On the occasion of Sir Roger Penrose’s 2020 Nobel Prize in Physics, we review the singularity theorems of General Relativity, as well as their recent extension to Lorentzian metrics of low regularity. The latter is motivated by the quest to explore the nature of the singularities predicted by the classical theorems. Aiming at the more mathematically minded reader, we give a pedagogical introduction to the classical theorems with an emphasis on the analytical side of the arguments. We especially concentrate on focusing results for causal geodesics under appropriate geometric and initial conditions, in the smooth and in the low regularity case. The latter comprise the main technical advance that leads to the proofs of  The theorem itself roughly says that, in a situation that physically amounts to the formation of a black hole—intuitively a region in spacetime of so strong gravity that not even light can escape—there necessarily exists an incomplete null geodesic, i.e., a light ray that ends suddenly.1The singularity theorems of General Relativity (GR) are commonly counted among the 20th century milestones in mathematical physics. They comprise a body of rigorous results in Lorentzian differential geometry that, under physically reasonable conditions, imply the occurrence of a “singularity” in the sense of causal geodesic incompleteness of the spacetime manifold. At the time of its appearance, the first singularity theorem proved by Roger Penrose in  came as big bang. Then, during the next couple of years, Robert Geroch, George Ellis and others helped to shape a body of results which we now call the classical singularity theorems—for some historical details see e.g. [A short time later, Stephen Hawking realised that one could apply a similar reasoning to an everywhere expanding universe and showed that in such a spacetime there has to be a timelike geodesic that is past incomplete. This theorem, known as the Hawking singularity theorem , is genesee e.g. , Sect. 5see e.g. , proved Over the decades the singularity theorems have not only become an integral part of GR, but are still an area of active research, cf. e.g. , Sect. 8In this work we offer a guided tour for the mathematically oriented reader to the classical singularity theorems and to their recent extensions for Lorentzian metrics of low regularity. The study of the latter is motivated by the discussion of the “character of the singularities”  if null if causal if it is timelike or null. These notions extend naturally to vector fields and, via their tangent vectors, to sufficiently smooth curves.The stage of the theory is an entclass1pt{minimatime orientation, i.e. a smooth choice of one of the two connected components of the set of causal vectors at any A spacetime then is a smooth Lorentzian manifold Given the existence of a timelike vector field on low regularity since the bulk of classical smooth Lorentzian geometry extends verbatim to smooth timelike vector field and we do not reduce the regularity of the differential structure itself, since this is no loss of generality: Any In this note we will also be interested in non-smooth spacetimes, that is smooth Lorentzian manifolds with a time oriented metric of regularity below Riemannian curvature tensor of Ricci tensorcurvature scalar, i.e., the contraction of the Ricci tensor, we arrive at the Einstein equationsenergy momentum tensor which encodes information on the matter and energy content of spacetime. We avoid a discussion of cosmological constant to vanish and set At the heart of GR are the field equations which connect the curvature of spacetime to the matter and energy it contains. To make this statement precise, recall the Minkowski space, i.e., Schwarzschild metric which gives the entire one-parameter family of spherically symmetric vacuum solutions of  wast-Bruhat . During t-Bruhat  for an osee e.g. ) and it see e.g. , see e.gsee e.g. .Space tells matter how to move. Matter tells space how to curve. While the latter statement refers to the field equations  parameter, that is geodesically complete it this is true for all geodesics.Geodesics are curves exponential map at normal neighbourhood of radial geodesic convex if it is normal for all its points, which implies the existence of a unique geodesic in Next we discuss the maximising the Lorentzian distance .Up to this point there do not occur any differences between Riemannian and Lorentzian manifolds. However, the Gauss Lemma, which states that the exponential map is a radial isometry, has quite different consequences in the two cases. While in the Riemannian case it implies that radial geodesics minimise the Riemannian distance in convex neighbourhoods, it here leads to radial causal geodesics causality theory, i.e., the theory of futures and pasts of points in a spacetime. For more details see [chronological and causal future of chronological and causal pastpush-up principle holds, which can bee seen as an improved transitivity of the causality relations: if To explain this statement in some detail, we need to introduce some basic notions from ails see  and the ails see . For \dolongest curve in Lorentzian distance on reverse triangle inequality, If Hopf-Rinow theorem asserts that a Riemannian manifold There are even more striking new phenomena in Lorentzian geometry when contrasted with the Riemannian case. Recall that the Riemannian distance globally hyperbolic ones. These are defined to be causalcausal diamondsAvez-Seifert theorem, globally hyperbolic spacetimes are causally geodesically connected, that is, any pair of points In the Lorentzian case, as discussed above, geodesic completeness in the Lorentzian case can be defined individually for spacelike, timelike, and null geodesics, giving rise to three independent notions. Also, causal geodesic completeness does not imply causal geodesic connectedness. So in some sense, global hyperbolicity is the Lorentzian counterpart of Riemannian completeness, while incompleteness is a central notion in the singularity theorems, as we shall discuss next.The notion of in spacetime a singularity should be seen as some kind of singular boundary point of spacetime.Despite vivid examples such as the Schwarzschild metric and cosmological models of expanding universes, it is a somewhat subtle matter to define the general notion of a “singularity” in GR. Intuitively it should be a point where the curvature becomes unbounded but such a scenario is literally incompatible with the dynamical picture of spacetime in GR: We can only ever speak of a point in spacetime, if we have solved the field equations around it and hence found the corresponding manifold and metric structure. So, rather than being a point singular, if it contains an incomplete causal geodesic.To cut a long story a condition on the curvature, also called energy condition,(C)a condition on the causality, and(I)an initial or boundary condition.Thenis causal geodesically incomplete.Without specifying these conditions in detail we can nevertheless give a first idea of how the corresponding proofs work, that is, we explain how the conditions (E), (C), and (I) conspire to contradict geodesic completeness.The initial condition (I) serves the purpose that some causal geodesics start to focus towards each other. The energy condition (E) then implies that this focusing goes on until eventually a causal geodesic develops a focal or conjugate point and consequently stops maximising the Lorentzian distance. On the other hand the causality condition (C) implies the existence of maximising geodesics, at least in some region of spacetime. Now to dissolve this contradiction we have to accept that some geodesics stop existing before they reach a conjugate or focal point, that is, they are incomplete.Let us now examine each of the three conditions in some more detail and introduce the corresponding notions in a meaningful and precise manner.We start with condition (E) which actually is a condition on the curvature of spacetime. It is, however, called energy condition since via the field equations  it correstrong energy condition (SEC), if null energy condition (NEC) if We say that a spacetime satisfies the ondition , if SEC\ondition  if NEC\dIt is worthwhile to note that the energy conditions are the only place where the field equations come into play, so that the validity of the singularity theorems widely exceeds GR and continue to hold for alternative theories, as long as they imply the corresponding curvature conditions.Jacobi fields, i.e., vector fields geodesic variations of longitudinal, and the transverse. The tangents of the transverse curves at the base curve give the variational vector fieldIn the context of the singularity theorems the curvature condition is needed to exert a focusing effect on geodesics and we shall explain this in some detail, for more information see e.g. , Ch. 9. conjugate points: two points Essential for the focusing of geodesics is the notion of A geodesic variation as above with A causal geodesic fails to maximise the Lorentzian distance after its first conjugate point.From here it is evident that the curvature can be linked to the global structure of a spacetime by means of conjugate points. In fact, in the Riemannian case the analogous observation leads e.g. to the theorems of Hadamard and Myers, while in the Lorentzian case it is intimately related to the singularity theorems, cf. e.g. .before they encounter their first conjugate point. However, since (a) is related to the global geometry of the manifold, there is no way to detect such points via estimates on the curvature. So the power of conjugate points lies the fact that they lead to geodesics no longer being maximising and that we can estimate when they occur.At this point it should perhaps be pointed out that a causal geodesic from a point stops being maximising if and only if either (a) there exists a distinct causal geodesic between the same endpoints of the same length (as argued above) or (b) the geodesic encounters a conjugate point. But the cut-locus of a point Motivated by this observation we now introduce the analytical tools to find conjugate points, for a detailed and systematic analysis based upon Lorentzian Morse index theory see , Ch. 10,To begin with, we observe that the relevant information on conjugate points is contained in the Next, to represent the information contained in Jacobi fields most economically we collect them into Jacobi tensor classes. That is, classes of matrix Riccati equationLagrange if its Wronskian vanishes, i.e., Raychaudhuri equation for the expansionGiven a Jacobi tensor class The key observation now is the following: The second and the third term on the right hand side of  are non-nditions  or NEC)\documentecisely, /(NEC) anecisely,  imply \dConjugate points from negative expansionLetbe an inextendible causal geodesic and suppose there is a Lagrange tensor classwith negative expansionat someIffor allthen there is a point conjugate toalongat someprovidedTo sum up, we have seen that if along a causal geodesic a negative expansion occurs, then given the energy conditions, eventually a conjugate point will arise implying the failure of the geodesic to be maximising beyond it. Now, to “generate” a point with negative expansion is the purpose of the initial conditions to which we turn next.We now proceed to condition (I), which is the ingredient that gets the focusing effect of geodesics started, i.e., which produces a negative mean