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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Splicing based body-temperature thermometer

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This work from the Lab of RNA Biochemistry at the Freie University Berlin shows just how sensitive splicing is to small changes in body temperature.

They looked at alternative splicing (AS) of U2af26 across a physiologically relevant temperature range (35-40oC). [U2af26 is a component of the essential splicing factor U2af (U2 auxiliary factor) where it can substitute for U2af35 in heterodimers with U2af65]

The authors show that U2af26 exon 6/7 skipping showed a very nice linear correlation with the temperature (see their figure below), suggesting that AS is able to react in a thermometer like way to read body temperature changes.

Screen Shot 2018-03-06 at 07.06.01

The paper goes on to show an involvement for SR proteins in temperature-regulated U2af26 AS, primarily via modulation of the phosphorylation state of SRs. The authors speculate that there will be a physiological role for temperature-controlled AS in other phenomena, such as hypothermia and fever.

 

 

Drawing Splicing 1

IMG_9188

Back to thinking about how to explain alternative splicing in an easy, graphical or pictorial way.

Here’s an attempt at sketching plant cells under a microscope. Grid like arrangement of cells, with chloroplasts (photosynthesis organelles) as greenish circles, and the cell nucleus as dark circles/blobs.

Not entirely sure where this is going…maybe a cartoon. Still hope to include Pandas somewhere along the line…

20 years of the the RNA Journal

RNA journal front cover cropped
Cover Art: Group in Sea, 1979, by Philip Guston

The RNA Journal is twenty years old and as part of their anniversary around 130 researchers in the field of RNA biology have contributed some of their personal reflections of working in this area. Contributors include Douglas Black, Michael Rosbash and Alberto Kornblihtt.

I’ve browsed through some of the essays and one that caught my attention was ‘Thoughts on NGS, alternative splicing and what we still need to know‘ by Kristen Lynch. Here she emphasises the need to determine the functional consequences of alternative splicing for an organism, and as she pointedly says ‘To truly appreciate the full impact of alternative splicing on biologic processes, and argue against those who wonder if it might all be “noise,” we need to do better. The question is how to achieve this goal’. [Note that NGS in the title of the article refers to Next Generation Sequencing]

As a relative newcomer to the field of AS, I think it’ll be useful for me to delve into these articles – they seem to be a refreshing way to learn how quickly research into AS has ‘evolved’ as well as providing an honest outlook as to what areas seem to be a priority for future work.

The cover art in interesting too – it is entitled ‘Group in Sea, 1979, by Philip Guston‘. He was an American abstract expressionist painter.

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