Imagine pre-messenger RNA as ….

Irn bru research

Ok, lets talk about pre-messenger RNA splicing, shall we?

Mmmm, na it’s a bit complicated. Hang on, it might be easier to use Scotland’s ‘other national drink’, the very orange ‘Irn Bru‘ to think about what’s going on here.

RNA pre-messages usually have strings of sequence made up of A’s, C’s, G’s and U’s. But let’s imagine that a pre-mRNA is the ode to Irn-Bru (see picture above), and that chunks of the message are removed – or spliced out – to leave a final useable message. See how the meaning of the original pre-message bears no resemblance to the final message? (and the ways messages are spliced can result in very different outcomes).

Irn Bru is the same colour as ethidium bromide DNA gel stain (…or SyBr safe, if you’re younger)]. Might test Irn-Bru for its DNA staining ability (it has mystical properties after all), but I’ll leave someone else to test whether EtBr/SyBr safe passes for Irn Bru*

* disclaimer no responsibility is hereby taken if anyone actually does this…

(Editor: did you know that Scotland is one of the few places in the Western world where Coke is not the no.1 top selling soft drink. In Scotland Irn Bru is the top selling soft drink.)

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Let’s eat grandma

lets eat grandma cropped

Thinking about how to encapsulate alternative splicing in one ‘killer’ picture.

The picture above is what I came up with….

The analogy is that the inclusion/exclusion of alternative exons during pre-mRNA splicing can have the same dramatic effects as the inclusion/exclusion of key punctuation in a sentence.

I sincerely apologise to all Grandmas seeing/reading this….

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Clocked: local SNPs in global pops

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R4RNA arc diagrams (top) for predicted secondary structure comparison of the (upper) G/G/U/G/C and (lower) A/U/G/C/A haplotypes, with (bottom) SNPs aligned along the LHY 5’UTR region (exons; boxes and introns; horizontal lines). [from Fig 1 (b and c) of James, Sullivan and Nimmo, PCE, 2018]
Our study on the correlation between ‘natural variation’ in a clock gene sequence with bioclimatic parameters is out now as OpenAccess in the journal Plant, Cell & Environment.

The paper is called ‘Global spatial analysis of Arabidopsis natural variants implicates 5′UTR splicing of LATE ELONGATED HYPOCOTYL in responses to temperature

The starting point for this work was the idea that the 5’UTR of the core clock gene LATE ELONGATED HYPOCOTYL, also known as LHY, could function as a thermosensor given that we previously saw temperature sensitive alternative splicing of LHY.

We tested our theory using the 1001 genomes resource, a whole-genome sequence database for at least 1001 strains of the reference plant, Arabidopsis thaliana. Arabidopsis is native to Europe, but can now be found in the United States, North Africa and temperate Asia. We examined subtle differences, or polymorphisms, in the DNA sequences of >1001 accessions. These are often referred to as single nucleotide polymorphisms (SNPs). We found that different strains tended to ‘shake out’ as particular ordered assemblies of the SNPs, called haplotypes [for example, in the picture above the G/G/U/G/C haplotype is compared to the A/U/G/C/A haplotype] .

We were interested to see if the distinct haplotypes aligned with particular features of where these plants were growing – maybe the haplotypes grouped according to latitude, longitude, or altitude? Or would they group according to climate, such as temperature? seasonality? or even rainfall? For this we made use of the WorlClim database – a free public resource offering global climate data for ecological modelling.

The key findings were that:

  1. One of the haplotypes has hallmarks of being a signature of ‘relict’ accessions (survivors of the last ice-age and the subsequent expansion of new populations). This version is the most distinct in the respect that, worldwide, the accessions bearing this haplotype are found in regions of low rainfall. They are also associated with the highest elevations with low mean annual temperatures and a wider range of maximum–minimum temperatures
  2. Two of the remaining three haplotypes seem to associate with milder annual mean temperatures and lower altitude and wetter habitats
  3. The fourth haplotype, seems to be a low temperature specialist. This haplotype is commonly found in the mountainous Pyrenees region of northern Spain and is prominent at the limit of Arabidopsis growth in northern Sweden
  4. By measuring the extent of LHY spliced upon cooling in representative strains from two haplotypes we established that haplotype does indeed affect the splicing of LHY transcripts in response to cooling
  5. We propose that the LHY haplotypes possess distinct 5′UTR pre‐mRNA folding thermodynamics and/or specific biological stabilities based around the binding of trans‐acting RNA splicing factors

There is much interest in identifying plant thermometers and how they have evolved to cope with new temperature environments. Our new work shows that subtle differences in the DNA sequence of global populations of Arabidopsis plants influences the scalable splicing sensitivity of the mRNA for this central clock component, thereby finely tuning the clock to specific temperature environments.

