Naturally ornate RNA-only complexes revealed by cryo-EM

General Science Discussion
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Rachael Kretsch has published an astonishing new preprint paper titled Naturally ornate RNA-only complexes revealed by cryo-EM where she reveals the 3D structure of three large RNA-only molecules. I’m blown away by what she found. All three are much more ordered and symmetrical than I’ve seen in other RNA structures.

I’ll present all three individually for players to view and perhaps use for inspiration in our current 240mer design lab project.

The Ornate Large Extremophilic (OLE) RNA consists of two identical pieces of RNA that connect at three sites.

Our OLE dimer map shows that it is organized as a series of parallel A-form helices, like a bundle of pipes. The exterior ends of these pipes from each chain are interconnected into a five-way junction…
An unusual but highly conserved symmetric interaction comprised of four A-A base pairs between two chains (L4, Fig. 1D), intermolecular base-pairing and stacking interactions connecting L5, L6, and L7 (Fig. 1E), and a kissing loop (L9.3, Fig. 1F) ‘weld’ the pipes together in the middle of the complex. Hereafter we denote these intermolecular interactions ‘bridges’ B1-B3, as used in ribosome nomenclature.

One thing I find useful to learn from this new structure map is how highly paired P5 and P7 are with P5 featuring GU pairs and P7 featuring AU pairs. This almost looks engineered!

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Do you by any chance have a copy of the sequences? I want to do modifications for the current lab.

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The sequences are listed on page 42 (Extended Data Table 1: Sequences used in this study).

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Moving on, the other two structures are multimeric, meaning several copies of identical RNA come together to form a structure, in this case a hollow sphere. The ROOL sphere consists of 8 copies and the GOLLD sphere consists of 14 copies. These appear to be an entirely new class of structures.

Symmetric multimers are common among proteins and rationally designed RNA molecules, but observations of natural RNA multimers are rare. When observed, natural RNA homomeric interactions typically involve a single contact.

Both of these structure have numerous intermolecular contacts, referred to as bridges in the paper, that connect the individual chains (copies). The intramolecular contacts are drawn as lines as normal, and the intermolecular contacts are listed as B1, B2, B3, etc. (All of this makes them look even more like a Death Star to me.) Both structures form a nanocage larger than the ribosome!

The Rumen-Originating, Ornate, Large (ROOL) RNA consists of two half-shells, each formed by four chains (copies) of the 659-base RNA.

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The mix of pairs in the long stems of the ROOL are more in line with what I typically see in natural RNA, although still a little heavy in AU pairs. I don’t have much more to say about the secondary structure except that AA non-canonical pairs are again more common than I would expect, and the lack of GU/UG crossing pairs makes sense since each RNA is essentially flat. (GU crossing pairs introduce flexibility.)

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@DigitalEmbrace, again a supercool explanation of the article!

I was wondering about if there were a protein equivalent of this RNA Death Star. Like a selfassembling hollow ball.

It hit me where I had seen it before. The protein capsid (shell) of a virus. Those viral coat proteins are typically assembled of repeat monomers of the same protein. An advantage as the virus then only needs one gene for a coat protein. It just makes more of it.

The natural question now is, if a virus exist with an RNA coat shell :wink:

Also I wonder if RNA capsids will generally be smaller than protein capsids.

Ok, I think I can answer the latter one myself. The nanocages of those RNA balls can be larger than ribosomes. Ribosomes are similar in size to or much smaller than viral capsids.

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The chaperonin GroEL GroES protein complex forms chambers to correct or assist protein folding. It’s ATP driven and has a pair of reciprocating chambers. While one GroEL chamber is occupied and closed off by a GroES cap, the other chamber can’t be used.

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Vaults are another example of compartments formed by protein. Not much seems to be known about them. Also composed of two halves. About 95% protein and 5% RNA.

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Potential function of an RNA nanocage

@DigitalEmbrace shared the CASP 16 preprint with several nanocage RNA’s including some PDB ids. I dug up the one for ROOL. (9J6Y)

Eterna style ROOL in Chimera

Eterna style ROOL in Chimera
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She said: Maybe you can solve the mystery of its function.

I have been speculating about if these RNA nanocages could be envelopes for viruses. But I had no idea which.

I read up on ROLL and I can see that they exist in both phages, bacteriophages and bacteria (the latter some which are not affected of the phage in question)

My hypothesis is that the nanocages are capsids/envelopes for phages and bacteriophages - as an alternative way for the virus to get out of a cell.

As Wikipedia says on prophages (Host integrated phages):

The cell may fill with new viruses until it lyses or bursts, or it may release the new viruses one at a time in an exocytotic process.

Why then would bacteria have ROLL sequences in their genome? I’m guessing it could be to keep phages handy for an attack on other bacteria.

So I need to figure out if the diameter of a phage could fit inside its roll cage.

Understanding the context of the ROOL sequence

I did a BLAST with the ROOL sequence. It looks like it mainly turns up in an organism L. salivarius with plasmids, specifically on the megaplasmid. Perhaps the nanocage is for the plasmid?

I read a paper reference in the ROOL family in RFAM. It was from before the lncRNA was named ROOL. But I could line it up with my ROOL with a few gaps. This paper says:

High-level lncRNA expression correlated with high megaplasmid copy number.

The presence of megaplasmids is a distinguishing and unifying feature of L. salivarius, and these plasmids range in size from 100 kb to approximately 400 kb, in linear or circular forms

From: A long and abundant non-coding RNA in Lactobacillus salivarius

From RFAM:

The GOLLD RNA in Lactobacillus brevis ATCC 367 was studied experimentally. This GOLLD RNA is apparently encoded by a prophage, and its transcription is increased during the phage lytic cycle.

Family: GOLLD (RF02032)

So here the lnRNA is also specifically expressed in relation to replication of the phage.

I suspect the nanocages are for safe transport of the phages or plasmid.

Calculation of the ROLL nanocage volumen

I dug up the diameter of the RNA cages in Rachel’s preprint.

ROOL - diameter of approximately 280 Å
GOOLD - diameter of 380 Å

Based on the above, I would expect phages with GOOLD genomes to be larger than ROOL phage genomes.

I wonder if an RNA nanocage can be inside or outside a protein envelope?

This is the formula for the volume of a sphere. I don’t have the inside diameter of ROOL so I have just assumed the outside diameter for now.

V = 4/3π^3

ROOL radius = 140Å → 14nm

RNA nanocage volume = 11494.04nm → 1.149404E+04

I’m imagining another packing style than the usual bacteriophage packaging inside a “syringe”. Rather a disposal of the nanocaged phage during the waste disposal system of the attacking bacteria as an extracellular vesicle. Then uptake as food by an unsuspecting nearby and phage susceptible bacteria, in the expectation that there is food.

So when the nanocage may be used by the phage genome itself, the phage perhaps has just extended the service to the bacterial plasmid that was kind enough to host it.

Here is the ROOL sequence and when Change region shown is changed to the whole sequence, one can see the megaplasmid size.

Ligilactobacillus salivarius strain BCRC 14759 plasmid unnamed1, complete sequence

Genome size of the plasmid with the ROOL = 405494 bp. Which is 405 kilobases (kb)

According to this capsid volume versus genome size slide, my ROOL nanocage is too small to contain the megaplasmid. So I think the phage is cutting itself out of the megaplasmid before it gets packed in the nanocage.

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Figure 2 1-s2.0-S258900422100420X-gr2_lrg
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Scaling relation between genome length and particle size of viruses provides insights into viral life history

Where may we find other RNA nanocages?

I found a whole paper dedicated to predicting potential protein capsids for smaller phages. They could be RNA as well.

Figure 4 The Missing Tailed Phages
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The Missing Tailed Phages: Prediction of Small Capsid Candidates

A good bid for where to look for other RNA nanocages could be in phages, plasmids and bacteria. In particular lnRNA’s belonging to phages.

Also RNA nanocage sequences seem to have a special base frequency. This may have something to do with avoiding misfolding with themselves and elsewhere. They have a general low content of C’s. C’s that would typically be involved in base pairing.

Sequence frequency in ornament RNA

Sequence frequency in ornament RNA
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This also holds in general for the OLD, ROOL and GOOLD sequences downloaded from RFAM. (Added in the next sheets.)

Perspective

Plasmids have antibiotic resistance genes. Bacteriophages can be used as therapy against bacteria with antibiotic resistance.

I wonder if RNAcaged plasmids/phages can be another chapter in the human story of the battle against disease

Thx to Rhiju for discussion