curvature vector of convergence of Beginning with the simplest case, consider a spacelike hypersurface Now, if we suppose that focal point. More precisely, a point To formulate a corresponding statement we still have to introduce the analogue of a conjugate point along a geodesic in the current “endmanifold” case, i.e., the concept of a Focal points from negative expansionLetbe an inextendible timelike geodesic starting atnormally from a spacelike hypersurfaceand supposefor allIfis negative  then there is a focal pointtofor someprovidedConsequently, stops maximising the Lorentzian distance tothe latest) atif it exists that long.trapped surface—to be introduced now—is one of the great innovations of the celebrated paper [The above focusing result will turn out to be at the analytical core of Hawking’s singularity theorem, and we now turn to the initial condition for Penrose’s theorem. In fact, the notion of a ed paper  and its both the families of normal future pointing null geodesics To begin with, we consider a spacelike submanifold This alone is not a useful property as is demonstrated by the trivial example of the intersection of two past lightcones in Minkowski space. However if we ask the hypersurface to be closed, i.e., compact without boundary (as suggested in the above example) it becomes a mighty tool as we shall see below.To get a vivid picture of the contraction property, we express it via the variation of the area We finally give the official definition of a closed trapped submanifold of arbitrary codimension Closed trapped submanifoldclosed future trapped submanifold if its mean curvature vector field is past pointing timelike on all of A compact without boundary, spacelike submanifold trapped set, i.e., a closed, achronal set future horismosThe idea of lightrays being trapped can also be expressed in purely causal terms via the notion of a future Now, using a focusing argument much like the one that lead to Proposition ion, see , Prop. 3Trapped set from trapped surfaceLetbe a future null complete spacetime where  closed (Lipschitz) hypersurface of spacetime that is hit by every inextendible causal curve. A central fact is that a spacetime admits a Cauchy surface iff it is globally hyperbolic, a statement which connects causal properties of spacetime with PDE-theory.To give a proper account we first introduce the notion of a Cauchy developmentNow global hyperbolicity is a strong condition and in many situations one wants certainly to do without it. Therefore the following generalisation comes in handy: We define the future Existence of maximiserLetbe a closed, achronal, spacelike hypersurface. Then to any pointin the future Cauchy developmentthere runs a future directed maximising geodesicfromMoreover, starts normal tohas no focal point beforeand it is timelike unlessCauchy horizon. More precisely, this notion is defined in causal terms as follows. The future Cauchy horizon of an achronal set Finally, we need to consider the boundary of the Cauchy development, called the It is now a basic fact that The first one of the classical statements we wish to discuss is the one by Hawking, which technically is the easiest, and we will be able to provide a sketch of its proof along the above discussion. In the literature one actually finds several (versions of) singularity theorems associated with the name of Hawking and we wish to discuss the one that supposes the existence of a compact Cauchy surface. It is, in particular, applicable to a spatially closed universe and hence gives evidence for a big bang in such models. It has, however, become a customHawkingLetbe a spacetime such that(E)for all timelike vectorsi.e., there is a compact spacelike Cauchy surfaceinwith(I)everywhere positive future convergenceThenis future timelike geodesically incomplete.By compactness there is a positive minimum trapped submanifolds of arbitrary codimension in [Next we turn to the theorem of Penrose. As already indicated above, its initial condition is the existence of a trapped surface, but the theorem has been generalised to nsion in , Thm. 1.At the heart of the proof of the Penrose theorem lies the following fact: The topological condition (existence of a trapped set) that is derived from the existence of a trapped surface, contradicts the existence of a non-compact Cauchy surface. The latter condition physically amounts to the fact that we are considering an isolated system and, in particular, the gravitational collapse of an isolated body.PenroseLetbe a spacetime such that(E)for all null vectorsi.e., there is a non-compact Cauchy surfaceand(I)there is an achronal closed future trapped surfaceThenis future null geodesically incomplete.Indirectly assuming completeness, we first establish that Now we take any timelike vector field genericity condition is introduced, which guarantees that all causal geodesics For the final part of this section we turn to the most sophisticated of the classical singularity theorems, namely to the one by Hawking and Penrose . It collachronal set without edge. Here the edge of an achronal set Also the Hawking-Penrose theorem features a third possible initial condition: a (future) trapped point,Later Dennis Gannon  and CharAnother more technical innovation of the Hawking-Penrose “singularity theorem par excellence”16Hawking & PenroseIn any spacetimethe following three statements cannot simultaneously hold: is chronological, i.e., it contains no closed timelike curve.Every inextendible causal geodesic incontains a pair of conjugate points.There is trapped setThis Lemma goes well beyond the causality arguments presented in this review so far and we will not attempt to sketch a proof here. Note that the causality condition (C1) is way more general than global hyperbolicity which was used in the above theorems. Nevertheless, it can be generalised somewhat further, see the discussion in , p. 793.We now give the analytic result in an extended form which, as already announced, includes yet another class of initial conditions, namely trapped submanifolds of co-dimension s due to , Thm. 3.Hawking & PenroseLetbe a spacetime such that(E)the strong energy condition it is chronological.Moreover, assume it contains at least one of the following: a compact achronal set without edge,a closed future trapped surfacea closed future trapped submanifoldof co-dimensionsuch that additionallyfor any future directed null geodesic withorthogonal toora future trapped point, i.e., such that on every future directed null geodesic fromthe expansionbecomes negative.Thenis causal geodesically incomplete.future causal incompleteness, which, however, holds in the respective future cases of the assumptions, see [Observe that we have not stated every inextendible causal geodesic in any of the initial conditions (I1)–(I4) imply that there is a trapped set, i.e., that (C3) holds.The line of arguments proving Theorem Let us elaborate somewhat on these two items. Starting with (1), the trick is done via the following advanced focusing result:Focal points from genericityLetbe a complete causal geodesic and assume  we haveFirst note that  and SECSEC give So assume that ain into  and usinain into  we obtaiequation  that theWe now turn to the discussion of (2) and begin with the following extension of Proposition Comapact horismosSuppose (and the existence of either (i) a closed future trapped surfaceor (ii) a closed future trapped submanifoldas in (I3), or (iii) a future trapped pointThenis compact, or the spacetime is null incomplete.Suppose  and the The proof uses again the focusing argument Proposition Now given (I4) condition (C3) follows immediately. In the cases (I2), (I3) one is only almost there, since in general uence of , GC), a, a\documFinally in case (I1) one shows that a compact achronal set This finishes our sketch of the arguments that establish the Hawking-Penrose theorem and also our journey into the classical singularity theorems. We next turn to their low-regularity extensions.In this section we wish to provide an overview of the extension of the classical singularity theorems to metrics of low regularity that have emerged over the last couple of years. Indeed the three key theorems discussed above have first been generalised to Lorentzian metrics of regularity ent}C1,1 –38 and t-metrics , 40 with-metrics . We shal-metrics . The cor-metrics  and thos-metrics  will be To begin with, we discuss the motivation behind this endeavor.Taking up the discussion from Sect. 18provided the metric is smooth, and, since the bulk of Lorentzian geometry remains valid there, if it is Taking a step back we also see that there is a regularity assumption which is implicit in the classical theorems. In fact, the theorems assert causal geodesic incompleteness of the spacetime If the regularity of the metric was just ing star , to giveing star –48.Moreover, if the regularity was even lower, one could be inclined to accept such a scenario as long as there is an analytic way to define the curvature and to make sense of the field equations . This isle sense , 51. Thenjecture , which dHowever, a first substantial conceptual problem arises with the very notion of geodesic incompleteness: Below olutions , which holutions , 55.A first step in this direction is to lower the differentiability of the metric to Of course, the regularity issue connected with the singularity theorems was already noted early on and extensively discussed in [Let us finally come back to the issue of extensions of spacetime. Certainly the classical theorems assert that the incomplete spacetime cannot be extended to a complete one keeping the assumptions and the Sbierski  who showSbierski  that timTo begin our technical account, note that the bulk of Lorentzian geometry remains valid for morphism , 60. Whiample in , which eample in . There i(A)The curvature is only locally bounded for (B)Normal neighbourhoods are not useful for (C)The geodesic equation fails to be uniquely solvable for Returning to the context of the singularity theorems, let us briefly collect the issues at hand and discuss the strategies we will employ in the sections to come. For a longer and more technically detailed list of places where the classical proofs rest on the lity see , Ch. 6.1Item (A) and, more precisely, the failure of the curvature to be defined pointwise causes several difficulties. First the energy conditions (E) have to be adapted, where especially  and GC)GC) turn Next, item (B) makes it necessary to revisit the whole machinery of causality theory and to extended it to the regularity at hand. We will refrain from going into any technical details here and just summarise the necessary background in Sect. 4.5Now, (C) first of all forces us to make a choice concerning the conclusion of the theorems, namely on the notion of incompleteness. It has turned out to be favourable to use the more stringent alternativeA However, despite the loss of uniqueness many properties of geodesics extend from the smooth to the non-branching assumption for maximising causal geodesics, to be detailed in Sect. One main issue will be to adjust the following vital aspect of the regularisation approach: We need to approximate maximising causal geodesics of the low regularity metric by maximising causal geodesics of the approximating metrics, which becomes a delicate matter in the absence of an exponential map (B) and unique solvability of the geodesic equation (C). This issue was addressed for globally hyperbolic