We anticipate that these findings will be of interest and relevant to crop breeding programs that aim to produce stable food crops in the face of changing climate. 

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RNAtips

RNAtips aqua

Here’s an interesting tool I came across recently. It’s called RNAtips (RNA (temperature-induced perturbation of structure). It’s a web based tool that aims to tell you regions of RNA that are likely to undergo structural alterations due to temperature change. Here’s the citation:

Screen Shot 2018-04-22 at 14.55.09

The input for RNAtips is either one or two RNA sequences of the same length (in FASTA format). You then specify two temperatures to define the temperature range for which the RNA structural perturbation should be calculated (the default range is 32–39oC).

One of the types of output plots is at the top of this post, where the density of the most temperature-sensitive positions over the whole RNA sequence together with localization of clusters (red blocks) and localisation of nucleotide substitutions (if any) are plotted.

How does it do this? RNAtips deciphers those nucleotides within the RNA sequence, which change the most in their probability to form Watson–Crick bonds in response to a given temperature change. One of the first steps appears to be the calculation of probabilities of nucleotides being paired in a double-stranded conformation  by using the RNAfold tool of the ViennaRNA package. The calculations performed are derived from those described in paper in RNA Biology in 2012. The web server output plots clusters of these temperature-sensitive positions within a sequence, therefore representing the most temperature-sensitive structural regions of that RNA.

This is a very interesting tool and it is useful to make interesting hypotheses relating to your RNA of interest. For example, how do the temperature sensitive regions of an RNA relate to possible alternative splice sites or predicted RNA binding site regions, or to particular secondary structure folds or sequence polymorphisms.

I wonder to what extent the temperature range selected when inputing the sequence data on the web-form influences things. The range 32-39oC is not really applicable to (most) plants. Does this mean the inferences made on RNA structure are ‘wrong’. It would be nice, also if the sequence plots (e.g. picture at the top of post) could be overlaid with multiple plots in order to compare multiple sequences.

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Free the roots

 

Been growin’ the Arabidopsis seedlings in Eppendorf tubes filled with agar media. The young roots do what they do best and grow downwards through the agar media (this is called ‘gravitropism‘). The race is on though….if we don’t help them now they will be trapped in a plastic hell of diminishing nutrients.

That’s when we come to the rescue and clip the bottom of the agar tubes so that the growing roots can escape to freedom. This part of the process of growing Arabidopsis hydroponically is between the two earlier posts ‘Seed spotting‘ + ‘Green shoots emerging‘ and this later one: ‘Looking for roots‘. We use a tube cutter from VWR.

 

Once all the tubes are clipped, a puddle of hydroponic ‘root juice’ is added to the tray. This is double the strength of the hydroponic media used to make the agar in the tubes. We do this to help tease the roots down and out of the tube…it’s their reward for their escape to freedom.

Got a good crop this time…hopefully that’ll mean we can do lots of interesting experiments.

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Looking for roots

 

Lets have a look at how our little Arabidopsis plants are getting on. These were the plants that were growing in the yellow ‘nursery’ boxes. The young seedlings are about two weeks old, now growing hydroponically in blackened boxes to keep the roots dark.

Here, were having a wee look to see if the roots have emerged from the bottom of the cut Eppendorf tubes. Yes ! there are roots coming out into the hydroponic media (a minimal medium without any sucrose).

The boxes are in environmentally controlled growth cabinets (Sniggers cabinets) where we can control light intensity and temperature and humidity. I have the plants growing in 12 hours of light and 12 hours of dark (12h LD) at 20oC. These are quite standard “lab” conditions. I suppose 12h LD would be equivalent to the equinoxes in Nature…..would we ever get 20oC in Scotland in March or September ? (I very much doubt!)

