5′ and 3′ modifications controlling RNA degradation: from safeguards to executioners

Abstract

RNA degradation is a key process in the regulation of gene expression. In all organisms, RNA degradation participates in controlling coding and non-coding RNA levels in response to developmental and environmental cues. RNA degradation is also crucial for the elimination of defective RNAs. Those defective RNAs are mostly produced by ‘mistakes’ made by the RNA processing machinery during the maturation of functional transcripts from their precursors. The constant control of RNA quality prevents potential deleterious effects caused by the accumulation of aberrant non-coding transcripts or by the translation of defective messenger RNAs (mRNAs). Prokaryotic and eukaryotic organisms are also under the constant threat of attacks from pathogens, mostly viruses, and one common line of defence involves the ribonucleolytic digestion of the invader's RNA. Finally, mutations in components involved in RNA degradation are associated with numerous diseases in humans, and this together with the multiplicity of its roles illustrates the biological importance of RNA degradation. RNA degradation is mostly viewed as a default pathway: any functional RNA (including a successful pathogenic RNA) must be protected from the scavenging RNA degradation machinery. Yet, this protection must be temporary, and it will be overcome at one point because the ultimate fate of any cellular RNA is to be eliminated. This special issue focuses on modifications deposited at the 5′ or the 3′ extremities of RNA, and how these modifications control RNA stability or degradation.

This article is part of the theme issue ‘5′ and 3′ modifications controlling RNA degradation’.

1. Introduction

The expression of genetic information is tightly regulated in all cells. In eukaryotes, this is a very complex process regulated at multiple levels from chromatin structure and transcription initiation, elongation and termination to pre-RNA processing, RNA localization and decay. Additional layers of regulation operate at the translational and post-translational levels.

Transcription produces both non-coding RNAs and messenger RNAs (mRNAs). Eukaryotic mature mRNA molecules are generated through mostly co-transcriptional processing reactions [1]. In the early stage of transcription, the 5′ end of mRNA is modified by a so-called m⁷G cap structure formed by 7-methylguanylate connected to mRNA via an unusual 5′ to 5′ triphosphate linkage [2]. Additional internal chemical modifications of nucleotides occur during mRNA biogenesis, such as the methylation of adenosines at position 6 (m6A) [3]. The exhaustive identification of the nature, extent and functions of all modifications targeting mRNAs currently constitutes a very dynamic and exciting field of investigations [4]. The body of the pre-messenger RNA is also spliced to remove introns [5], which in some organisms constitute the predominant part of pre-mRNA molecules. Finally, 3′-end processing generates a mature 3′ terminus, which is polyadenylated by canonical poly(A) polymerases (ncPAPs) [6]. The only eukaryotic mRNAs whose 3′ end processing does not involve polyadenylation are the replication-dependant histone mRNAs in mammals [7]. Those particular mRNAs contain an RNA stem-loop structure close to the 3′ end of the mature RNA and are processed by a specific mechanism. In addition to producing pre-mRNA, independent transcription units produce precursors of several other functional RNA classes (e.g. pre-rRNA, pre-snRNA, pre-miRNA), as well as pervasive transcripts such as long intergenic non-coding RNAs, many of which play important regulatory roles [8]. Such RNAs are processed by a variety of different mechanisms.

Although the decay of eukaryotic RNAs can be initiated by endoribonucleolytic cleavages, it is mainly carried out by exoribonucleases [9]. Therefore, 5′ and 3′ ends of RNA molecules need to be protected. In the case of non-coding RNAs, this protection is mostly achieved by shielding 5′ and 3′ extremities within the ribonucleoparticle, which is formed by the non-coding RNA and its associated proteins. A diversity of processes can also contribute to the protection of extremities of non-coding RNAs. For instance, 2-O’-methylation of the last 3′ nucleotide stabilizes all small RNAs in plants [10,11] and piRNAs in animals [12]. In the case of mRNA, the 5′ end is blocked by the m⁷G cap structure while the 3′ end contains a poly(A) tail. Nucleotides adjacent to the m⁷G cap can be further methylated to either stabilize transcripts or increase translation of certain mRNAs [13]. The m⁷G cap initially is recognized by the nuclear cap binding complex, which facilitates mRNA export from the nucleus and is later replaced by the essential translation initiation factor, eIF4E. Interestingly, in addition to the classical m⁷G cap, a new type of cap, the nicotinamide adenine dinucleotide (NAD⁺) cap was recently identified in bacteria, yeast and humans [14,15]. In yeast and humans, about 1–5% of mRNAs contain NAD⁺ rather than m⁷G cap. In contrast to m⁷G cap, the NAD⁺ cap cannot support translation in human cells and destabilizes mRNAs through the deNADding activity of the DXO proteins [16]. The discovery of alternative caps and the elucidation of their function are certainly exciting areas of research.