times in , Sect. 2 theorem  in SectsHere we briefly discuss the general distributional framework in which to understand the curvature of metrics consult , 63–65.distributions of orderdistributional-tensor fields of orderThe space of  distributional connection is a map -connection, if A Moreover, if When deriving the surrogate energy conditions for Since we want to use the classical arguments on smooth approximations nd Grant , that prWe will have to relate the geodesics of the approximating metrics to the geodesics of As already indicated above our method of choice to deal with the analytic arguments in the proofs of the singularity theorems is regularisation and we will detail our convolution based approach below. Prior, we outline our overall strategy to implement an analytic machinery that forces causal geodesics of the rough metric to stop maximising: We will formulate suitable energy conditions for the low regularity metric nditions  and (NEC e.g. in .) This wWe proceed by introducing the regularisations to be used in detail. The basic ingredient is chartwise convolution and we begin by choosing a mollifier, i.e., a smooth, nonnegative function 26 in Just as is the case for smoothing via convolution in the local setting we obtain optimal convergence, that is Regularisations and convergenceLetbe aspacetime. Then for anythere exist smooth Lorentzian metricsonsatisfyingMoreover we have control on the speed of convergence ofand compatibility betweenandas follows: For any compactthere issuch that for small enoughAn analogous statement holds foras well as for the inverse metricsandWith these preparations we may now have a look at the distributional energy conditions for metrics In this section we introduce the energy conditions akin to  and NECNEC for \We will be more detailed in case of the  where weTo begin with, recall that a scalar distribution distributional strong energy condition  the main problem is that the convergence of volution  implies To establish these statements, we have to estimate the convolution of a product Friedrichs lemmaLetand letwithon compact sets. Theninsmooth metrics satisfying  and iffor somethen there is a focal pointtofor someprovidedConsequentlystops maximising the Lorentzian distance tothe latest) atif it exists that long.This focusing result will play a mayor role in the proof of the ation of  needs soull, cf. , Sect. 5distributional null energy condition the timelike Ricci curvature is nonnegative ini.e., there is a compact spacelike Cauchy surfaceinwith(I)everywhere positive future convergenceThenis future timelike geodesically incomplete.First we assume by contradiction that By Lemma 4.2Next we want to apply Proposition Denote by uch that  holds onTherefore, as in the proof of the classical Hawking theorem curves between any pair of points Note that we have neither shown nor needed that also }g∈C0 in , establiesics by , Thm. 6 esics by  for moreFor the proof of the Penrose theorem we will need the following version for null maximisers from Letbe a globally hyperbolicspacetime and letbe a closed, spacelikedimensional submanifold. Then for anythere exists at least one null geodesic fromtomaximising the distance toFurther, such a geodesic can be obtained as alimit of a sequence ofnullgeodesicsmaximising thedistance toWe now proceed to the Penrose theorem which was generalised to Letbe aspacetime such that(E)the null Ricci curvature is nonnegative ini.e., there is a non-compact Cauchy surfaceand(I)there is an achronal future trapped surfaceThenis future null geodesically incomplete.We assume null completeness and first establish that the future horismos So let’s assume by contradiction that there is So, In the following we begin to turn towards the low regularity extension of the third of the classical theorems, namely the one of Hawking-Penrose. We start with the causal result, i.e., Lemma In anyspacetimethe following statements cannot simultaneously hold: is chronological,29i.e., it contains no closed timelike curve.No inextendible timelike geodesic in an open globally hyperbolic subset is maximising.No inextendible null geodesic is maximising.There is trapped setWhile in establishing the We now wish to introduce an appropriate distributional version of the genericity condition which will allow us to prove the advanced focusing results needed to establish the As in the case of the  we will lds, cf. .distributional genericity condition is aorthonormal frame alongand(ii)inwhereis an orthonormal frame alongFinally, there areandsuch that alongthe tidal force operator for small enoughsatisfiesd assume  at\docuThe announced focusing result asserting the existence of a pair of conjugate points along causal geodesics of smooth metrics given an estimate of the form  is [40, 31Advanced smooth focusingLetbe smooth. Given someandthere existandsuch that any causal geodesicdefined onfor which the following hold(i)onand(ii)there exists a smooth parallel orthonormal frame forand somesuch that w.r.t. this frame the tidal force operator satisfiespossesses a pair of conjugate points onWe proceed indirectly, assuming that for any Recall from Sect. eorem of  implies One may actually find such an initial condition in the form equation  for \docFinally, since both Now, as discussed at the end of Sect. To motivate the introduction of the new non-branching condition we wish to sketch the essential argument that allows us to ng under  and (DSEDGC) and  resp.  solution of the original i.v.p. by (unique) solutions of the regularised problem seems unfeasible unless a specific regularisation is constructed to force exactly the desired convergence. Such an approach is of course completely unsuited to the situation at hand, where we want to approximate a given but arbitrary maximising geodesic.First, note that in the present situation, i.e., given an ODE with merely continuous r.h.s. and possibly different solutions to the same initial data, the following issue arises in non-branching condition for maximising geodesics, which is well motivated by similar conditions used in (Riemanian) metric geometry. There, in absence of a differentiable structure, geodesics are defined as  minimisers of the length functional and local uniqueness of geodesics is expressed in the form of a non-branching condition. Here, a branch point is defined as an element of a minimiser at which the curve splits into two minimisers that on some positive parameter interval do not have another point in common, cf., e.g., [Instead we will employ a ., e.g., , 77. Sim., e.g., , the rol., e.g., , 76.are locally maximising and to preclude them from branching. We now explicitly introduce our (rather weak) non-branching conditions, where we do not require the second branch to be maximising as well:While in the present to hold  A geodesic A found in , Ex. 3.2We may now finally proceed to establish the existence of appropriate approximating sequences of maximising geodesics, which is the missing link in our argument for . We onlyApproximating geodesics in non-branching spacetimesLetbe a globally hyperbolic MTNBspacetime. Ifis a maximising, timelikegeodesic then for any smallthere exists a subsequenceofand maximising, timelikegeodesicsconverging intolines—in non-branching Now applying essentially the argument from the beginning of this section (and its corresponding null version) we may establish the nonexistence of inextendible maximising geodesics—commonly called No lines) Letbe aspacetime satisfying ((i)Ifis globally hyperbolic and MTNB and if Ifis causal and MNNB and if If\We remark on the following subtleties concerning the distinction between the timelike and the null case in the above result: We may assume the (very strong) condition of global hyperbolicity in the timelike case, since Theorem causal instead of merely chronological, as was sufficient in the classical results.But, fortunately in the null case there is a sharper distinction between maximising and non-maximising geodesics because a null geodesic stops maximising if and only if it leaves the boundary of a lightcone, and we have already exploited the structure of such boundaries, cf. the proof of Theorem In this last of the preparatory sections we generalise the four distinct initial conditions of the Hawking-Penrose Theorem to the present The easiest case to deal with is the first, i.e., (I1), since it needs no special attention: Given the extension of causality theory discussed in Sect. support sense.Next we deal with the trapped submanifold-cases, i.e., (I2) and (I3). In the present regularity class it is natural to extend the corresponding initial conditions to trapped future trapped submanifold if any point the mean curvature vector field is past pointing timelike in the sense of support submanifolds on all of (Closed trapped future support submanifoldCondition (2) means that for any Now, to show that, given a closed trapped ow under  suppose suppose  and introduce trapped points in the current setting. In [Trapped points in future trapped, if for any future pointing null vector A point Now the techniques established above allow one to also prove the distributional strong energy condition it is causal, and(B)it is maximally causally non-branching (MCNB).Moreover, assume it contains at least one of the following: a compact achronal set without edge,a closed future trappedsurfacea closed future trappedsubmanifoldof co-dimensionsuch that  are faithful generalisations of the classical conditions. The causality condition (C) is slightly stronger than the condition of chronology used in the classical result and we have commented on our use of causality at the end of Sect. Finally, we come to condition (B), which is entirely new. As discussed in Sect. Of course, there is still the alternative that the regularity of the metric drops even further, i.e., below Assuming causal geodesic completeness, we once again deduce a contradiction, now using Lemma First note that (C) implies (C1) there. Then Theorem The case for (I1) has been made at the beginning of Sect. With these arguments we have finished our account on the extension of all three of the major singularity theorems of GR to regularity We begin this final section with a summary and some conclusions drawn from the results presented here. Then we will discuss some further perspectives of the approach at hand as well as alternative approaches to singularity theorems beyond the smooth setting.In this review we have discussed the extension to eorem in , which wThe proofs are, at the one hand, based on the recent extensions of causality theory to low regularity Lorentzian metrics and, on the other hand, rely on an extension of the focusing results for causal geodesics. Indeed the latter, which is achieved via a regularisation approach, is the main technical advance presented here.causally plain ones, cf. [Next we briefly discuss the further prospects of this approach. It is generally expected that the causality parts of the results extend to an issue , and theOn the analytic side, it seems also feasible to extend the recent techniques to locally Lipschitz metrics. Here, one primary task is to extend the Friedrichs Lemma olutions , see alsStill this is actually a long way from the largest possible class where the curvature can be (stably) defined in an analytical  way, i.e. the GT-class A somewhat related topic is singularities in semi-classical and quantum theories of gravity. There the energy conditions are expected to be violated and