Post-translational modifications of histones play a key role in DNA-based processes, like transcription, by modulating chromatin structure. N-terminal acetylation is unique among the numerous histone modifications because it is deposited on the N-alpha amino group of the first residue instead of the side-chain of amino acids. The function of this modification and its interplay with other internal histone marks has not been previously addressed. Here, we identified N-terminal acetylation of H4 (N-acH4) as a novel regulator of arginine methylation and chromatin silencing in Saccharomyces cerevisiae. Lack of the H4 N-alpha acetyltransferase (Nat4) activity results specifically in increased deposition of asymmetric dimethylation of histone H4 arginine 3 (H4R3me2a) and in enhanced ribosomal-DNA silencing. Consistent with this, H4 N-terminal acetylation impairs the activity of the Hmt1 methyltransferase towards H4R3 in vitro. Furthermore, combinatorial loss of N-acH4 with internal histone acetylation at lysines 5, 8 and 12 has a synergistic induction of H4R3me2a deposition and rDNA silencing that leads to a severe growth defect. This defect is completely rescued by mutating arginine 3 to lysine (H4R3K), suggesting that abnormal deposition of a single histone modification, H4R3me2a, can impact on cell growth. Notably, the cross-talk between N-acH4 and H4R3me2a, which regulates rDNA silencing, is induced under calorie restriction conditions. Collectively, these findings unveil a molecular and biological function for H4 N-terminal acetylation, identify its interplay with internal histone modifications, and provide general mechanistic implications for N-alpha-terminal acetylation, one of the most common protein modifications in eukaryotes.

Viroids, due to their small size and lack of protein-coding capacity, must rely essentially on their hosts for replication. Intriguingly, viroids have evolved the ability to replicate in two cellular organella, the nucleus (family Pospiviroidae) and the chloroplast (family Avsunviroidae). Viroid replication proceeds through an RNA-based rolling-circle mechanism with three steps that, with some variations, operate in both polarity strands: i) synthesis of longer-than-unit strands catalyzed by either the nuclear RNA polymerase II or a nuclear-encoded chloroplastic RNA polymerase, in both instances redirected to transcribe RNA templates, ii) cleavage to unit-length, which in the family Avsunviroidae is mediated by hammerhead ribozymes embedded in both polarity strands, while in the family Pospiviroidae the oligomeric RNAs provide the proper conformation but not the catalytic activity, and iii) circularization. The host RNA polymerases, most likely assisted by additional host proteins, start transcription from specific sites, thus implying the existence of viroid promoters. Cleavage and ligation in the family Pospiviroidae is probably catalyzed by an RNase III-like enzyme and an RNA ligase able to circularize the resulting 5′ and 3′ termini. Whether a chloroplastic RNA ligase mediates circularization in the family Avsunviroidae, or this reaction is autocatalytic, remains an open issue.

Avocado sunblotch viroid, peach latent mosaic viroid, chrysanthemum chlorotic mottle viroid, and eggplant latent viroid (ELVd), the four recognized members of the family Avsunviroidae, replicate through the symmetric pathway of an RNA-to-RNA rolling-circle mechanism in chloroplasts of infected cells. Viroid oligomeric transcripts of both polarities contain embedded hammerhead ribozymes that, during replication, mediate their self-cleavage to monomeric-length RNAs with 5=-hydroxyl and 2=,3=-phosphodiester termini that are subsequently circularized. We report that a recombinant version of the chloroplastic iso- V iroids are plant pathogens constituted by a small circular noncoding RNA (12,18,19,45). Most viroid species are gathered within the family Pospiviroidae and characteristically contain a central conserved region (CCR) in the middle of their molecules, which are predicted to fold in rod-or quasi-rod-like minimum free-energy conformations, and replicate in the nuclei of infected cells. However, four viroid species-Avocado sunblotch viroid (ASBVd), Peach latent mosaic viroid (PLMVd), Chrysanthemum chlorotic mottle viroid (CChMVd), and Eggplant latent viroid (ELVd)-that do not contain a CCR in their molecules are grouped into the family Avsunviroidae (15, 17). These four species contain hammerhead ribozymes embedded in both polarities of their RNA strands that catalyze self-cleavage of the oligomeric viroid RNA intermediates resulting from replication that occurs in the chloroplasts of infected cells (17). In this family, replication follows the symmetric variant of an RNA-based rolling-circle mechanism (4, 9, 25). In this variant, the circular viroid strand of plus [(ϩ)] polarity-which is arbitrarily assigned to the viroid RNA strand most abundant in the infected tissue-is reiteratively transcribed by an RNA polymerase to produce oligomeric strands of complementary or minus [(Ϫ)] polarity. These RNAs are selfcleaved by the ribozymes to produce monomeric linear RNAs that are circularized. Then, in a second and symmetrical part of the cycle, the monomeric (Ϫ) circular RNA is transcribed to oligomers that are self-cleaved and the resulting linear monomers subsequently circularized to finally produce the monomeric (ϩ) circular RNA.The effect of the inhibitor tagetitoxin on RNA synthesis in chloroplastic preparations of ASBVd-infected avocado (Persea americana Mill.) leaves suggests that the nucleus-encoded chloroplastic RNA polymerase (NEP), and not the plastid-encoded RNA polymerase (PEP), is the enzyme that transcribes the viroid RNAs in the infected tissue (37). This notion is sustained by the intense PLMVd replication in peach [Prunus persica (L.) Batsch] leaves expressing a PLMVd-incited albinism in which PEP-dependent transcription is basically absent (41). Consistent with this view, ASBVd double-stranded RNAs, regarded as the replication intermediates, have been detected in chloroplasts of infected cells (36). Concerning the second replication step, characterization of the termini of linear ASBVd and CChMV...