At the other end of the mRNA, the poly(A) tail is bound by poly(A) binding proteins (PABPs), which facilitate mRNA export from the nucleus and enhance protein synthesis through interactions with translation initiation factors [17]. Poly(A) also stabilizes mRNA molecules by preventing exoribonucleolytic decay. Consequently, the deadenylation rate largely determines mRNA half-life. Shortening a poly(A) tail in the cytoplasm to fewer than 15–20 nt destabilizes its interaction with the last PABP [18,19]. Once this last PABP is released, the mRNA becomes translationally inactive and susceptible to degradation, either through the decapping and 5′ to 3′ decay or by 3′ to 5′ decay pathway. However, it is now appreciated that poly(A) tail dynamics is more complex than previously suspected. Deadenylated mRNAs can be uridylated [20–22] or stored in a dormant state to be later re-adenylated to activate protein synthesis [23–26]. Such reactions are mediated by terminal uridylyltransferases (TUTases) and non-canonical poly(A) polymerases. Interestingly, the non-canonical addition of non-templated nucleotides is important for the physiology of not only mRNAs but also other RNA classes such as pre-miRNAs, mature miRNAs or nuclear non-coding RNAs. Such modifications can stabilize RNA molecules or induce their decay. Of note, the destabilization of RNA by nucleotide tailing is a conserved process across organisms and it was identified in Escherichia coli [27]. Although in bacteria, bulk RNA decay is initiated by endonucleolytic events, the ends of RNA molecules also play an important role in controlling RNA fate. Secondary structures may impede the progression of 3′ to 5′ exoribonucleases and polyadenylation accelerates the degradation of RNA decay intermediates facilitating the action of 3′ to 5′ exoribonucleases. Moreover, the triphosphate moiety present at the 5′ end of the primary transcripts restricts endonucleolytic cleavage by the RNase E and its conversion to monophosphate accelerates the decay rates of many, but not all, transcripts [28].

Because of the impact of the ends of RNA molecules on their stability and biological functions, all RNA modifications must be tightly controlled, and complex regulatory networks of protein–protein and protein–RNA interactions are involved in capping/decapping, polyadenylation/deadenylation and uridylation. This field of research is currently expanding, and several key discoveries were made in recent years. This theme issue of Philosophical Transactions B entitled ‘5′ and 3′ modifications controlling RNA degradation’ gathers articles explaining how 5′ and 3′ RNA modifications control the decay of coding and non-coding RNAs. The first three articles focus on the cap structure, followed by seven articles about various aspects of RNA tailing by non-canonical poly(A) polymerases and TUTases.

Charenton et al. [29] describe how the removal of the 5′ end cap structure is regulated, focusing on the mechanistic aspect. Although the elimination of the mRNA m7GpppN cap is a simple chemical reaction catalysed by the Nudix family hydrolase Dcp2, this enzyme has intrinsically very weak activity [30]. Thus, it requires several accessory factors that enhance its activity. In recent years, there has been considerable progress in our understanding of how all these proteins such as Dcp1, Lsm1–7 complexes, Pat1, Edc1–Edc2 and/or Edc3 cooperate with Dcp2 to ensure that removal of the mRNA cap structure is tightly controlled.

Toczydlowska-Socha et al. [31] describe RNA helicase DDX15 as a novel regulator of cap methylation. In contrast to yeast, the vertebrate m7GpppN cap can be post-transcriptionally modified at the first nucleotide by RNA cap1 methyltransferase (CMTr1) [32,33]. Such modification is believed to play a role in antiviral defence [34]. Very little is known about the regulation of CMTr1-mediated cap modification. The authors identified RNA helicase DHX15 as a CMTr1 interactor. They further show that CMTr1 activity is hindered towards RNA substrates with highly structured 5′-termini and DHX15 facilitates methylation of such RNA species. The physiological role of this effect remains to be established, but this article describes the first example of regulated 2'-O-ribose methylation of the mRNA cap structure.

