With the discovery of the trp attenuator in the 1970s, followed shortly by the ColE1 plasmid RNA I, RNA became a much more interesting personality. The biomolecule turned out to wear far more hats than mRNA, rRNA, and tRNA. In all its multitudes, RNA seems to boast an infinite functional repertoire, from modulating mRNA stability to catalyzing chemical reactions to sensing small molecules to more.
We've had a smattering of regulatory RNA appreciation on the blog. First, we had a guest author's bird's-eye view of the world of small RNAs. Last week, Christoph wrote of a multitalented RNA molecule involved in regulating V. cholerae virulence. We've also covered the tmRNA in two posts, first in an enthusiastic Twitter-based overview and then in a look at its remarkable cross-kingdom role in the Vibrio fischeri-Hawaiian bobtail squid symbiosis. In the forty-odd years since the discovery of the trp attenuator and RNA I, a smorgasbord of such regulatory RNAs has been characterized especially in familiar faces like E. coli and Pseudomonas, but symbionts – endosymbionts, even more so – haven't received as much scrutiny.
Now we highlight another fascinating case of cross-kingdom RNA exchange that features an endosymbiosis instead (marking the first time the blog has covered a eukaryote-in-eukaryote endosymbiosis). The microbial duo in this recent study by Jenkins et al. consists of Paramecium bursaria and its green algal endosymbionts, Chlorella spp. The two are engaged in a facultative photosymbiosis, the algae providing sugars produced via photosynthesis and the Paramecium providing organic nitrogen to meet its end of the evolutionary deal. This all sounds very peaceful and pleasant, but what Jenkins et al. find is that a dog-eat-dog (cell-eat-cell?) mechanism may underlie this symbiosis. A punchy line from the paper: "Conflict is an inevitable outcome of all symbioses, the resolution of which can significantly impact the stability of an interaction. Enforcement mechanisms that punish misbehavior can act to stabilize symbioses."
The authors ask, what drives the transition from mere engulfed cell to a more stable intracellular resident, and then what keeps that resident from being digested? It turns out that part of the narrative may be driven by cross-kingdom RNA interactions, and the "punishment" for the protist host arises when it digests its endosymbionts.
P. bursaria digestion of endosymbionts can be triggered by exposure to cycloheximide. The ribosomal translational inhibitor sets off a surge in lysosomal activity within P. bursaria, which digests and wipes out the endosymbiotic algae. This loss throws a wrench in the protist's growth (Fig. 2), so the presence of its endosymbionts is indeed important.
This endosymbiont breakdown is like a hybrid of bursting a piñata and opening Pandora's box: it's true that digesting endosymbionts may have tasty benefits like nutrients, but the other molecular detritus left in the wake of the destroyed endosymbionts can't be forgotten. That includes RNA. Jenkins et al. demonstrate that in cycloheximide-treated P. bursaria, the host RNAi machinery moving about in the cytoplasm slice and dice released endosymbiont mRNAs to produce 23-nt sRNAs. This recognition and processing of the RNA is done by a band of proteins like Dicer and the Argonaute proteins. The resulting sRNAs go on to silence P. bursaria genes, interacting with transcripts such as elongation factor 1α (EF-1α), tubulin β chain (tub-β), and a heat-shock protein (HSP90) and ultimately restricting host growth.
P. bursaria RNAi machinery is thus capable of processing both endogenous and exogenous mRNA, confirmed by the normal growth restored by a Dicer knockdown. This was double-checked by exposing P. bursaria to synthetic fragments of algal mRNA, which again hampered growth. And just as before, this growth defect was relieved by knockdown of host RNAi machinery. Both synthetic dsRNA and the more biologically relevant synthetic ssRNA did the trick – but in the latter case, only for ssRNA in the sense orientation, the same orientation as the source mRNA transcripts in vivo.
So, the host's digestion of algal endosymbionts releases algal RNA that slips into the host's RNAi circuits and then hampers host growth. The endosymbiont's mRNAs are molecular sleeper agents, kicking into retributory action on the host only if touched by host RNAi machinery: a canny way of enforcing "endosymbionts are friends, not food." Jenkins et al. dub these interactions "RNAi collisions." It's a tit-for-tat game, except the game pieces here are made of RNA and it spans evolutionary history.
Cost-benefit analyses like these play into endosymbiont population density across species. Endosymbionts provide nutrients and defense, but too high of an endosymbiont titer can siphon away host resources, for example. The host must fine tune its resident population at different life stages, in different ambient conditions. In aphids, endosymbiont titer affects resistance to heat, and in Wolbachia–infected Drosophila, it affects host phenotypes like oocyte development. P. bursaria itself has been found to tinker with its endosymbiont load in varying light conditions, too. Some endosymbiont digestion is necessary at times, so what RNAi collisions accomplish, the authors suggest, is to prevent elimination of endosymbionts en masse.
This is also not the first RNA rodeo of its kind: endosymbiont-host RNA-RNA interplay has previously been observed in other cellular Matryoshka doll set-ups. For example, tRNA-derived sRNAs of several nitrogen-fixing rhizobial endosymbionts are exported to the legume host through a yet unknown mechanism, where they regulate host genes involved in root nodule formation by co-opting their hosts' RNAi machinery. There, silencing target genes promotes root nodulation. Wolbachia, too, taps into RNA as a tool for manipulating its host. Some Wolbachia sRNAs regulate not only Wolbachia transcript levels, but also regulate transcript levels of its Drosophila host's genes – including dynein heavy chain mRNA, thereby possibly regulating its own intracellular trafficking.
What's unknown in these two previous examples is whether endosymbiont degradation has anything to do with them. The Wolbachia sRNAs were identified through simple fractionation of whole endosymbiont-containing mosquito cells and Northern blotting, and the rhizobial sRNAs were identified by comparing RNA-seq data from free-living Rhizobia, colonized root nodules, and uncolonized root nodules. Perhaps here, too, RNA-RNA interactions are interlinked with endosymbiont digestion.
What other regulatory RNAs will be discovered that might regulate endosymbioses? Definitely, regulation by RNA has advantages particularly relevant to the reduced–genome contexts of many endosymbionts. Partial base-pairing of sRNAs with target mRNAs allows for broad regulation by a single RNA molecule, which is useful when a cell must be economical with its genome's coding capacity. RNA-based regulation is also less metabolically costly than protein-based regulation, which frees up a cell's resources for key symbiotic duties like churning out defensive secondary metabolites. It's also quick: regulatory RNA doesn't need to be translated, so it's inherently a speedy means of adapting to changing conditions like host life stage and nutrient demand. And the potential for chemical modifications of RNA leads to the opportunity for staggering molecular diversity.
So, we keep a vigilant eye out for more cases of microbial creativity with RNA!