Post-transcriptional Coordination Of

492 VOLUME 15 NUMBER 6 JUNE 2014 NATURE IMMUNOLOGY For many years, the post-transcriptional regulation (PTR) of immuno- logical mRNAs was studied in isolated, transcript-restricted settings and thus it was assumed to be a process lacking the coordinative ability of immunological transcription. The identification of non- coding RNAs such as microRNAs (miRNAs) and long noncoding RNAs as specific entities able to concurrently regulate several mRNAs proved to som
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  492 VOLUME 15 NUMBER 6 JUNE 2014 NATURE IMMUNOLOGY For many years, the post-transcriptional regulation (PTR) of immuno-logical mRNAs was studied in isolated, transcript-restricted settings and thus it was assumed to be a process lacking the coordinative ability of immunological transcription. The identification of non-coding RNAs such as microRNAs (miRNAs) and long noncoding RNAs as specific entities able to concurrently regulate several mRNAs proved to some extent that such viewpoints were unwarranted. We propose here that immunologists should additionally consider three fundamental features of PTR that are often neglected. First, most PTR events involve the interaction of RNA with RNA-binding proteins (RBPs) that are abundant in mammalian cells 1 . Second, RBPs bear many structural modules (beyond their RNA-recognition domains) that facilitate protein-protein interactions and catalytic events; thus, they are integrated into many intracellular events 2 . Third, RBPs, mRNAs and, in specific cases, noncoding RNAs assemble in ribonucleoprotein (RNP) complexes, the actual functional units of PTR. The composition of such complexes is in constant flux, with some RBPs gaining or losing access during the subcellular journey of an RNA and others remaining bound to their RNA target through-out its life 3 . The potential importance of changes in RNP complexes emerged through data obtained with technological platforms used to assess RBP-RNA interactions at the nucleotide level 4 . Analysis of such data suggests that the composition of RNP complexes can depend on the ability of constituent RBPs to ‘read’ regulatory RNA sequences or structures (generally called ‘elements’; Box 1 ) present in the untranslated termini or body of each RNA. The concomitant presence of such elements in mRNAs that encode functionally related factors seems to allow their coordinated regulation and use 5 . Moreover, the ever-expanding list of mutant mouse lines with modifi-cations in RBP-encoding genes ( Table 1 ) demonstrates the importance of RBPs in immunological homeostasis and connects the functions of RBPs to signal-induced immunological expression programs. Such connections suggest that PTR has not evolved solely as a ‘proofing’ extension of immunological transcription but instead has evolved as an additional determinant of immunological reactions able to alter the srcinal transcriptomic definition of types of cells of the immune system. Here we discuss key examples of post-transcriptional events in such cells and highlight the importance of changes in RNP com-plexes during immunological physiology and pathology. Mechanics of PTR Genomic studies have provided evidence of the execution of nuclear and cytoplasmic PTR events in cells of the immune system that alter the maturation, destruction and protein synthesis of mRNA 6–11 . Such events are executed by core post-transcriptional machinery that can be engaged differently by regulatory RNP complexes. The sum of PTR events that occur in cells of the immune system is very broad; here we focus on four core post-transcriptional processes to exem-plify the regulatory functions of RBPs: splicing, editing, decay and translation ( Fig. 1a ).The first such process involves the excision of intronic sequences from pre-mRNA; in eukaryotes this can occur concurrently with transcription 6,9  and after the addition of a 5 ′  7-methylguanosine cap that