Because of the importance of the cap structure for mRNA stability and protein synthesis, there is a clear need for the development of novel tools for the analysis of mRNA cap metabolism [35]. Bednarek et al. [36] invented several novel biotin-labelled cap analogues modified within the triphosphate bridge to increase their stability in cellular conditions. They are efficiently incorporated into RNA in vitro and can be applied to a variety of different experiments including protein affinity purification, pull-down assays, in vivo visualization, cellular delivery, etc.

The first contribution of the seven articles that focus on the tailing of RNA 3′ ends is by Hajnsdorf and Kaberdin [37], who describe how oligoadenylation facilitates RNA decay in bacteria. Hajnsdorf and Kaberdin present the machinery responsible for the oligoadenylation of bacterial RNAs and their degradation. They focus mostly on the knowledge obtained in E. coli, because the seminal discoveries in this field were made using this model organism. Yet, work performed on evolutionarily distant bacteria is also mentioned. Hajnsdorf and Kaberdin summarize the known RNA targets of oligoadenylation in E. coli to define the impact of oligoadenylation on gene expression.

By reading the following review by Tudek et al. [38], an interesting comparison can be made between the complexity of roles played by oligo/polyadenylation in bacteria versus eukaryotes. Tudek et al. focus on the intricate role of poly(A) tails in controlling RNA fate in the nucleus of budding yeast and human cells. Although polyadenylation of eukaryotic mRNAs is known to promote stability, export from the nucleus and translation, oligoadenylation also operates as an RNA destabilizing tag in eukaryotic nuclei. This RNA destabilizing role of oligoadenylation is therefore conserved across evolution, from bacteria to man, and it actually represents the primordial role of oligoadenylation. Tudek et al. provide a detailed view of the different mechanisms and factors leading to the polyadenylation of eukaryotic RNAs. One could think that the distinction between the processes leading to destabilization or stabilization by polyadenylation is straightforward. On the contrary, Tudek et al. demonstrate that this boundary is becoming increasingly uncertain, with both pathways sharing common factors.

The next review article is by Warkocki et al. [39], who summarizes our current knowledge about all mammalian ncPAPs and TUTases. Previously, 7 ncPAPs and TUTases have been annotated in the human genome. Recently, a new TENT5 (FAM46) family of four members has been identified [40,41]. Thus, the human genome encodes at least 11 ncPAPs and TUTases. Our knowledge about these enzymes is very fragmentary and in many cases there are conflicting reports about their substrate specificity. Nonetheless, it is an exciting field of investigation and several recent reports indicate that they have important, although diverse, physiological roles. For instance, uridylation by TUT7/TUT4 in the cytoplasm induces decay of various RNA species and protects cells against viruses and LINE-1 retrotransposons. Nuclear TUTase (TUT1) is involved in U6 snRNA 3′ end formation [42] and was also suggested to polyadenylate a subset of mRNAs in a phosphatidylinositol bisphosphate-dependent manner [43]. Cytoplasmic polyadenylation by TENT5C stabilizes mRNA and enhances protein synthesis [40]. Nuclear non-canonical poly(A) polymerases TENT4A/B were initially implicated in the induction of exosome-mediated RNA decay, but a recent report suggests that they can also stabilize mRNAs [44]. This is because of TENT4A/B's sightly promiscuous nucleotide specificity, which leads to incorporation of guanine residues inhibiting deadenylation in the cytoplasm.

Zigáčková & Vaňáčová [45] describe in detail our current knowledge on the role of uridylation in RNA metabolism and physiology of the cell. These authors cite the key factors involved in the uridylation and degradation of RNAs, i.e. TUTases and 3′–5′ exoribonucleases recognizing uridylated RNAs. Zigáčková & Vaňáčová review all known types of RNA substrates uridylated by TUTases. Those RNA substrates include nuclear and cytosolic RNAs, coding and non-coding, and also viral RNAs. The different roles of RNA uridylation are then explained. This description is not restricted to various degradative mechanisms but also includes the role of uridylation in promoting maturation, and possibly controlling localization or translation.

