Top 10 signs RNA is awesome

by helenajambor

(sort of reply to Raj lab)

A buzzfeed like list of “top 10 signs that a field is bogus” guarantees a fun read, makes audiences crack up and all of us agree with at least one point. But, sometimes it hits close to home. And in this case not even home, but right into my heart, touching on my one big love, the RNA. And RNA localization even scored the number one spot! To illustrate the cellular role of RNA localization and more generally describe the importance of RNAs I have compiled the Top 10 Signs that RNAs and their localizations are awesome!

In short:

1. Degree of RNA localization is not overrated but essentially unknown!

2. RNAs not always randomly distributed!

3. Why should RNAs be localized?

  • For starters, diffusion is in fact very limited!
  • Then also: RNAs don’t diffuse well at all!
  • And: the cytoplasm is not water!
  • Cells are not small and round.
  • Are all localized RNAs encoding localized proteins?
  • Localized RNAs = silenced pool?
  • Localized RNAs = not translated?
  • Evolution!

 

  1. Degree of RNA localization is not overrated but essentially unknown!

To assess if RNA localization is overrated or underappreciated will require a good number of studies. And RNA localization has simply not been studied very much at genome-scale. Most genome-wide studies were performed on neuron that have clear polarity and extensions that can be separated from the cell body for RNA isolation/sequencing. Less is known for non-neuronal cells; In Drosophila embryos a total number of up to 70% of RNAs can localize – yet if this number seems overrated remember it is a sum of the entire embryogenesis and all cell types present in an embryo. In adult tissue such as the ovary we saw a great degree of variability: the absolute numbers of localized RNAs varied from 0.1 to 10% of the expressed transcripts per cell type. But, more importantly, the percentage of localized RNAs also varied in one cell over time suggesting that RNA distributions are context specific: we therefore suggest to categorize RNAs as ubiquitous versus localization-competent – these RNAs can enrich subcellularly but are not always localized.

The reason why genome-wide analyses of RNA distributions are rare is simply the huge amount of work each one still takes – for most cell types cutting off pieces does not work, cell fractionation to retrieve subcellular fractions is notoriously erroneous and “standard” in situ hybridization at genome scale is a lot of work. Even for single genes in situ hybridization often go wrong, are often done with improper probes (too long, not clean), old-fashioned detection method (NBT, BCIP) that don’t allow subcellular resolution etc.

Two things need to happen to get us ahead in the field: we need probes for assessing RNA distributions in living tissues and we need topological sequencing methods. Both methods are being currently developed, but its still early days to say if they are the breakthrough. So in my book, time will tell how widespread RNA localization really is. Until then lets postpone discussion of numbers – in the end does it make a difference if it is “only” 5% of RNAs? That is still a lot of transcripts! It will be much more interesting how localization-competent RNAs are regulated over time and in space!

  1. Are RNAs randomly distributed?

Well, we already know that many are not randomly distributed. And during my screen I often observed that RNAs encoding a known localized proteins, were also localized. In many instances (references upon request 😉 the authors had reported the RNA to be ubiquitous using a less sensitive approach.

RNAs also change their subcellular distribution as many others and we reported. They change localization over time, under stress, when the cell undergoes other dramatic changes that also result in global changes of cellular organization such as entering the cell cycle, becoming migratory or by viral infections.

Whether one can see RNAs in their localized states depends thus on a number of factors: the right detection method and integrity of the probes used but also on cell type. All this is important for if the RNA of interest is constitutively localized and even more important if the RNA is one of the localization-competent RNAs that have dual states!

  1. Why should RNAs be localized?

For starters, diffusion is in fact very limited!

I also like Bionumbers a lot, just got the fantastic book, and it gives you the answer! While diffusion works really well at the scale of bacterial cells, its effectiveness rapidly declines with an increase in cell size: doubling of the distance results in four times the diffusion time.