renders mRNA competent for cap-dependent translation. In its canonical form, splicing requires recognition of ‘intronic boundaries’ on the pre-mRNA by the spliceosome, a macromolecular RNP nucle-ated by small nuclear RNP complexes of the ‘U’ class. The assembly of such complexes on individual introns results in their removal and exon joining. In eukaryotes, protein production and function is diver-sified further via alternative splicing; i.e., the selective inclusion or Division of Immunology, Biomedical Sciences Research Center “Alexander Fleming”, Vari, Greece. Correspondence should be addressed to D.L.K. ( 18 February; accepted 1 April; published online 19 May 2014; doi:10.1038/ni.2884 Post-transcriptional coordination of immunological responses by RNA-binding proteins Panagiota Kafasla, Antonis Skliris & Dimitris L Kontoyiannis Immunological reactions are propelled by ever-changing signals that alter the translational ability of the RNA in the cells involved. Such alterations are considered to be consequential modifications in the transcriptomic decoding of the genetic blueprint. The identification of RNA-binding protein (RBP) assemblies engaged in the coordinative regulation of state-specific RNAs indicates alternative and exclusive means for determining the activation, plasticity and tolerance of cells of the immune system. Here we review current knowledge about RBP-regulated post-transcriptional events involved in the reactivity of cells of the immune system and the importance of their alteration during chronic inflammatory pathology and autoimmunity. POST-TRANSCRIPTIONAL AND POST-TRANSLATIONAL CONTROL OF IMMUNITY  REVIEW  NATURE IMMUNOLOGY   VOLUME 15 NUMBER 6 JUNE 2014 493 exclusion of introns, exons and regulatory regions in the final mRNA product 12 . This can occur in response to peripheral signals and is usually orchestrated by auxiliary and regulatory RBPs. Examples include the many heterogeneous nuclear RNPs (hnRNPs; hnRNPA–hnRNPU), which usually bind splicing silencers ( Box 1 ), and the serine-arginine–rich (SR) RBPs, which usually recognize splicing-enhancer motifs 12,13  ( Fig. 1c ).A second nuclear process that can diversify immunological mRNAs is RNA editing; this acts to enforce nucleotide conversions that alter protein coding or affect RNA structure. In mammals, the most com-mon form of editing is the deamination of adenine to produce inosine; this is facilitated by members of the ADAR (‘adenosine deaminases acting on RNA’) family of double-stranded RNA (dsRNA)-binding enzymes 10  ( Fig. 1b ).As eukaryotic mRNA matures and becomes stable with the addition of the poly(A) tail, nuclear RBPs with access to additional RNA ele-ments remain bound for further use 5,12 . Those multifunctional RBPs have individual functions but also act as scaffolds ( Fig. 1a ) and nucle-ate the assembly of novel regulatory RNP entities; thus, they allow interactions with cytoskeletal and signal-transduction machinery to control the export of mRNA from the nucleus and prime mRNA for subsequent use in the cytoplasm. Prominent examples include vari-ous hnRNPs 14  as well as the so-called ‘ARE-BPs’ (‘AU-rich element (ARE)-binding proteins’) that recognize AREs ( Box 1 ) located in the 3 ′  untranslated regions (UTRs) of many immunological mRNAs. In the cytoplasm, regulatory RNPs can be remodeled further with the inclusion of RBPs that define the balance between two major PTR events: decay of mRNA and translation of mRNA.Three pathways of mRNA decay  15  seem to predominate in cells of the immune system ( Fig. 1a , d ). The first, exonucleolytic decay, occurs through the progressive removal of the poly(A) tail (dead-enylation) from exonuclease complexes such as PARN or the large CCR4-NOT complex ( Fig. 1a ). Subsequently, mRNA bodies can either be degraded from the 3 ′  end by the large exonucleolytic exo-some complex or lose their 5 ′  cap through the action of decapping enzymes such as Dcp1 and Dcp2 and then