to hold only in some averaged sense, see e.g. . The quecausal cone structures, that is a theory of upper semi-continuous distributions of cones over manifolds . In this setting it is indeed possible to establish the causal core of some of the singularity theorems: One may see the analytic concepts like the energy conditions, focusing results, etc. used throughout this note merely as tools that produce subsets in Now shifting away from the more analytic parts of the singularity theorems we first turn to causality theory. It has long been clear that causality theory is very robust in general. Most arguments are rather topological in nature and can actually be seen as belonging to frameworks more abstract than Lorentzian differential geometry. Indeed, Ettore Minguzzi in  has receCausal Penrose TheoremLetbe a globally hyperbolic closed cone structure admitting a non-compact stable Cauchy hypersurface. Then there are no compact future trapped sets and ifis non-empty and compact there is an inextendible future null geodesic entirely contained inLorentzian length spaces introduced there are the analogue of metric length spaces, which have long been used as an essential tool to extract the metric core of many notions and results in Riemannian geometry, see e.g. [Another and quite different approach to singularity theorems has been opened up in the context of the recently developed synthetic approach to Lorentzian geometry put forward by Michael Kunzinger and Clemens Sämann in . The Lorsee e.g. . Lorentzh spaces , 84, 85 In this setting it becomes possible to formulate synthetic versions of the singularity theorems, in the sense that the energy conditions are implemented as synthetic curvature bounds. A first theorem in Lorentzian length spaces that are warped products and which uses suitable sectional curvature bounds (implying Ricci curvature bounds in such geometries), based on triangle comparison is the following version of the Hawking theorem, cf. [Synthetic Hawking TheoremLetbe a geodesic length space,34and letbe a warped procduct withandsmooth.35Assume thathas timelike sectional curvature bounded below by 0 and thatis non-constant. Thenorand henceis past or future timelike geodesically incomplete.smooth Lorentzian setting in [Of course, implementing the classical energy conditions  or NEC)NEC) rathtting in , 88 and tting in . The basTMCP-Hawking TheoremLetbe a timelike non-branching, globally hyperbolic Lorentzian pre-length space satisfying a TMCP-property. Letbe a Borel achronal future timelike complete subset with mean curvature bounded above. Then every future timelike geodesic starting inhas a bounded maximal domain of existence.To sum up, in this review we have discussed the classical singularity theorems of GR and sketched the main arguments leading to their proofs. One may actually identify two main lines in these arguments, the analytical and the causal one. The former is concerned with providing focusing results for causal geodesics using the energy conditions which lead to the occurrence of conjugate or focal points and hence provide estimates on when causal geodesics stop maximising the Lorentzian length. The second, causal line of arguments gives criteria for maximising causal geodesic to exist. Confronting these two threads leads to a contradiction unless some of the causal geodesics become incomplete.In the main part of this work we have extended the causal and the analytic line of arguments to Lorentzian metrics of regularity In the final discussion we have complemented these results with an overview of recent versions of singularity theorems in a purely causal setting as well as in a synthetic setting. It is clear that these results and techniques are still fresh, and that many interesting lines of research emerge from here. Also, it is unclear to date how the synthetic results precisely relate to the analytical approach put forward here and its possible extension to even lower regularity as discussed above, but see  for a fiIn any case, it can firmly be stated that the singularity theorems are not only an integral part of GR and Lorentzian geometry but, even more than half a century after they first emerged, they are still an interesting field of research holding many quests to be resolved in the future—both from the physical side, see e.g. , Sect. 8


Archaeoglobus fulgidus. We demonstrate that AfAgo associates with small RNA molecules carrying 5′-terminal AUU nucleotides in vivo, and characterize its affinity to various RNA and DNA guide/target strands in vitro. We also present X-ray structures of AfAgo bound to oligoduplex DNAs that provide atomic details for base-specific AfAgo interactions with both guide and target strands. Our findings broaden the range of currently known Argonaute-nucleic acid recognition mechanisms.Argonaute (Ago) proteins are found in all three domains of life. The best-characterized group is eukaryotic Argonautes (eAgos). Being the structural core of RNA interference machinery, they use guide RNA molecules for RNA targeting. Prokaryotic Argonautes (pAgos) are more diverse, both in terms of structure (there are eAgo-like ‘long’ and truncated ‘short’ pAgos) and mechanism, as many pAgos are specific for DNA, not RNA guide and/or target strands. Some long pAgos act as antiviral defence systems. Their defensive role was recently demonstrated for short pAgo-encoding systems SPARTA and GsSir2/Ago, but the function and action mechanisms of all other short pAgos remain unknown. In this work, we focus on the guide and target strand preferences of AfAgo, a truncated long-B Argonaute protein encoded by an archaeon  From the structural and mechanistic point of view, all characterized eAgos are very similar, as they all use small (~ 13–30 nt) RNA molecules as guides for sequence-specific recognition of RNA targets, and are monomeric proteins sharing four major conserved functional domains and two linker domains, which are organized in a bilobed structure5. The N-terminal lobe consists of N-domain that separates guide and target strands6, and PAZ domain responsible for binding the 3′-terminus of the guide RNA; the C-terminal lobe consists of MID domain, which binds the 5′-terminus of the guide RNA, and PIWI domain, an RNase H homologue8. The two linker domains, L1 and L2 connect N and PAZ and PAZ and MID domains, respectively. Upon recognition of the RNA target, eAgos may either cleave it employing catalytic activity of the PIWI domain or, particularly eAgo proteins that encode catalytically inactive PIWI domains, recruit partner proteins leading to degradation of the target RNA or repression of its translation9. Target specificity of eAgos is determined solely by correct base pairing between guide and target RNA strands. Nevertheless, all eAgos associate only with 5′-phosphorylated guide RNAs, and many have intrinsic specificity for the 5′-terminal nucleotide of the guide RNA (gRNA). For example, A. thaliana AGO1, K. polysporus KpAgo, and H. sapiens hAgo2 prefer guide RNAs with a 5′-terminal uridine, A. thaliana AGO2 prefers 5′-A, and A. thaliana AGO5 prefers 5′-C13. The 5′-phosphate and the 5′-terminal nucleotide are recognized in conserved pockets of the MID domain.Argonaute (Ago) proteins are found in all three domains of life . The best-characterized group is eukaryotic Ago (eAgo) proteins. Being the functional core of RNA interference machinery, eAgos are involved in the regulation of gene expression, silencing of mobile genome elements, and defence against viruses14. Unlike eAgos, prokaryotic Agos (pAgos) are diverse in terms of their structure, mechanism, and function18. The best understood are the so-called full-length or long pAgos, which are composed of N, PAZ, MID and PIWI domains, and thus closely resemble eAgos. There is mounting evidence that long pAgos function as prokaryotic antiviral systems, with the PIWI domain performing cleavage of invading nucleic acids13. However, unlike eAgos, which canonically use RNA guides for recognition of RNA targets in a process called RNA interference (RNAi)21, different long pAgos may use either RNA or DNA guides and/or targets18, and in vitro may associate with phosphorylated 13, non-phosphorylated guide strands  or lack preference for 5′-phosphorylation 24. Interestingly, the recently described KmAgo from Kurthia massiliensis can utilize both DNA and RNA guides to cleave DNA and RNA targets in vitro, albeit with different efficiencies26. Some long pAgos also specifically recognize the 5′-terminal nucleotide of the guide strand, e.g., CbAgo from Clostridium butyricum prefers 5′-terminal deoxyadenosine, and TtAgo prefers 5′-terminal dC27, while RsAgo from Rhodobacter sphaeroides prefers guide RNA with 5′-U29. The majority (~ 60%) of identified pAgos are ‘short’, as they encode just MID and PIWI domains, the latter being catalytically inactive due to active site mutations. The mechanism, guide/target preferences and function of short pAgos is an emerging topic in the Argonaute field, as evidenced by recent characterization of SPARTA and GsSir2/Ago antiviral systems30.Ago proteins are also identified in 9% and 32% of sequenced bacterial and archaeal genomes, respectively8 prokaryotic Argonaute AfAgo  encoded by a hyperthermophilic archaeon Archaeoglobus fulgidus8. Although phylogenetically classified as a truncated long-B pAgo, AfAgo contains only MID and catalytically inactive PIWI domains in a single polypeptide chain, akin to typical short pAgos, and could therefore be considered a pseudo-short pAgo33. Even though AfAgo is one of the first and one of the best structurally characterized prokaryotic Argonautes, with an apo-, DNA-, and two RNA-bound structures currently available35, its guide/target preferences remain undefined. We show here that AfAgo co-purifies with small RNA molecules carrying 5′-terminal AU nucleotides in vivo and investigate its affinity to various RNA and DNA guide/target strands in vitro. We also present X-ray structures of AfAgo bound to DNA oligoduplexes with 5′-ATT and 5′-ATC terminal sequences that provide structural details on the base-specific AfAgo interactions with the termini of both guide and target strands. Our findings broaden the range of currently known Argonaute-nucleic acid recognition mechanisms.In this work we focus on the truncated long-BE. coli, AfAgo co-purifies with tightly bound nucleic acids, predominantly RNA . Surprisingly, AfAgo had a strong preference for two 5′-terminal RNA nucleotides, A at the first and U at the second position , and a discernible preference for U at the third position  and synthetic single-stranded (ss) RNA and DNA oligonucleotides containing phosphorylated 5′-AUU and 5′-ATT termini, respectively (Supplementary Table S1). The experiments revealed that under our experimental conditions (see Methods), AfAgo preferentially binds ssRNA over ssDNA. Binding of ssDNA was detected only at exceedingly high (> 0.5 µM) AfAgo concentrations  and a 5′P-ATC terminus, which is analogous to a suboptimal ssRNA terminus 5′-AUC (PDB ID 6XUP and 6XU0) (Supplementary Table 35 (RMSD 1–1.64 Å when overlaid by residues 11–427).The AfAgo complex with the suboptimal 5′P-ATC DNA formed crystals of P1 symmetry that contained two DNA-bound protein subunits per asymmetric unit. The main conformations of both subunits are nearly identical, the most significant difference being the main chain conformation of the loop formed by residues 144–149  or recruitment of partner proteins . However, the