In addition, diffusion is not equal in all cell types: macromolecular crowding, large immobile protein structures, and interactions with other molecules influence diffusion. And finally, molecules themselves do not all have equal diffusibility: this depends on protein type, size, if it is in heavy particles etc. While a GFP molecule can traverse a eukaryotic cell in as little as 1 second, for cellular proteins this is much slower: even small proteins like transcription factors already require 3-30 seconds. The larger the protein gets and the more interactions it has with other proteins, the lower its diffusion coefficient becomes. For example it would take a ribosomes ~8 minutes to cross a cell!

  1. Then also: RNAs don’t diffuse well at all!

First of all, RNAs are big! By definition, already the open reading frame is 3 times longer than the protein they encode for, but they have additionally 5’UTR and 3’UTR and introns and long Poly(A)tails. The length of an unwound 1kb RNA in the cell is 300nm! And even in Drosophila 1kb is just the length of the UTR, in vertebrates they are much longer! Then this beast is highly negatively charged, i.e. likely tons of interactions are inhibiting its diffusibility. Then to overcome the charge, they are covered by spermidine, polyamines, proteins and what not – each molecule making the RNA less likely to diffuse fast. And even though proteins and amines are small and bundle the large RNA up – in the end it still has a four times bigger spherical expansion than the proteins.

  1. And: the cytoplasm is not water!

Diffusion is fast in water, but alas, the cytoplasm does not resemble water much. It is heavily crowded making it really hard for any molecule larger than a GFP to just randomly move around. In addition, recent papers suggest that the cytoplasm under starvation, during the cell cycle and in changing pH etc can “freeze” and become a gel. (Search glass-like cytoplasm and any paper from Simon Alberti lab!)

  1. Cells are not small and round.

While we like to think of cells as little round balls as they appear in cell culture and in textbooks, they in fact are hardly ever round. Most cells in tissues are polarized, they have extension, filopodia, asymmetries, form extensive surface interactions and protrusion, bulges… Even cells that in old microscopes appeared round, apparently look almost like neurons when observed with higher resolution! The role of RNA localization for establishing and maintain such highly polarized structures in neurons is well established and could easily be more widely used (but to show this more people would need to work on it! We don’t have much data on cross-tissue comparisons of mRNA localization).

  1. Are all localized RNAs encoding localized proteins?

Probably not. Do we know for sure? No, so far we have not one good dataset globally comparing RNA and protein localizations (coming, provided I get the funding!). Even if there was little correlation between RNA localization and protein distribution: that could be interesting too and we could understand more about the diverse roles of RNA in cells! Co-localization could enable complex formation, facilitate reactions, or serve as a backup mechanism for protein localization: for oskar RNA over the years more localization steps were discovered that individually were not critical in sum ensured germ cells could form (arguably, germ cell formation might be a more backed-up mechanism than RNA localization in somatic cells).

  1. Localized RNAs = silenced pool?

The localized state of RNA could also be a mechanism for translational silencing – similar to sequestration of RNAs into sponge/nuage/P-body type RNA-protein complexes. Do we know? No, again we have no genome-wide data. But most localized RNAs that have been studied in great detail so far are also under translational control at least for a period of their lifetime.

  1. Localized RNAs = not translated?

One exciting possibility is also that RNAs could have dual roles – protein coding and a structural role. This is in fact the case for oskar RNA in the fly: its early, 5-day long localization has nothing to do with encoding the protein, but is absolutely necessary for survival of the oocyte. In fact, for the early stages only ~100 nucleotides of the UTR are necessary – but they need to be localized!!!

  1. And finally, RNAs are localized in all life forms, algae, bacteria, yeast, many cell types, and also, RNA world… evolution, duh!

 

You see, RNAs are great and good for many things in cells! I look forward to a chance to discuss in much more detail over beer! In the end, we all agree bogus science is science that is crappily done, but no field itself is pointless to pursue – only time can tell what impact it will have.

 

 

Disclaimer: this list most likely is not complete! Am happy to update my list any time!

 

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