become degraded by the 5 ′ -to-3 ′  exonuclease Xrn1 ( Fig. 1a ). Regulatory RNPs bound to ele-ments in untranslated termini (such as AREs or constitutive decay elements (CDEs);  Box 1 ) and miRNA-loaded RNA-induced silencing complexes (RISCs) 16  containing the translation-initiation factor Ago1 can recruit deadenylation and exonucleolytic machinery for induc-ible degradation. In the second mechanism, endonucleolytic cleav-age, site-specific RNases induce internal cleavages that yield RNA fragments that are then susceptible to exonucleolytic degradation. In cells of the immune system, this form of degradation is engaged either by inducible endonucleases or by RISCs containing the slicer Ago2. The third mechanism, nonsense-mediated decay, is a surveillance mechanism that eliminates aberrant mRNAs with premature termina-tion codons and thus prohibits the generation of abnormal proteins. During the first round of translation, premature termination codons are detected by a protein complex that contains upstream frameshift proteins, which orchestrate the stalling of subsequent rounds of trans-lation and recruit degradation machinery  15 .The translation of immunological mRNAs is controlled via restric-tions in the assembly of the 80S ribosome on mRNA from its constitu-ent 40S and 60S subunits (initiation) ( Fig. 1e ) or the construction of the protein sequence from the RNA template (elongation). The former seems to involve several regulatory RNP configurations, whereas the latter is directly affected by signal transducers 17 . Initiation control occurs mainly during the interaction of eIF4F (a complex consisting of the RNA helicase eIF4A, the cap-binding protein eIF4E and the scaffold eIF4G) with the 5 ′  cap of mRNA. That interaction is inhibited by the eIF4E-binding proteins 4E-BP1 and 4E-BP2, which are in turn deactivated by the metabolic checkpoint kinase complex mTORC1 (ref. 18). Deprivation of nutrients, infection and inflammation can block the translation regulator mTOR by activating its negative regulators (TSC proteins) and can thus inhibit protein synthesis. Alternatively, inflam-matory signals may block the recruitment of eIF2–GTP–methionyl initiator tRNA ternary complex onto the 40S ribosomal subunit. This stalls the formation of the 43S complex and its interactions with Box 1 The nature and recognition of regulatory RNA elements A wide collection of regulatory RNA cis   elements can be specifically recognized by trans  -acting factors, such as RBPs or noncoding RNA. They are believed to operate as primary structures (sequences), secondary conformations (for example, stem loops and internal bulges) and/or tertiary multidomain architectures. Rigid structures such as internal ribosome entry sites can be recognized ‘tightly’ by factors that bring with them essential components such as ribosomal subunits. Alternatively, structures may exist in less-rigid conformations, in close or distal proximity with each other within the RNA body. Paradigms of elements targeted by RBPs include the following: elements on the pre-mRNA that, upon recognition by relevant RBPs enhance or silence the definition of ‘introns’ and ‘exons’ by the spliceosome (and thus act as either exonic splicing enhancers or exonic splicing silencers that reside in an exon and regulate its inclusion or skipping, respectively) or intronic splicing enhancers or splicing silencers that reside in an intron and affect the use of proximal splice sites or exons; variable tandems of loosely defined AU- (and U-rich) motifs (AREs) located in the UTRs of unstable mRNAs 110,111 ; spatially restricted secondary structures that control degradation and cap-mediated translation, such as CDEs, GAIT elements and the recognition domain of regnase-1 (refs. 34,45,67); and domains bound by miRNA. Regulatory RBPs contain multiple RNA-binding domains arranged also in tandem or distantly (RRMs, zinc fingers, KH motifs and dsRNA-binding domains) ( Table 1 ). The