function and action mechanisms of most prokaryotic Ago proteins, in particular catalytically inactive ‘short’ pAgos, remain unknown. In this work, we analyzed guide and target strand preferences of a truncated long-B8 prokaryotic Argonaute AfAgo from a hyperthermophilic archaeon A. fulgidus, and revealed its sequence specificity to the 5′-terminal nucleotides of the guide strand, and to the complementary fragment of the target strand.Argonaute proteins use a RNA or DNA guide strand for specific recognition of RNA or DNA target strands36. The tight interaction of AfAgo with RNA seemingly contradicts previous studies34, in which authors described preferential binding of AfAgo to single- and double-stranded DNA, but not RNA. Presumably, this discrepancy arose due to non-optimal 5′-terminal RNA and DNA sequences (5′-U or 5′-C) used, as we find that in vitro AfAgo also preferentially binds RNA with a 5′-AUU terminus and substitutions at the 5′-terminus reduce affinity . However, this should not invalidate our conclusions related to AfAgo preferences for ssRNA and ssDNA as the optimal guide and target strands, respectively. Indeed, pre-incubation the AfAgo-ssRNA binding reaction mixtures at elevated temperatures (70 °C) prior to EMSA did not alter the ability of AfAgo to discriminate the 5′-terminal ssRNA sequences, albeit it decreased the observed AfAgo binding affinities to all ssRNA variants , and to the near-optimal RNA-RNA duplex .We have also solved four crystal structures of AfAgo bound to DNA-DNA oligoduplexes with the 5′-AT terminal sequences, which mimic the 5′-AU terminus of the in vivo bound RNAs. Two structures were obtained with different optimal-like 5′P-ATT oligoduplexes  and two structures with a suboptimal-like 5′-ATC oligoduplex (PDB ID 6XUP and 6XU0). Although interactions with 5′-ATT oligoduplexes may not directly translate to possible interactions with 5′-AUU RNA guides, where AfAgo might adopt a slightly different conformation and potentially interact with the RNA guide less strongly, our structural data suggest that AfAgo employs base-specific readout of the terminal nucleotides of the bound guide and target strands Figs. . This in34 (bases at other positions of these structures differ and therefore can not be directly compared). This similarity of base-specific contacts observed with gRNA34 and DNA [this work] indicates that the terminus of the gDNA/tDNA duplex used in our study may provide an adequate mimic for the optimal gRNA/tDNA heteroduplex. Similar disruption of the equivalent base pair was also observed in the AfAgo structures with non-optimal 5′-terminal nucleotides (PDB ID 2W4235 and 2BGG32). In this case, the flipped gT/U1 base in the 5′-end binding pocket is unable to form adenine-specific contacts observed in our crystal structures, including H-bonds with gA1 base made by Asn119 main chain N, Thr120 OH and water-mediated H-bond between 124 OH group and N7 atom of gA1  and the complementary target strand thymine tT1’, which is disrupted, and the bases are placed into separate protein pockets (Extended Data Table 29), or to the target strand39. Another unique feature of AfAgo is that it is a homodimeric protein that can bring together two copies of the guide-target duplex40. In crystal structures presented in this study we observed the same dimerization mode , raising further questions regarding possible AfAgo functions in vivo. To understand its role in bacterial cells, we are currently performing further structural and functional studies of AfAgo and its putative partner proteins encoded in the same operon.Specific recognition of both guide and target strand nucleotides distinguishes AfAgo from previously characterized Argonaute proteins, which limit specific recognition of terminal nucleotides either to the guide strand , 500 mM NaCl, 5 mM 2-mercaptoethanol, supplemented with 2 mM phenylmethylsulfonyl fluoride, incubated for 20 min at 50 °C; cell debris was removed by centrifugation at 48,400 × g for 1 h. The supernatant was loaded onto a HiTrap chelating HP column charged with Ni2+  and eluted with a linear gradient (15–500 mM) of imidazole in the lysis buffer. Protein-containing fractions were pooled, diluted to 0.2 M of NaCl with a buffer containing 20 mM Tris–HCl (pH 8.0 at 25 °C), 10% (v/v) glycerol, 5 mM 2-mercaptoethanol and incubated for 1 h at 37 °C with 1 mM EDTA (ethylenediaminetetraacetic acid) and RNase A/T1 (ThermoFisher Scientific) (1:100). Next, the protein solution was centrifuged at 48,400 g for 30 min, the supernatant containing RNA-free AfAgo was loaded onto a HiTrap Heparin HP column  and eluted using a 0.2–1.0 M NaCl gradient. Finally, the protein was run through the HiLoad 16/600 Superdex 200 column  in lysis buffer supplemented with NaCl to 1 M and dialyzed against 20 mM Tris–HCl (pH 8.0 at 25 °C), 500 mM NaCl, 50% (v/v) glycerol. AfAgo with bound RNA was purified as above, omitting RNase treatment.Archaeoglobus fulgidus DSM 4304  was used in this work. AfAgo-bound RNA was purified using phenol isolation from an RNase-untreated AfAgo preparation. RNA sequencing was performed as described in41. The raw reads were first processed by trimming adapter sequences using AdapterRemoval42. Reads then were aligned to the reference genome with BWA-MEM43. After the alignment, only the aligned reads were retrieved from the alignment file using the ‘bam2fastq’ program from the SAMtools toolkit44. The processed reads were analysed using a Unix ‘awk’ filter to extract RNA sequences and a Perl program that counted the occurrence of each RNA base in the first 50 positions of the reads. The weblogo45 program was used to plot these nucleotide frequencies. The most frequently occurring nucleotide letter in the first position was A (86.2%) and in the second position was U (84.6%); these positions were also among the most reliable positions in all reads.The pETDuet-1 plasmid with the cloned hypothetical protein AfAgo from The raw RNA reads are deposited to the Sequence Read Archive as the file “LT_02.fq.bz2” (sample name “LT_02”) under the BioProject accession number PRJNA763829.32P-labelled nucleic acids, also pre-diluted to a 2 × final concentration in the same buffer. Nucleic acids used for binding studies were MZ-1480 and MZ-1698-1708 for ssRNA and MZ-1447 for ssDNA. Duplexes were prepared by annealing MZ-1480 to MZ-1481 for dsRNA, MZ-1447 to MZ-1455 for dsDNA, and MZ-1480 to MZ-1455 for RNA/DNA heteroduplex. Nucleic acids were diluted such that the final NA-protein mixture would contain 1 nM 5′32P-labelled and 4 nM unlabelled 5′P-NA. The binding reaction mixture was incubated for 10 min at room temperature (25 °C) and loaded onto an 8% PAAG gel (29:1 acrylamide/bis-acrylamide) prepared with 40 mM Tris–acetate (pH 8.4 at 25 °C), 1 mM EDTA, 100 mM KAc. Additional experiments with MZ-1480 and MZ-1707 were conducted by incubating the binding reaction mixture for 10 min at 70 °C. Electrophoresis was run at room temperature in all cases. To study the RNA-guided nucleic acid targeting mechanism, AfAgo was pre-mixed with MZ-1480, a 5′P-ssRNA guide, at 0.4:0.8 µM ratio of AfAgo:guide, incubated for 10 min at room temperature and diluted to 2 × final binding reaction concentration in the same buffer as above. Diluted target NAs were added to the reaction mixture at a 1:1 volumetric ratio to a final reaction concentration of 5 nM (1 nM 5′32P + 4 nM 5′P), the mixture was incubated for 10 min at room temperature and analyzed as described above. Target NAs used were MZ-1556 and MZ-1557 as 8- and 4-nucleotide RNA targets, respectively, and MZ-1560 and MZ-1561 as analogous DNA targets, respectively. For the heparin-supplemented reactions, heparin was pre-mixed with the target NAs before adding them to the binding reaction mixture so that the final heparin concentration was 100 ng/µl. Radiolabelled substrates were detected and quantified using a phosphor imager. The results were analysed with OptiQuant and OriginPro software. The Kd was calculated from the following formula:NB unbound substrate, nM; S0 initial substrate concentration, nM; E0 initial protein complex concentration, nM; Kd dissociation constant, A1 nonbinding fraction of substrate, %.Nucleic acid-free AfAgo was diluted to a 2 × final concentration in a binding buffer consisting of 40 mM Tris–acetate (pH 8.4 at 25 °C), 1 mM EDTA, 100 mM potassium acetate (KAc), 0.1 mg/ml bovine serum albumin, 1 mM DTT and 10% (v/v) glycerol, and mixed in different ratios with 5′2. Glycerol was removed using NAP columns  equilibrated with 20 mM Tris–HCl (pH 7.5 at 25 °C), 150 mM NaCl, 5 mM MgCl2 and 2 mM DTT. Complexes were concentrated by ultrafiltration. The concentration of the complexes used in crystallization trials was in the range of 90–120 µM (as monomer). Crystallization experiments were prepared by mixing the protein solution with equal volumes of crystallization buffers : 2 µl of RNase Mix was added to 2 ml of 1.9 mg/ml AfAgo in a storage buffer and incubated for 30 min at ambient temperature. Complexes of AfAgo with DNA were prepared by mixing protein solution in a storage buffer with an equimolar amount of oligoduplex in the presence of 2 mM DTT and 5 mM MgCl46 and by CCP4 software47. The structures were solved by molecular replacement using MOLREP v11.6.0448 with PDB entries 1YTU and 2W42 as models. Structures were refined with REFMAC v5.8.023049 and PHENIX v1.1350 and remodelled using COOT v0.8.9.151. The data collection and refinement statistics are presented in Supplementary Table Four datasets were collected at EMBL P14 and P13 beamlines on the PETRA III ring of DESY synchrotron in Hamburg (Germany. The datasets were processed by XDSSupplementary Information 1.Supplementary Information 2.Supplementary Information 3.


The adenosine deaminase actingon RNA (ADAR)enzymes that catalyzethe conversion of adenosine to inosine in double-stranded (ds)RNAare evolutionarily conserved and are essential for many biologicalfunctions including nervous system function, hematopoiesis, and innateimmunity. Initially it was assumed that the wide-ranging biologicalroles of ADARs are due to inosine in mRNA being read as guanosineby the translational machinery, allowing incomplete RNA editing ina target codon to generate two different proteins from the same primarytranscript. In humans, there are approximately seventy-six positionsthat undergo site-specific editing in tissues at greater than 20%efficiency that result in recoding. Many of these transcripts areexpressed in the central nervous system (CNS) and edited by ADAR2.Exploiting mouse genetic models revealed that transgenic mice lackingthe gene encoding Adar2 die within 3 weeks of birth. Therefore, therole of ADAR2 in generating protein diversity in the nervous systemis clear, but why is ADAR RNA editing activity essential in otherbiological processes, particularly editing mainly involving ADAR1?ADAR1 edits human transcripts having embedded Alu element invertedrepeats (AluIRs), but the link from this activity to innate immunityactivation was elusive. Mice lacking the gene encoding Adar1 are embryonicallylethal, and a major breakthrough was the discovery that the role ofAdar1 in innate immunity is due to its ability to edit such repetitiveelement inverted repeats which have the ability to form dsRNA in transcripts.The presence of inosine prevents activation of the dsRNA sensor melanomadifferentiation-associated protein 5 (Mda5). Thus, inosine helps thecell discriminate self from non-self