 

 

 

Are ye’ a cold kinase? Or are ye’ no a cold kinase?

Screen Shot 2018-03-26 at 17.08.22Screen Shot 2018-03-26 at 17.08.41

I’ve been wrestling with a couple of ‘kinase’ papers of recent. They were both published in December 2017 in the journal Developmental Cell.

Firstly there is Zhao et al. with ‘MAP Kinase Cascades Regulate the Cold Response by Modulating ICE1 Protein Stability

…and the other paper is Li et al. with ‘MPK3- and MPK6-Mediated ICE1 Phosphorylation Negatively Regulates ICE1 Stability and Freezing Tolerance in Arabidopsis

Both papers examine the early response of plants to temperature and the involvement of protein kinases – principally the mitogen-activated protein kinases (MAPKs) e.g. the MAP kinase kinase kinases (MAP3Ks; also called MAPKKKs or MEKKs), MAP kinase kinases (MAP2Ks; also called MKKs or MEKs), and MAPKs…..you can already see how complicated this can get.

When plants adapt to cold there are large changes in the expression of thousands of genes, and its now well established that these changes are mediated by what are known as the CBF genes and they do this by regulating another subset of genes known as the COR genes. Its a bit like a domino effect. Once cold is triggered the dominoes start falling leading to COR gene expression and the plants physiological response to temperature.

Whereas quite a lot is now known about the later stages of the domino line at the molecular level, less is known about the ‘who’ and ‘what’ starts to triggering the domino line. Moving slightly up from the CBFs are the ICEs (ICE1 and ICE2).  ICE1 is a transcription factor that binds to CBF promoters and activates their expression. ICE is therefore seen as a very interesting ‘domino’ and how it is knocked over (..or activated) in this cascade is of great interest.  Several suspects were on the wanted list, including the MAPKs: MPK3, MPK4 and MPK6 and MEKK1 and MKK2, and these are the focus of both these papers.

I won’t go into much more detail. However, here are the highlights from both papers:

Li et al.:

1. Cold activates mitogen-activated protein kinases MPK3 and MPK6

2. MPK3/MPK6 phosphorylate and destabilises ICE1 protein, and

3. MPK3/MPK6 activation attenuates plant freezing tolerance

Zhao et al. :

1. The MKK4/5-MPK3/6 cascade negatively regulates freezing tolerance

2. The MEKK1-MKK2-MPK4 cascade positively regulates freezing tolerance

3. MPK3/6-mediated phosphorylation of ICE1 promotes ICE1 degradation

I think the other important thing is that Zhao et al. show that MPK4 positively regulates the cold response by constitutively suppressing MPK3 and MPK6 activity i.e. MPK4 blocks the ‘destruction’ of ICE1 by MPK3 and MPK6.

Anyway there is also a commentary article in the same issue in Developmental Cell on the background and features of these two papers called ‘MAP kinase Signaling Turns to ICE’

Must check it out. I think it will articulate these results much better than I can…

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C’est Chic

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Caught a piece on Nile Roger’s new ‘Chic’ album on GMTV this morning…what a inspiration!

He explained that his new album is called ‘It’s About Time

Wow…talk about great minds thinking alike! Our blog, and research is also ‘about’…’time’.

Only we are all about circadian clocks and how plants tell the time.

Just how do plants anticipate the light at dawn for photosynthesis, and how do they time the breakdown of the starch that they’ve made during the day for surviving the dark of the night? How do they do this?

It’s their circadian clocks – molecular DNA and protein ‘cogs’ that link together to make a molecular timer. They just keep ticking – even during the night – maybe it even allows plants to ‘stay up all night to get lucky!’

Pretty funky, no?

We have circadian clocks too. Nile Rogers is an international jet set superstar. I’m sure he’ll feel his clock ‘squeal’ at times with all the globe trotting and jet lag. That’s when the internal clock becomes de-synchronised with its new environment, and it takes a day or two to readjust. Understanding circadian biology is a really ‘cool’ area of research just now. You only have to look at last years Nobel Prize for Physiology or Medicine – it went to circadian clock research!

All this funk makes me want to get down to our research song! Check it out here:

https://abouttimeresearch.com/2015/05/22/its-nice-2-alt-splice/

Good times………

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