various domains in an RBP combine individual weak interactions with specific protein-RNA complexes of high affinity 2 . When bound to RNA, an RBP can stabilize weak secondary structures and force the formation of secondary RNA structures by bringing distantly located motifs in closer proximity. In addition, homo- or heteropolymerization of RBPs on their RNA targets can take place and thus expose or hide regulatory RNA elements, prevent or allow the binding of other RBPs and connect to essential machinery and signaling modules. In this context, differences in the engagement of RBPs on selective combinations of RNA elements in RNPs provides specificity and coordination in PTR 5 . REVIEW  494 VOLUME 15 NUMBER 6 JUNE 2014 NATURE IMMUNOLOGY REVIEW eIF4F, other eIF complexes and poly(A)-binding protein, which are all required for assembly of the 48S initiation complex 19 .In conditions of infectious stress 20  or inflammatory stress, auxiliary ribosomal factors, transcript-restricted RNPs and Ago1-containing RISCs 21  may hinder initiation and divert stalled preinitiation complexes to subcellular foci known as ‘stress granules’, where they await signal-induced ‘decisions’ 19 . In the presence of permissive signals, preinitiation complexes are released; conversely, restrictive signals drive RNPs to sites enriched for degrading factors such as processing bodies 19 .The engagement or inhibition of core post-transcriptional machin-ery by regulatory RNPs can ‘fine tune’ complex reactions and diver-sify cellular phenotypes. Below we discuss prominent examples of RBP-mediated regulation and highlight the extensive degree of post- transcriptional networking that underlies immunological reactivity. RBP-mediated controls in innate immunity Cells of the innate immune system (for example, monocytes- macrophages, myeloid dendritic cells and polymorphonuclear cells) evolved to recognize damaging agents and noxious substances; clear dying cells and infectious organisms; provide support to the adaptive arm of immunity; and aid tissue regeneration 20 . To accommodate such functions, cells of the innate immune system undergo a con-tinuum of changes in RNP configurations and post-transcriptional programs induced by the inflammatory microenvironment ( Fig. 2 ). RBPs in the recognition and clearance of pathogens Pathogens, infected cells and damaged or transformed tissues are detected by germline-encoded pattern-recognition receptors (PRRs) such as Toll-like receptors (TLRs) and RIG-I-like receptors 22,23 , which are expressed either on cell surfaces or intracellularly. PRRs use a  variety of adaptor complexes to activate intracellular signaling cas-cades that drive proinflammatory biosynthesis (for example, the tran-scription factor NF- κ  B, mitogen-activated protein kinases (MAPKs) or interferon-regulatory factors (IRFs)) and/or cellular execution. RBPs can alter the signaling thresholds of PRRs in many ways. For example, prolonged TLR signaling via the adaptor MyD88 activates nuclear SR proteins to promote the skipping of an exon in  Myd88  pre-mRNA 24,25 . The resulting variant MyD88 protein lacks a region required for assembly of the IRAK proinflammatory signaling com-plex and prohibits signal transmission. Transcript-specific controls can also enforce signaling via the NF- κ  B inhibitor IKK and MAPKs; for example, through changes in the translation of mRNA encod-ing the kinase Tak1 facilitated by ARE-BPs 26 . Interestingly, RBPs can also act as protein components of innate receptors. CIRP is an RBP that aids the translation of mRNA during cold shock and hypoxia. In hypoxic macrophages and brain microglia, CIRP is transported to the cell surface and interacts with MD2 (the coreceptor of TLR4) and enhances its proinflammatory activity  27,28 . The regulatory effect of surface CIRP is suggested by the effect of its depletion mediated by antibodies or genetic means, which counteracts acute inflammatory assaults in rodents 27 .RNA