RNA, acting like a barcode onmRNA. As innate immunity is key to many different biological processes,the basis for this widespread biological role of the ADAR1 enzymebecame evident.Drosophila genetic models to study ADAR functions and how these were crucialin determining the biological functions of the ADAR proteins. Finally,we describe the severe consequences of rare mutations found in thehuman genes encoding ADAR1 and ADAR2.Our group has been studying ADARs from the outsetof research onthese enzymes. In this Account, we give a historical perspective,moving from the initial purification of ADAR1 and ADAR2 and cloningof their encoding genes up to the current research focus in the fieldand what questions still remain to be addressed. We discuss the characterizationsof the proteins, their localizations, posttranslational modifications,and dimerization, and how all of these affect their biological activities.Another aspect we explore is the use of mouse and  GalloA.; KeeganL. P.; RingG. M.; O’ConnellM. A.An ADAR that edits transcripts encoding ion channelsubunits functions as a dimer. EMBO J.2003, 22, 3421–343012840004.1Forcatalytic activity, Drosophila Adar protein has to bind to dsRNA asa dimer.HealeB. S.; KeeganL. P.; McGurkL.; MichlewskiG.; BrindleJ.; StantonC. M.; CaceresJ. F.; O’ConnellM. A.Editing independent effects of ADARson the miRNA/siRNA pathways. EMBO J.2009, 28, 3145–315619713932.2ADARenzymes have editing independent functions as they can bind RNA havingdsRNA regions and inhibit other RNA processing events.MannionN. M.; GreenwoodS. M.; YoungR.; CoxS.; BrindleJ.; ReadD.; NellåkerC.; VeselyC.; PontingC. P.; McLaughlinP. J.; JantschM. F.; DorinJ.; AdamsI.R.; ScaddenA. D. J.; ÖhmanM.; KeeganL. P.; O’ConnellM. A.The RNA-Editing Enzyme ADAR1 Controls Innate Immune Responses toRNA. Cell Reports2014, 9, 1482–149425456137.3Adar null mutantin mice can be rescued to allow live birth by combining this mutationwith one in Mavs as this prevents downstream signaling via the RLRpathway.DengP.; KhanA.; JacobsonD.; SambraniN.; McGurkL.; LiX.; JayasreeA.; HejatkoJ.; Shohat-OphirG.; O’ConnellM.A.; LiJ.B.; KeeganL.P.DrosophilaAdar RNA editing-dependentand -independent effects are required for brain and innate immunefunctions in . Nat. Commun.2020, 11, 158032221286.4DespiteDrosphila having a very divergent innate immune pathway, the roleof Adar in innate immunity is evolutionarily conserved.5 Most RNA modifications occur in tRNAs; however,recent excitement in this research field has been generated by theoccurrence of RNA modifications in mRNA. Deamination of adenosineto inosine is among the most abundant modifications in mRNA with overa hundred million sites in the human genome.6 The ADAR enzymes that catalyze adenosine deamination in dsRNA recognizethe A-form structure and have little sequence specificity, with basepreferences limited to the edited adenosine and the bases on eitherside of it. Inosine in tRNA anticodons is generated by the adenosinedeaminase acting on tRNA (ADAT) enzymes, whose catalytic domains aresmaller than those of ADARs but with similar deaminase active sites.7With the advent of newtechnologies, the world of RNA modificationshas been gradually revealed. Currently, there are approximately 170different RNA modifications known in RNA in the three domains of life.N6-methyladenosine(m6A) modification, there are no epigenetic erasers forinosine; also,there are no particular classes of readers that bind to it and performa particular function such as increasing or decreasing the stabilityof inosine containing mRNA. Instead, inosine itself within dsRNA isa signal for the innate immune sensors that the RNA is “self”and to not initiate an immune response ; it cou6A modificationis how it is identified. m6A requires identification andimmunoprecipitation first by m6A antibodies followed byRNA sequencing, miCLIP or meRIP . This c see ref ). IntrigIn this review, we will focus on the contributionour group hasmade to the field of RNA editing over the past 30 years.Xenopus oocytes, a strand-separating apparent unwinding activity was detected.15 Further investigation showed that this activity did not unwind dsRNAbut instead converted adenosine to inosine so that it no longer base-pairedwell with uracil, allowing edited dsRNA strands to separate.16 When the initial discovery of this modificationwas made, it was noted that in some viral infections there was a conversionof adenosine to guanosine in the sequences of some cDNAs encodingviral proteins after prolonged virus infection.17 This is the hallmark of RNA editing, and thus, it was presumedthat the same editing activity was responsible. Therefore, the questbegan to purify the enzyme responsible and to clone the encoding gene.20When performing experiments with antisense RNA in E. coli to useas an antigen for injection into rabbits to generate polyclonal antibodies.These antibodies were tested for their capacity to inhibit the enzymaticactivity that was used for the purification of the protein; it wasonly at this stage that one knew if one was successful in purifyingthe original enzyme and cloning the correct gene. This scheme wasused to purify and clone mammalian ADAR1.21 There are two isoforms of the protein, a predominantly nuclear isoform(p110) and a cytoplasmic interferon (IFN) inducible isoform that hasan extended amino-terminus (p150).22As there was very little cDNA sequence data available in theearly1990s, one first had to purify the enzymatic activity to homogeneityfrom abundant inexpensive sources such as calf thymus and then scaleup the purification to generate a sufficient amount of protein sothat peptide sequences could be obtained by mass spectrometry 1. RedundGRIA2 Q/R site, where a glutamine residue is converted intoan arginine in the mammalian GRIA2 transcript encodingthe glutamate 2 (GluA2) subunit of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionicacid (AMPA) receptors.23 RNA editing isessential at this position as it regulates the calcium permeabilityof AMPA receptors; heterotetrameric channels containing a GluA2 subunitwith glutamine in the pore are permeable to calcium, whereas thosewith arginine are not. This position is edited to almost 100% andwhen it does not occur, there is an influx of calcium resulting inseizures and neuronal cell death.The first RNA editing site identified was the critical GRIA2 Q/R position,and when we did not observe it withpurified ADAR1, we assumed that a specificity factor was lacking thatbound to ADAR1. In an attempt to characterize this specificity factor,a sizing column was used to estimate the molecular weight of the complexrequired to edit the GRIA2 Q/R editing site. Surprisingly,the adenosine deaminase activity that edited the Q/R site had a smallermolecular weight than ADAR1, and thus there was another RNA editingenzyme: ADAR2.24It was presumed that ADAR1would catalyze the editing at the 25 We used HeLa nuclearextracts rather than calf thymus as we were aware that the secondADAR activity was enriched in it.24 Whereaspurified ADAR1 was stable during purification and subsequent studieson the enzymatic activity, ADAR2 was extremely labile and very sensitiveto freeze–thawing. Subsequently the ADARB1 gene encoding ADAR2 was cloned, and it was demonstrated that itis responsible for editing the Q/R site in the GRIA2 transcript;27 the recombinant ADAR2 no longershowed the lability of endogenous ADAR2, which might be due to posttranslationalmodifications present only on the endogenous protein.A scheme similarto that described above was used for ADAR2. WhereasADAR1 was purified from 7 kg of calf thymus, ADAR2 was purified from14.67 g of HeLa nuclear extract, which is the equivalent of approximately1500 L of HeLa cells.28 Therefore, we reasonedthat perhaps the ADAR enzymes formed dimers similar to other membersof their family of cytidine deaminases (CDAs) and ADATs. We presumedthat this stable binding was required for ADAR deamination activityat selected sites and that the ADAR enzymes scanned dsRNA for thesespecific sites. This turned out to be true as we demonstrated that Drosophila Adar protein indeed requires dimerization ondsRNA for enzymatic activity.1 This resultwas in contrast to publications from two other groups, one statingthat ADAR2 dsRNA binding domains (dsRBDs) interact and that ADAR2did not form dimers.29 The other publicationstated that ADAR proteins formed dimers but does not require bindingto dsRNA for their formation.30 This controversysurrounding ADAR dimerization was finally resolved by the structureof an ADAR2–dsRNA cocrystal having a truncated ADAR2 composedof the deaminase domain and dsRBDII binding to an edited dsRNA substrate.Indeed, ADAR2 forms dimers on dsRNA, and surprisingly, they are asymmetric;one deaminase domain uses its active site and dsRNA-binding face tobind to the back of another deaminase domain, which performs the editing.31 This was in contrast to our findings that Drosophila Adar,1 which is anorthologue of ADAR2, requires its amino terminus for dimer formation.As currently there is no structure for the full length ADAR2 protein,it is possible that the amino terminus and perhaps dsRBD1 are alsoinvolved in dimerization. Another possibility is that the result isan artifact of the yeast two-hybrid system used to demonstrate theinvolvement of the amino terminus of Drosophila Adarin dimerization.A key question was how ADAR enzymes recognizetheir target transcripts.It was demonstrated that ADAR2 had stable binding at edited siteswhereas only transient binding occurred at nonselective, promiscuousediting sites.32 When ADAR1p150 was expressed from transfectedplasmids in cells, it generated both p110 and p150 isoforms due tothe use of alternative starting methionines. When the M296 AUG forp110 was mutated, the ribosome scanned downstream to the next in-framemethionine at M337.As rabbit polyclonal antibodies were generatedin the process ofcloning the genes encoding ADAR1 and ADAR2, they were used to characterizethe localizations of both endogenous and overexpressed ADAR proteins.22 whereas ADAR2is only present in the nucleus.32 However,when ADAR1 p110 or ADAR2 is overexpressed then both nuclear proteinsare in constant flux in and out of the nucleolus.33 When a plasmid encoding an editing-competent substrate was transfectedinto the cells, then these ADAR proteins relocalized from the nucleolusto a nuclear site where the substrate was being transcribed; thisdid not occur if the plasmid encoded an RNA that did not form dsRNA.Thus, the nucleolus stores excess ADAR proteins, which is not surprising,as the nucleolus contains high levels of Alu RNAs.34 The ADAR proteins can relocalize from the nucleolus andbe recruited to a site for RNA editing when required.As previously reported, ADAR1 p110 is mainlynuclear and ADAR1p150 is mainly cytoplasmic,35 ,on lysine 41835 2. This m37 Pin1 binds to a consensussequence (which is a phosphorylated serine/threonine residue precedinga proline) and catalyzes the cis/trans isomerization of the peptidebond at proline. Pin1 binds to the amino-terminal region of ADAR2and in its absence ADAR2 mis-localizes to the cytoplasm lacking Adar (the gene encoding mouse Adar1), ADAR2Δ4–72 is active in the cytoplasm and compensates forthe lack of the mouse Adar1 protein.3 ADAR2Δ4–72 is stable in the cytoplasm as it has only one copyof the PPxY motif and two are required for its degradation by WWP2.38ADAR2 is regulated by the phosphorylation-dependentpeptidyl-prolylcis/trans