

Department of Animal and Plant Sciences, University of Sheffield, Western Bank, Sheffield S10 2TN, UK

Department of Molecular Biology, Max Planck Institute for Developmental Biology, Spemannstrasse 35, 72076 Tubingen

doi:   10.1111/tpj.12754

PMID:  25557441

This paper is from the most recent issue of The Plant Journal, and I thought it made some rather interesting points.  The paper focused on small RNA seq of several tissues from Capsella rubella, a member of the Camelineae tribe and frequent outgroup to the Arabidopsis genus.  With sRNA annotations from A. thaliana and A. lyrata, the authors look at the evolution of miRNAs within closely related species.

First of all, I was interested in the bioinformatics suite that this group chose to perform their annotation.  After aligning unique reads with bowtie, they used several clustering softwares (miR-deep 1.3, DSAP, UEA sRNA toolkit), resulting in some wildly different loci and annotations.  Within figure 1c, it appears that only half of known miRNA loci annotated by DSAP or miR-deep are found in common with each other, though this is higher when looking at miRNA families.  Is this because of failings within these softwares (the authors mention a high false negative rate in miR-deep)?

The article goes on to look at the variation in miRNAs in relation to their target genes between the 3 species.  They found that unique miRNA-target pairs were highly species divergent, with most pairings being unique to the different species.  Of the non-divergent pairs, almost all are more ancient pairings that are present outside of brassicaceae, leading the authors to hypothesize that there are two differentially evolving subsets of miRNAs: “young, evolutionarily dynamic miRNAs, and older miRNAs with a conserved subset of mRNA targets”.

The authors go on to look at the levels of polymorphism in miRNAs and their targets throughout A. thaliana.  This analysis ultimately lead to higher mutation rate in miRNA sequences themselves, forcing the authors to conclude that the target sites are undergoing stronger selection.  I thought this was a bit confusing, as you might expect higher conservation in target sites which could be in the CDS of genes, a point mentioned by the authors but not elaborated upon.

– Nate

Exosome is not related to small RNA or RdDM based silencing in plants

DOI: 10.1371/journal.pgen.1003411   PMID:  23555312

I looked at the paper The Role of the Arabidopsis Exosome in siRNA– Independent Silencing of Heterochromatic Loci.  This was an interesting topic for me, as it connected some aspects of RNA metabolism and use I hadn’t originally thought as interacting.  The paper is examining the premise that the exosome might be indirectly involved in the regulation of small RNA production and RdDM in plants, as has been reported in yeast.  This effect was previously examined in Bühler M et al. 2008, in the yeast model organism S. pombe, where exosome deficient mutants were found to have vastly altered levels of siRNAs.  This is thought to be caused by a buildup of aberrant non-degraded ncRNAs interacting with small RNAs and siRNA machinery.

The authors used small RNA sequencing to determine the global make-up of annotated small RNAs in Arabidopsis, looking for a difference in exosome deficient plants.  The authors couldn’t find an effect in small RNA quantity or distribution, considering both the type of small RNA (miRNA, genic RNA, ncRNA…) as well as the possible target location (TEs, inverted/tandem/double repeats..)

Despite the apparent lack of exosome mutation on small RNAs, the authors did find a downstream effect, where mutant plants had an increase in the quantities of RdDM-regulated heterochromatic loci.  They examined this in the context of POL IV and V mutants, in which exosome deficiency lead to a dramatic loss of regulation in these (2) loci.  Following data shows that this is not from a decrease in methylation of these sites.  Histone association is seen to be lower in these loci in exosome deficient plants, as well as an association between exosome and flanking scaffold regions, leading the group to speculate that there is a cooperative effect between these structures, acting independently of RdDM.

Looking at the data in this paper, there are still several enigmatic results that are poorly explained by their model.  Their data indicate that there is a combinatorial effect between the exosome and RNA pol V in silencing heterochromatin loci, but later data contradicts this, showing that mutant plants containing mutations in both have higher enrichment when pulled down by histone (figure 6a).  I struggled to find reasoning for this observation in the paper, but perhaps I missed it in my readings.  Despite some of these confusing points, I thought the results presented provided an interesting context for alternative forms of heterochromatin regulation, rather than RdDM. Overall a broad and interesting read.  My take-away points are 1) that the exosome in plants (as opposed to yeasts) must have some layer of insulation between RNA degradation and RdDM machinery and 2)  there are alternative forms of RNA directed heterochromatin regulation.

Hope this isn’t too far off topic, just thought it was interesting.