viruses are detected via endoplasmic or cytoplasmic PRRs that bind to bacterial single-stranded RNA (ssRNA) (TLR7 and TLR8) or viral dsRNA (TLR3, TLR9 and the DExD–H-box helicases RIG-I, Mda5 and DHX33) 22,23,29  to promote the production of type I interfer-ons. Interferons also alert the host’s degradation machinery to attack  viral RNA and mask its translation machinery from exploitation by Table 1 Recognition of RNA by RBPs, and features of mouse RBP mutants RBPRBDOther domainsRNA elementPTRImmunological relevance of mutantRefs.Upf1 (Rent1)ZnFST-[Q]NMDDefective thymopoiesis a 80Upf2 (Rent2)ZnFMIF4GNMDDefective thymopoiesis b 81Zfp36 (TIS11, TTP)ZnFAREEXD, TRAcute or chronic inflammation a,b 41–44Zfp36L1 (TIS11b, BRF1)ZnFAREEXDDefective thymopoiesis, T-ALL b 77Zfp36L2 (TIS11d, BRF2)ZnFAREEXDDefective thymopoiesis, T-ALL b 77Elavl1 (HuR)RRMsARE (U-rich)SPL, pA, EXD, TR, miRNAAcute or chronic inflammation; cancer; defective thymopoiesis; inhibition of autoreactivity a,b 61,64–66, 85,101TIA-1RRMsARE (U-rich)SPL, TRSensitivity to acute or chronic inflammation a 51–53hnRNPD (AUF1)RRMsG-richARE (U-rich)EXD, TRAcute or chronic inflammation a 54,56,57Khsrp (KSRP, FUBP2)KHG-richARE, miRNAEXD, TR, miRNAEnhanced antiviral responses b 58hnRNPLRRMG-, P- richCA-richSPLDefective thymopoiesis b 90hnRNPLLRRMCA-richSPLDefective peripheral T cell responses b 95Rc3h1 (roquin, roquin-1)ROQ, ZnFRING, P-richCDEEXDSystemic autoimmunity a,b 49,97Rc3h2 (roquin-2)ROQ, ZnFRING, P-richCDEEXDSystemic autoimmunity a,b 49,97DicerDsRBDsHelicase, PAZ, RNaseIIIdsRNAmiRNADefective thymopoiesis b 92,93NF-90DsRBDsdsRNATRDefective peripheral T cell responses a 100PKRDsRBDsdsRNARCDefective viral clearance a 30ADAR1DsRBDsdsRNARC, EDDefective viral clearance a 31L13aRibosomalGAITTRAcute or chronic inflammation b 69ASF (SF2, SRSF1)RRMRS, G-richAG-richSPLSystemic autoimmunity c 88Zc3h12a (MCPIP1, regnase-1)ZnFPINStem-loopENDSystemic autoimmunity a,b 34,96Arid5aARIDStem-loopENDResistance to acute inflammation and autoimmunity a 35CIRBP (Cirp, hnRNPA18)RRMG-richUnknownTRResistance to acute inflammation a 27SRSF2 (SC35)RRMRS, G-richUnknownSPLDefective thymopoiesis b 89S6 (S6R)RibosomalTRDefective thymopoiesis a 82Rpl22RibosomalTRDefective thymopoiesis a 83eIF4ERibosomalTRResistance to infection and inflammation; T cell anergy d 71,73eEF2RibosomalTRResistance to acute inflammation d 17 Mode for the recognition of RNA by RBPs (synonyms in parentheses) with a proven function in immunological responses, as well as characteristics and phenotypes of mice with mutant RBPs. ARID, AT-rich–interaction domain; ED, editing; END, endonucleolytic decay; EXD, exonucleolytic decay; KH, K-homology domain; MIF4G, middle domain of EIF4G; NMD, nonsense-mediated decay; PAZ, Piwi, Argonaute and Zwille domain; PIN, ssRNA-cleavage domain of upstream frameshift protein Upfs; RC, receptor function; RING, RING-finger domain; ROQ, RNA-binding domain of roquin proteins; RRM, RNA-recognition motif; RS, arginine-serine–rich domain; SPL, splicing; pA, polyadenylation; TR, translation; ZnF, CCCH or other zinc finger; ribosomal, connections via the ribosome; T-ALL, T cell acute lymphoblastic leukemia. a Germline-encoded mutations. b Myeloid cell– or T cell–restricted mutations via Cre- lox  P–mediated somatic recombination. c Human permutation. d Inferred by deletion of regulators.  NATURE IMMUNOLOGY   VOLUME 15 NUMBER 6 JUNE 2014 495 REVIEW      e      l      F      4      A Cytoplasm NucleusCytoplasm Nucleus 40S subunitelF2-GTP and met-tRNAi ↓ 43S complexCytoplasmNucleus 31341 12314 3421 4    1 PP P P P PPPPSF TLRMyD88TCRdsRNAvirusILocalization  A A A A AAAAAAAAAAAAAAAAAAAA Editing and splicingStressgranulesExosomecomplexRBP1RBP2Introns ExonsRBP3 (MF)RBP4 (MF)RNA elements ab c NMDp38ErkPKC- θ  IKK α and IKK β AREAREGAIT TTP AdaptorRegnase-1AMD or CDETNFRTLR4Endonucleolytic d e Cap A AAAAAAAAAAAAAAAAAAAA AAAA TranslationDecay AAAAAAAAAAAA CCR4-NOTDCP1 and DCP2 Xrn1 AA A A  A