isomerase Pin1 (peptidyl-prolyl isomerase NIMA interactingprotein 1).ytoplasm 2. There 40 as are the two ADAD proteins(adenosine deaminase domain containing proteins 1 and 2) that areexpressed in the testis and are homologous to ADAR proteins.41 So, in mammals, there are two active enzymesand three inactive paralogues that are all dsRNA binding proteinsin this extended protein family.The ADAR proteinsare dsRNA binding proteins in addition to beingRNA editing enzymes. Therefore, ADARs could have biological functionsthat are independent of RNA editing. This hypothesis was supportedby the observation that ADAR3 that is expressed in the brain is enzymaticallyinactivemiR-376 cluster in vitro,2 as it was previously demonstrated that RNA editing by ADARscould retarget miRNAs of this cluster.42 We demonstrated that inactive ADAR2 could bind pri-mir-376a2 and inhibit Drosha cleavage. In the Drosophila model,the expression of inactive human ADAR1 also inhibited the siRNA pathway.Thus, by binding to dsRNA, the ADAR proteins can antagonize otherpathways, and this is independent of catalytic activity. This wasthe first demonstration of an editing-independent function of ADARproteins.We chose to study the propertiesof ADAR proteins that are importantfor micro-RNA (miRNA) processing; specifically, processing of the Adar are embryonic lethaland do not survive beyond E12.5.44 They displayan overproduction of IFN, liver disintegration, and loss of hematopoieticcells, as well as widespread apoptosis. For more than ten years, manygroups sought Adar1-edited targets to rescue this lethal phenotype,as they presumed that it might be rescued by the edited isoform ofits key target transcript. This would be similar to the rescue ofthe Adarb1 (gene name for Adar2) by knocking-in theedited isoform (R) of Gria2,45 implying that in a mouse model the most important target of RNAediting by Adar2 is the Gria2 transcript at the Q/Rsite.Mice that arenull for Adar null mice. Wetook a genetic approach and crossed Adar null micewith mouse mutants lacking the mitochondrial antiviral-signaling protein(Mavs), which is downstream of two antiviral receptors for dsRNA,retinoic acid-inducible gene I (Rig-I) and melanoma differentiation-associatedprotein 5 (Mda5). The mutation removing Mavs blocks all downstreamsignaling for IFN induction and the Adar Mavs doublemutant mice survive to live birth3 46 double mutant,demonstrating that Mda5 is the main dsRNA receptor that binds uneditedRNA, and activating the downstream IFN response. Mouse embryos expressinginactive Adar1 E912A live about 2 days longer than Adar null mouse embryos, and when they are combined with Ifih1, they have a normal lifespan.46 Thisvalidated in vivo what we had previously reported,that ADARs have functions that are independent of RNA editing.2However, no transcript or miRNA was identified in whicheditingby Adar1 was essential for mouse embryonic development. ADAR1 editstranscripts encoding short interspersed nuclear element (SINE) invertedrepeats, such as AluIRs in humans . When te birth3 3. AnalysAdar Trp53 null MEFs were created,as Adar MEFs could not be cultured, and derivativestable celllines were generated that expressed either ADAR1 p110 or ADAR1 p150,inactive ADAR1 or an ADAR2 N-terminal deletion that localizes to thecytoplasm.3 A robust immune response waselicited in Adar Trp53 null MEFs by serum starvation,and derivative stable cell lines expressing active ADAR1 or cytoplasmicADAR2 effectively suppressed the ISG inductions in this immune response,whereas enzymatically inactive ADAR1 was the least effective, thoughthere was some effect. This result demonstrates that cytoplasmic ADAR2is capable of reducing the innate immune response and that a significantreduction by ADAR1 requires editing activity.Adar Trp53 MEFsby transfecting them with an in vitro transcribedRNA3 which gives an immune response likelydue to incomplete capping of the RNA.47 This immune induction was reduced by transfecting into the cellsdouble-stranded oligos containing four inosine–uracil pairsin the middle. This demonstrates that the cell uses inosine in dsRNAto discriminate self from non-self RNA and to turn off innate immunity.It is possible that inosine dsRNA acts directly as an inhibitor ofMDA5, preventing its oligomerization and activation despite the presenceof other immunogenic dsRNAs in the cell. This hypothesis is contraryto the idea that RNA editing destabilizes and unwinds dsRNA in AluIRs;however, it has been demonstrated that RNA editing can stabilize dsRNAstructures as inosine improves base paring (I:C) at many editing sites.48 Perhaps in the future, dsRNA oligos containinginosine or other modified nucleosides could be used to reduce chronicinflammation.An immune responsewas induced in Adar gene in Drosophila,49 and it is an orthologue of human ADARB1 (ADAR2).50 Mutant flieslacking Adar activity are obtained in reduced numbers from crossesbecause some of the mutants die as larvae, Adar mutantflies have locomotion defects and ataxia and suffer from age-relatedneurodegeneration49 57 near the deaminase active site. Most editing sites have editingsite complementary sequences (ECSs) in the next downstream intronto form the editing substrate; however, the dsRNA required for RNAediting at the Adar S/G site ispresent within the coding exon so that it does not have to be editedco-transcriptionally before splicing.58 The level of this Adar S/G self-editingis low in embryos and rises to 40% in adult flies. The edited AdarG isoform is less active than the genome-encoded Adar S isoform, both in vivo and in vitro. Therefore, by regulatingalternative splicing as well as by self-editing of the Adar transcript, Drosophila can generate different isoformsof proteins expressed in the nervous system that change enzymaticactivity during development.Adar edits over 1300 specific positions, includingabout a thousandrecoding sites, in approximately six hundred transcripts in 57 An unedited, genome-encoded Adar S isoform inthe 3/4 splice isoform cDNA was generated in which the serine codonis changed to remove the adenosine that can be edited. Gal4/UAS-driven Adar 3/4 S overexpression is lethal in embryosand larvae, so it was placed under the control of Gal80ts and a genetic screen was performed to identify suppressors of ADARoverexpression (Adar OE) lethality at higher temperatures where theGal80ts is inactive and Gal4 drives Adar 3/4 S overexpression.59 Onesuppressor identified was the resistance to dieldrin (Rdl) gene, which encodes an inhibitory GABA-gated chloride channel.Electrophysiological examination of larval motor neuron axons showedthat Adar5G1 loss offunction mutants have increased excitability, whereas Adar OE motorneuron axons show decreased excitability which is corrected by halvingthe dosage of the Rdl gene under screening conditions.Overexpressing the active genomeencoded Adar S isoform prematurelyand at high levels in embryos and larvae is lethal as there is anincrease of editing in embryos to levels more like those in adults.Adar mutant Drosophila rescues all Adar mutant phenotypes tested and editsthe range of Drosophila editing sites similarly to Drosophila Adar.50 Human ADAR1expressed in Adar mutant Drosophila also edits many, but not all, of the fly editing sites but the orderof site preference is different and ADAR1 does not rescue Adar mutant phenotypes. ADAR1 p150 and p110 are both toxicin Drosophila and affect RNAi and probably also miRNAprocessing.2Human ADAR2 expressed in AdarE374A mutant was generatedby CRISPR/Cas9.4 This AdarE374A mutanthas a similar phenotype as the Adar null mutant,implying that Adar editing activity is required for locomotion andprevention of age-related neurodegeneration. However, when the inactiveAdar E374A isoform was expressed from a UAS-AdarE374A construct to approximately four timesthe normal physiological level, it rescued the neurodegenerative phenotypebut not locomotion. This again is evidence that Adar protein has editing-independenteffects.2An inactive Drosophila, despite the Drosophila enzyme being the ortholog of ADAR2 and not ADAR1.4 This suggests that the one Drosophila proteinhas characteristics of both mammalian ADAR1 and ADAR2 and may editwider sets of substrates, some of which affect CNS while others affectimmune function. Innate immunity in Drosophila isvery different from that in mammals as it lacks IFN and the RIG-Ilike receptor (RLR) pathway; however, it is significant that the roleof ADAR in immunity is evolutionarily conserved. The innate immuneinduction in Adar mutant Drosophila is suppressed by silencing Dicer-2, which is a dsRNA sensor in Drosophila that has a similar helicase domain to MDA5.Surprisingly, the lossof Adar activity also leads to immune inductionin ADAR cancause Aicardi-Goutièressyndrome (AGS6);60ADAR was the sixth gene identified in which human mutations cause thissyndrome , an autosomal dominant disorder withmilder IFN induction and a childhood onset of hypo- and hyperpigmentedmacules on the extremities,62 which hasbeen reported mainly in Japan and China. The mutations causing DSHare frameshifts and translation stops, leading to truncated ADAR1proteins; however, DSH1 symptoms arise as a dominant phenotype andmay be due to haploinsufficiency in human ADAR heterozygotes.Mutationsin ADARB1 (gene encoding ADAR2).64 These patients show severe developmental delay, microcephaly, andintellectual disability, and some have intractable early infantile-onsetseizures  antibodies69 as reduced RNA editingactivates the RLR pathway and IFN response thus overcoming immunotherapyresistance. However, there are still many open questions.ADAR1’s role in innate immunity isnow well established,and ADAR1 has also been shown to be an important therapeutic targetin cancer treatment, in particular in those types of cancer that havean increased ISG transcript signature.Adar mutants.ADAR1 edits AluIRs in transcripts; however, the level of this editingis low, often just one percent;6 therefore99 percent of adenosines remain unedited, so it is unclear how thislow level of editing can prevent activation of the innate immune response.Two scenarios could possibly explain this; highly duplex transcriptsexist that have hyperediting, which is highly efficient editing ofmultiple adenosines in a short region to prevent it from being immunogenic.Hyperedited sites would likely be species-specific and are difficultto find in sequence data, and these have not yet been identified.Another possibility is that inosine in dsRNA prevents the activationof MDA5, locking it in an off position so that only a few inosinecontaining transcripts are required. Another major question is whatare the editing independent functions of ADARs?Onepuzzling question is whether there are specific unedited transcriptsthat trigger the innate immune response in ADAR1 has acentral and pivotal role in innate immunity. As ADAR1controls the level of unedited cellular dsRNA, it regulates the homeostasiswithin the cell. Lowered levels of edited dsRNA will trigger an innateimmune response, whereas high levels of edited dsRNA turn it off.Therefore, if we can regulate the level of RNA editing by ADAR1, wecan regulate particular immune responses, increase them to fight aninfection, and decrease them during chronic inflammation. This couldalso be game changing in relation to infectious diseases. Harvestingthe therapeutic potential of these “oddball” enzymeswill likely have major beneficial consequences.


