Small RNAs Considered

by Janie
 

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.
 

Figure 1. P. bursaria with algal endo­sym­bionts. Source. Fron­­tis­piece.

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.
 

Figure 2. From Figure 1 of the paper. (A) P. bursaria cell number (gray) versus algal chlorophyll fluorescent in­tensity (pink) in stationary phase cultures treated with cy­clo­heximide compared to untreated con­trols. Loss of algal fluorescent intensity indicates algal death/loss of function in response to cycloheximide treatment. (B) Stages of endosymbiont elimination in rep­re­sentative P. bursaria cells over 3 days of cyclohex­imide treatment. LysoTracker Green fluorescence (green; ’) indicates in­creas­ed host lysosomal activity in res­ponse to cyclo­hexi­mide treatment. Al­gal chlorophyll fluorescence (pink; ’’) highlights endosymbiotic algae within the P. bursaria cell. Source.
 

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!