ADARIFNARGSK3hnRNP LL hnRNP LSF3bCytoplasmLPSSF3a M     y   D    8    8     Ub AAAACDE AAAAAAAA CBPelF3 AUG PTC EJ EJC60SUpf2SMG1Upf1 RoquinRoquin IFN- γ  R4E-BPs4E-BPsmTORC1TSCs  eEF2K    K  a   t   i  e   V   i  c  a  r   i   /   N  a   t  u  r  e   P  u   b   l   i  s   h   i  n  g   G  r  o  u  p Stress, nutrient deprivation, infectioneIF4E e  I  F  4  E   PPPPPelF4GPABP AAAAAAAAAAA elF3   60SeEF2EPRSL13 α  GAPDH TIA-1and TIAR Stressgranules Figure 1  Immune system–related PTR events. ( a ) RBPs accompany RNA from the very early steps of its life, via the recognition of relevant RNA elements, and form RNP complexes. Nuclear RNPs orchestrate early post-transcriptional events, such as pre-mRNA splicing, editing and the nucleocytoplasmic trafficking of mRNA. Multifunctional (MF) RBPs remain bound on regulatory elements and selectively coordinate regulatory RNP configurations in the cytoplasm. Cytoplasmic RNPs drive mRNA through translation by the ribosome. In contrast, RNPs remodeled because of external signals guide their target mRNA to destructive machinery that acts to promote deadenylation (for example, CCR4-NOT), decapping (for example, DCP1 and DCP22), 3 ′ -to-5 ′  exonucleolytic degradation (for example, the exosome complex) or 5 ′ -to-3 ′  exonucleolytic degradation (for example, Xrn1). ( b – d ) Paradigms of the involvement of RNPs in immunological reactions include the following: the type I interferon–induced editing of viral dsRNA by ADAR1, which converts adenine to inosine and compromises viral integrity ( b ); the TLR- or TCR-induced activation of alternative splicing mediated by hnRNPL, hnRNP LL and PSF or the SR proteins SF3a and SF3b, all of which affect the inclusion or skipping of exons ( c ); and basal and inducible mRNA-degradation mechanisms that occur in cells of the immune system ( d ). These exonucleolytic events are promoted by TTP and roquin after they bind onto the relevant elements (AREs or CDEs) of target mRNA and recruit degradation machinery. The inducible functions of TTP can be inhibited during inflammatory activation via phosphorylation by MAPKs or SAPKs. Alternatively, the endonucleolytic cleavage of target mRNA is promoted by regnase-1, which is in turn inhibited by signaling via IKK α  and IKK β  and protein kinase C- θ  (PKC- θ ) and proteosomal degradation. Finally, aberrant transcripts containing premature termination codons are sensed by Upf-containing complexes and are selectively degraded by nonsense-mediated decay (NMD). IFNAR, receptor for IFN- α ; GSK3, glycogen synthase kinase 3; LPS, lipopolysaccharide; TNFR, receptor for TNF; Erk, MAPK; CBP, cap-binding complex; SMG1, phosphatidylinositol 3-kinase-related kinase; PTC, premature termination codon; EJ, exon junction; EJC, exon-junction complex. ( e ) Impediments to the translation of specific mRNAs can be imposed via inducible blockade of mTORC by TSC proteins and mTOR’s inability to deactivate 4E-BP1 and 4E-BP2 by phosphorylation, which otherwise prevent eIF4E from reaching capped mRNAs. Alternatively, stress signals can stall the formation of eIF2-containing preinitiation complexes via inhibitory phosphorylation of eIF2 and sequestration of these complexes in cytoplasmic stress granules by ARE-BPs like TIA-1. Moreover, IFN- γ   can lead to the phosphorylation of L13a and can be diverted from ribosomal subunits to mRNA containing GAIT elements and block the translation of that mRNA. Finally, translation elongation can be controlled by the stress-induced inhibitory phosphorylation of eEF2 kinase, which releases eEF2 to allow its recruitment to ribosomes. Such processes may also involve the generation and function of noncoding RNA populations; these have been intentionally omitted here to emphasize the functions of RBPs. IFN- γ  R, receptor for IFN- γ  ; PABP, poly(A)-binding protein; EPRS, glutamyl-prolyl-tRNA synthetase; Met-tRNAi, methionyl initiator tRNA.
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