Awareness of RNA-based therapies has increased after the widespread adoption of mRNA vaccines against SARS-CoV-2 during the COVID-19 pandemic. These mRNA vaccines had a significant impact on reducing lung disease and mortality. They highlighted the potential for rapid development of RNA-based therapies and advances in nanoparticle delivery systems. Along with the rapid advancement in RNA biology, including the description of noncoding RNAs as major products of the genome, this success presents an opportunity to highlight the potential of RNA as a therapeutic modality. Here, we review the expanding compendium of RNA-based therapies, their mechanisms of action and examples of application in the lung. The airways provide a convenient conduit for drug delivery to the lungs with decreased systemic exposure. This review will also describe other delivery methods, including local delivery to the pleura and delivery vehicles that can target the lung after systemic administration, each providing access options that are advantageous for a specific application. We present clinical trials of RNA-based therapy in lung disease and potential areas for future directions. This review aims to provide an overview that will bring together researchers and clinicians to advance this burgeoning field. The COVID-19 pandemic has publicized the value of RNA-based therapies because of the rapid development and widespread use of mRNA vaccines . HoweverA major attraction of RNA-based therapies is that the primary nucleotide sequence forms the basis of the therapeutic effect. This principle has been best demonstrated by the development of COVID-19 mRNA vaccines created by Moderna and Acuitas/BioNTech/Pfizer, which took months rather than the years typically required for drug development . Not onlSince early descriptions of RNA in the late 1950s , the fiein vivo injections of mRNA (The field of RNA therapeutics dates back as far as 1978 when Stephenson and Zamecnik designed an antisense oligonucleotide (ASO) that utilized RNA base-pairing to inhibit viral replication of the Rous sarcoma virus . By 1990 of mRNA . Further of mRNA . In 1998 of mRNA . Subsequ of mRNA . Despite of mRNA . These t of mRNA .etc.) can be delivered therapeutically. In practice, current RNA-therapeutics fall into three broad categories: 1) RNA designed to inhibit target gene expression, often described as antisense RNA in reference to their mechanism of action via base-pairing  RNA -pairing , and 3) -pairing . In this-pairing .Most antisense therapies consist of short RNA molecules, often <30 nucleotides, termed oligonucleotides, and include short interfering RNAs (siRNAs), microRNAs (miRNAs), and antisense oligonucleotides (ASOs) . AntisenCurrent antisense therapies typically consist of short nucleic acid molecules less than 30 nucleotides in length and can be produced through solid phase synthesis, which is performed in a base-by-base fashion . Thus, tRNAi leverages the endogenous RNAi pathway by which mRNA cleavage and degradation is initiated by the RNA-induced silencing complex (RISC) . EndogenmiRNAs are endogenous small, non-coding RNA molecules that play a crucial role in regulating gene expression. There are currently thought to be 2,656 human miRNAs   predicteAlthough the seed sequence is central to miRNA function, other factors also influence which mRNA targets are regulated. Many miRNAs are expressed in a highly tissue-specific fashion and contribute to tissue-specific expression patterns . There iSeveral types of RNA-based therapy can leverage the biology of miRNAs and can either potentiate or inhibit miRNA function. miRNA mimics are synthetic miRNAs that can be used to repress a set of miRNA-regulated genes, whereas antisense oligonucleotides such as antagomiRs or blockmiRs are synthetic oligonucleotides that bind to miRNAs or compete for their target sites on mRNAs respectively . miRNA smiRNA-based therapy has been assessed in non-malignant and malignant lung disease in preclinical studies. miRNAs in the miR-29 family are downregulated in pulmonary fibrosis . MRG-229siRNAs are a class of double-stranded RNA molecules that can silence specific genes by binding to complementary sequences in the mRNA and triggering degradation or inhibition of translation. Endogenous siRNAs exist and differ from miRNAs in that they are not derived from primary miRNA transcripts, and they do not undergo canonical miRNA processing. Rather, they can be derived from long double-stranded RNA (dsRNA) which have a variety of sources and are cleaved by Dicer to form siRNAs . In thissiRNAs are also derived exogenously and are used for gene knockdown in research and RNA-based therapeutics. In contrast to miRNAs, siRNAs typically have 100% sequence complementarity with their target genes . As a reDespite this theoretical single-sequence specificity, siRNA can still have off-target effects . One prosiRNAs are a powerful tool in the knockdown of target RNAs and have been a frequently used tool in the research setting to assess gene function in a variety of biological systems . Like miASOs are a more chemically and mechanistically diverse group of molecules compared to miRNAs and siRNAs, both of which act through the RNAi pathway. ASOs are single-stranded molecules that can alter mRNA expression through a variety of mechanisms, including ribonuclease H (RNAse H) mediated decay, or steric hindrance of splice-sites or translation initiation . DependiThe mechanism of action can be modulated by ASO biochemistry is a determinant of the mechanism of action. RNAs H is an endogenous enzyme that cleaves the RNA strand in an RNA-DNA duplex . Thus, RASOs that act as steric blockers in an RNAse H-independent fashion  consist ASOs that bind to intron-exon junctions in pre-mRNA destabilize splicing sites or displace/recruit splicing factors, and result in the exclusion or inclusion of certain exons . Open reThe FDA has approved several ASOs outside of lung disease, such as Nusinersen and Mipomersen . In non-The most widespread applications of mRNA therapeutics are the mRNA-based vaccines against SARS-CoV2 . mRNAs aWhen introducing genetic information, such as in vaccines or CRISPR-Cas9-based approaches , an advaex vivo with mRNA coding for tumour-associated antigens and administered back to patients to elicit an immune response  are a group of noncoding RNAs that have covalently joined ends and are present in all studied eukaryotic organisms . AnotherAs with vaccines, genetic information traditionally delivered via DNA-based molecules can be delivered via RNA-based therapies. CRISPR-Cas systems were first identified as prokaryotic adaptive immunity that protects against phages and has been widely adopted in research for genome editing . CRISPR-Double-stranded RNA (dsRNA) is a potent activator of innate immune responses that form a natural defence against viral RNA and transposable elements . Thus, tSeveral strategies have been employed to mitigate the issues that can arise and lead to unintended immune stimulation. One is to avoid using sequences with known immunostimulatory motifs, for example, 5′-UGU-3′, 5′-UGUGU-3′ , and 5′-in vivo  and competitively inhibit the function of endogenous miRNAs. In this way, exogenous administration of siRNA or miRNA can lead to the upregulation of endogenous miRNA target mRNAs . Saturatin vivo . Therefoin vivo .RNA is susceptible to degradation by nucleases . SpecifiThe potential versatility of RNA therapies presents great opportunities for disease treatment but requires that the RNA payload is delivered to the appropriate cells. Because of their negative charge, RNA molecules will encounter barriers in crossing cell membranes, and native RNA is susceptible to the ubiquitous ribonucleases (RNases) present in the body . ChemicaThe development of nanotechnology-based delivery strategies is a critical component of effective RNA-based treatments . NanotecThe most established form of nanodelivery agents are lipid-based . Lipid-bBecause systemic delivery of RNA often leads to accumulation in the liver , variousSpecific cell types within the lung including epithelial cells, endothelial cells, and immune cells along with cells outside the lung, such as hematopoietic stem cells can be targeted by specific LNP formulations . A stratin vivo to lung epithelium via inhalation  acid (PLGA) nanoparticles have been used to deliver peptide nucleic acids (PNAs) to correct F508del CFTR mutation ne model . Airway ne model . Furtherne model .Peptide nanoparticles leverage amino acid sequences to impart properties such as structure, charge, solubility, and polarity, which can facilitate interactions with RNA and cellular uptake . KL4 pepin vivo  are released from the net-positively charged hydrogel matrix to the adjacent tissues. The design of the peptide-miRNA nanoparticle, including its surface charge state, size, miRNA encapsulation efficiency, and conformation, are important factors in clathrin-mediated cell entry and endosomal trafficking. This platform has been applied in models of malignant pleural mesothelioma (MPM), an aggressive cancer that arises from the mesothelial surface of the pleural cavities . There iWith progress and promise shown in pre-clinical studies, RNA-based therapies are now being explored in clinical trials across several domains of lung disease: infectious disease, cancer, and airway disease . RNA pay+ and CD8+ T-cell responses  mRNA of the respiratory syncytial virus (RSV). A proof-of-concept randomized, double-blind, placebo-controlled trial compared saline nasal spray with a nasal spray of ALN-RSV01 2 days before and 3 days after experimental RSV infection in 88 healthy subjects and showed a 38% decrease in culture-defined RSV infections. ALN-RSV01 had a similar safety profile to placebo, and the antiviral effect was independent of pre-existing antibodies and intranasal cytokines . RSV infIn thoracic cancers, a range of RNA-based approaches, from antisense therapies to cancer vaccines, have been assessed in clinical trials. Early reports of RNA-based therapy in lung malignancy focused on ASOs . In an ein vivo was proposed as a reason for the lack of efficacy.Other groups have assessed ASOs in small cell lung cancer (SCLC). G3139 (oblimersen) is an ASO directed against Bcl-2 and underwent a pilot clinical trial , and a sThe first clinical experience for miRNA-based therapy in thoracic malignancy was the assessment of mIR-16-based miRNA mimics in malignant pleural mesothelioma (MPM). A Phase I dose escalation open-label trial assessed TargomiRs, a term describing proprietary miRNA mimics based on the miR-16 seed sequence that are encapsulated in a bacterial-derived mini-cell vector targeted to the epidermal growth factor receptor (EGFR) by panitumumab-based antibodies. Of 22 patients who were assessed for response, 1 achieved an objective response to therapy by CT .Cancer immunotherapy is designed to elicit an immune response to tumour antigens. In stage IIIB/IV NSCLC, a phase I/IIa trial assessed the safety of CV9201, an mRNA-based immunotherapy encoding five NSCLC tumour antigens  . This apMost approved RNA-based therapies target monogenic disorders . In the In theory, mRNA coding for CFTR could be helpful with any CFTR mutation. MRT5005, a codon-optimized CFTR mRNA delivered by inhaled lipid nanoparticles, is being evaluated in a randomized, double-blind, placebo-controlled Phase I/II study. Interim analysis of 42 subjects found 14 febrile reactions over 28 days, most of which resolved within 1–2 days and allowed ongoing treatment. FEV1 remained stable, but no beneficial effects on FEV1 were observed .ASOs have also been used to target specific factors contributing to airways inflammation in asthma . Early dThe evolution of chemistry for RNA-based therapeutic payloads and for nanotechnology-based delivery vehicles have advanced the applicability of RNA-based treatments to human disease. By leveraging the ability to selectively target different compartments within the lungs  and specific cell types , RNA therapy offers enhanced precision in targeting lung disease . UniversIn obstructive airway diseases, such as asthma and chronic obstructive pulmonary disease (COPD), treatment delivered by inhalation can enhance local delivery while decreasing systemic exposure . Naked RLung transplant recipients face the challenge of maintaining graft function and preventing rejection. Specific intragraft immune cell populations are of prognostic and likely mechanistic importance in the development of chronic lung allograft dysfunction . Here, aLocal delivery of RNA-based therapy to the lung via inhalation or to the pleural space are attractive options, yet RNA delivered via these routes may not effectively reach the pulmonary endothelium to benefit pulmonary vascular disorders. However, delivery vehicles have been engineered to target lung endothelium, even with systemic delivery . Loss-ofAdditionally, critical care conditions like acute respiratory distress syndrome (ARDS) require comprehensive treatment approaches that address both local lung inflammation and systemic consequences. RNA therapy delivered systemically holds promise in treating ARDS  by targeRecently, the treatment of interstitial lung disease (ILD) has been driven by the recognition of progressive fibrosis as a feature of several ILDs. However, multiple clinical, radiographic, and histologic patterns exist, reflecting distinct pathologic processes . MultiplNoncoding RNAs have emerged as major products of the human genome and are involved in all cellular functions. Many classes of noncoding RNA exist, many of which have specific functions and mechanisms, such as miRNAs. Long noncoding RNAs (lncRNAs) have traditionally been defined as noncoding transcripts of more than 200 nucleotides, though a new consensus statement proposes that they be defined as >500 nt to better distinguish from other noncoding RNAs . NotablyEmerging evidence is defining roles for lncRNAs in lung development, homeostasis and disease. For example, the lncRNA LL18/NANCI contributes to saccule formation/alveolarization in mouse lungs . In hypoIn conclusion, the future of RNA therapy in lung diseases is promising and offers new avenues for effective treatment. The ability to target specific compartments within the lungs, in combination with various RNA payloads, provides versatile options for precise and personalized therapy. We have briefly overviewed various RNA payloads, delivery vehicles and the clinical state of RNA-based therapy in the lung. Few clinical trials have been completed, and vaccines directed at SARS-CoV-2 are the only FDA-approved treatments. Thus, continued research and development in this field are crucial to fully realize the transformative potential of RNA therapy in the management of lung diseases.