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…
Are RNA thermosensors more common than we thought? Interesting article in the Journal of Experimental Biology speculating whether RNA thermometers (RNATs), well-studied in Prokaryotes, are prevalent in the other Kingdoms of Life.
Changes in the conformation of RNATs (see their Figure 1, above) typically involve melting of short regions of the mRNA, for example hairpin structures, in response to elevated temperature (a ‘zipper’ mechanism) or a shift between alternative conformations of the mRNA that involve larger regions of the molecule (a ‘switch’ mechanism).
The RNAT contains the Shine–Dalgarno (S–D) sequence (AGGAGG) that, when fully exposed, can bind to the small (30S) ribosomal subunit and allow translation to commence. The start codon (AUG) is often located eight nucleotides downstream from the S–D sequence. Thus melting of the ‘thermometer’ allows the S–D sequence and start codon to interact with the 30S subunit, promoting translation of the mRNA.
Interesting read – I wasn’t familiar with the concept of ‘marginal stability’ – the idea that for RNA secondary and tertiary structures, thermosensor regions must have the right stability – or ‘balancing act’ – to allow temperature-driven changes in shape to take place when (and only when) a signalling function is required.
I particularly liked the section on ‘Differential translation of allelic mRNAs: another way to modulate the proteome?‘ – the concept that natural variants (allozymes) with different thermal optima can provide a species with an opportunity to establish populations with adaptively different thermal optima in regions of its biogeographic range where temperatures differ. Thus a cold-optimised allozyme might be more common in populations living in colder regions of a species’ range, whereas the warm-optimised allozyme would be dominant in warmer regions, and therefore crucially that slight changes in base composition likely alter the thermally sensitive mRNA structures that govern translational ability in a way that ensures differential translation of distinct allelic messages.
The author, George Somero, make an interesting point that we might assume that “changes in temperature often are regarded as having negative influences on macromolecular stability” adding “However, there is also a ‘good’ side to this thermal perturbation: the alteration in conformation of the macromolecule that is caused by a change in temperature can function as a thermosensing mechanism and lead to downstream changes that are adaptive to the cell.”
Exciting times lie ahead for RNA structure and temperature sensing….
Trying to re-learn the language of GATEWAY cloning. ‘BP reactions’, ‘LR cloning’, ‘attB sites’, ‘entry clones’, ‘destination vectors’, ‘binary vectors’…I could go on. Thinking of trying a series of ‘Improved Gateway Binary Vectors (ImpGWBs)‘ to make plants express our genes of interest (GOIs) fused to markers. These are described in this paper:
As for the choice of marker, there is a dizzying choice, so as well as trying to get to grips with GATEWAY, there is the question of what best to use as marker. I’ve had disappointing results fusing our GOIs to green fluorescent protein (GFP) in the past, so maybe time to try something else. As you can see, there is no shortage of choice: