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.)
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:
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
Two of the remaining three haplotypes seem to associate with milder annual mean temperatures and lower altitude and wetter habitats
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
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
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.
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.
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.
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…
Starting on making GATEWAY gene constructs for making RNA binding protein-GFP fusions for iCLIP and RIP, along the lines of work by the Staiger group (with nice link to ‘Living in an RNA World‘ blog post). Been trying out SnapGene to manipulate and visualise DNA sequences….so far so good (way better than Vector NTI).
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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I’ve been working with Ally Wallace to develop cartoon-like drawings that illustrate our research – here is some work in progress. I like the lab soundscape in the background. The aim was to develop something that might appeal to a younger audience, and tries to take an imaginative approach to the subject. Maybe a bit too wordy, so I’m now doing much more stripped back drawings – these were all done on an iPad using the ProCreate app. Then again, maybe it would be better as a short pamphlet/book….let’s see.
I was asked the other day if I could explain our research in 45 seconds, and after fumbling about with cumbersome nuggets such as ‘post-transcriptional mechanism’ and ‘spliceosome‘ and ‘exon-intron junctions’ decided it probably needed a drastic change of tact!
Anyway, it struck me that one of the key things to get across about alternative splicing is how important the inclusion (or exclusion) of an exon in a pre-mRNA has on how the mRNA is read or interpreted. If you substitute reading RNA messages with English grammar it reminds me of a funny Panda-related sentence that Hugh introduced me to a while back. It emphasises just how important a comma (or alternate exon, for example) has on the whole interpretation of the message. Compare these two sentences describing Pandas:
Eats shoots and leaves OR Eats, shoots and leaves
Notice how the comma completely changes the whole meaning and interpretation of the statement.
I think that this could be a good way to try to put across the key feature of splicing. Can it be done in 45 secs? Watch this space!
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