De Almeida, Scheer et al. [46] focus on RNA uridylation in plants. The uridylation of various RNA substrates has been investigated in plants, from small RNAs and other non-coding RNAs, to mRNAs. Most of the knowledge on plant RNA uridylation was obtained using the flowering plant Arabidopsis thaliana, but seminal work was also done in the green algae Chlamydomonas reinhardtii. Both forward and reverse genetic strategies led to the identification of two distinctive TUTases in these model plant species: MUT68/HESO1 and URT1. De Almeida, Scheer et al. report a detailed evolutionary history of both TUTases in Archaeplastida (i.e. all plants). HESO1 and URT1 homologues each form a monophyletic group, and the presence of both TUTases has been maintained in almost all species of the green lineage, indicating specific and critical functions. De Almeida, Scheer et al. then recapitulate the diverse molecular functions of both TUTases during RNA degradation processes in plants and conclude with key points of future research.

The article by Meaux and co-authors [47] combines a review of our current knowledge on how uridylation induces decay of replication-dependent histone mRNAs in mammals, with new information on two molecular players involved in this process: the serine/threonine protein kinase Smg1 and the RNA helicase Upf1. Replication-dependent histone mRNAs in mammals are the only mRNAs that are not polyadenylated. Instead of a poly(A) tail, they end with a short stem-loop structure. The levels of those mRNAs are tightly regulated, with a massive degradation at the end of the S-phase or in response to inhibition of DNA synthesis. This degradation involves uridylation and, besides TUTases, key factors like the stem-loop binding protein (SLBP) or the exoribonuclease, 3′hExo (ERI1). Meaux et al. bring new information on the involvement of Upf1 domains in this process and report that the kinase Smg1 is required for histone mRNA degradation when DNA replication is inhibited. In addition, they show by using a strategy based on high-throughput sequencing, that the pathway of rapid histone mRNA degradation is identical at the end of S-phase or when DNA replication is inhibited in S-phase.

The final article in this issue by Ugolini and Halic [48] gives an overview of the mechanisms of RNA-based defence against transposable elements (TEs). TEs are major drivers in the evolution and diversity of genomes. New mutations caused by TEs may turn out to be beneficial in terms of gene regulation or RNA processing. Yet, organisms must evolve defence mechanisms to restrict transposition. One potent mechanism that organisms use to discriminate their own DNA (self) from foreign transposable DNA (non-self) is based on small RNA pathways. In addition to reviewing the processes that restrict transposition in various organisms, Ugolini and Halic point out that primal small RNAs must be degraded to avoid uncontrolled silencing. This degradation frequently involves the tailing of small RNAs and those processes are detailed.

Altogether, these reviews and experimental reports illustrate the functional diversity of 5′ and 3′ modifications in controlling RNA stability and degradation. One important conclusion that must be drawn from this compilation is that the list of modifications detailed in this theme issue is not comprehensive. We are likely far from having an exhaustive view of the types of modifications decorating the 5′ and 3′ extremities of RNA, in addition to internal nucleotide modifications. Innovative technologies will keep fuelling unexpected discoveries in years to come, and we must be aware that we are just beginning to fully appreciate the real impact of terminal RNA modifications in gene regulation.

Biographies

Authors' profile

Dominique Gagliardi is head of the ‘RNA degradation’ group at the Institut de Biologie Moléculaire des Plantes (IBMP), a CNRS-driven research institute dedicated to plant biology and located at the University of Strasbourg, France. His group focuses on the identification and characterization of novel actors of RNA degradation pathways in plants, to determine their impact on genome expression, on development and upon RNA virus infections. He and his colleagues are particularly interested in understanding how 3′ modifications by adenylation or uridylation control RNA's fate.

Andrzej Dziembowski is a Professor of Molecular Biology at the Institute of Biochemistry and Biophysics, Polish Academy of Sciences and at the Faculty of Biology, University of Warsaw. His research focuses on the regulation of gene expression at the post-transcriptional level. He is particularly interested in the mechanisms of RNA decay, and the role of enzymes modifying RNA 3′ ends in the determination of mRNA stability and translation potential. In his research, he uses various model systems and advanced biochemical, transcriptomic and proteomic approaches.

Data accessibility

This article has no additional data.

Competing interests

We have no competing interests.

Funding

D.G. acknowledges support from the Centre National de la Recherche Scientifique (CNRS) and research grants from the Agence Nationale de Recherche (ANR) as part of the ‘Investments for the Future’ program in the frame of the LABEX ANR-10-LABX- 0036_NETRNA and ANR-15-CE12-0008-01. A.D. acknowledges support from the ERC StG 309419 and NCN UMO-2013/10/M/NZ4/00299 grant.