Epigenetic modifications, including those occurring on DNA and on histone proteins, control gene expression by establishing and maintaining different chromatin states. In recent years, it has become apparent that epigenetic modifications do not function alone, but work together in various combinations, and cross-regulate each other in a manner that diversifies their functional states. Arginine methylation is one of the numerous PTMs (post-translational modifications) occurring on histones, catalysed by a family of PRMTs (protein arginine methyltransferases). This modification is involved in the regulation of the epigenome largely by controlling the recruitment of effector molecules to chromatin. Histone arginine methylation associates with both active and repressed chromatin states depending on the residue involved and the configuration of the deposited methyl groups. The present review focuses on the increasing number of cross-talks between histone arginine methylation and other epigenetic modifications, and describe how these cross-talks influence factor binding to regulate transcription. Furthermore, we present models of general cross-talk mechanisms that emerge from the examples of histone arginine methylation and allude to various techniques that help decipher the interplay among epigenetic modifications.

Members of the family Pospiviroidae, like Citrus exocortis viroid (CEVd), replicate through an RNA-based asymmetric rolling-circle mechanism in which oligomeric plus-strand [(؉)] RNA intermediates are cleaved to monomeric linear (ml) RNA and then circularized. Here we show, by rapid amplification of 5 and 3 cDNA ends and in vitro ligation assays, that ml CEVd (؉) RNA resulting from cleavage of a dimeric transcript transgenically expressed in Arabidopsis thaliana contains 5-phosphomonoester and 3-hydroxyl termini. The nature of these termini and the double-stranded structure previously proposed as the substrate for cleavage in vivo suggest that a type III RNase catalyzes cleavage and an RNA ligase distinct from tRNA ligase promotes circularization.Viroids are plant pathogens consisting of a non-proteincoding small circular RNA (246 to 401 nucleotides [nt]) (5,8,13,26). The approximately 30 viroid species known are classified into two families, Pospiviroidae and Avsunviroidae, whose members replicate in the nucleus and chloroplast, respectively (13). Viroid replication occurs through an RNA rolling-circle mechanism characterized by the reiterative RNA-RNA transcription of circular templates and processing of the resulting oligomeric RNAs of one or both polarities (2). In members of the family Pospiviroidae, like Potato spindle tuber viroid (PSTVd), the most abundant circular RNA in vivo to which plus-strand [(ϩ)] polarity is assigned arbitrarily, is transcribed into complementary oligomeric minus-strand [(Ϫ)] RNAs. These strands serve directly as templates for the synthesis of oligomeric (ϩ) RNAs that are processed into monomeric linear (ml) and monomeric circular (mc) RNAs (1). In members of the family Avsunviroidae, like Avocado sunblotch viroid, the oligomeric (Ϫ) RNAs are processed into mc (Ϫ) RNAs, which through a second rolling circle direct the synthesis of oligomeric (ϩ) RNAs, with subsequent processing producing the mc (ϩ) RNAs (7). Whereas in the family Avsunviroidae, cleavage of the oligomeric RNAs of both polarities is mediated by hammerhead ribozymes that generate ml RNAs with 5Ј-hydroxyl (5Ј-OH) and 2Ј,3Ј-cyclic phosphodiester (2Ј,3ЈϾP) termini (11), how this step happens in the family Pospiviroidae is unclear. Previous work showed that viroid RNA processing occurs accurately in transgenic lines of the viroid nonhost Arabidopsis thaliana expressing dimeric transcripts of representative members of the family Pospiviroidae (6). With this experimental system, the processing site of oligomeric (ϩ) RNA intermediates has been mapped at a position within a conserved double-stranded structure that two consecutive hairpin I motifs can promote ( Fig. 1) (14).Here, using transgenic A. thaliana expressing a dimeric transcript of Citrus exocortis viroid (CEVd) (genus Pospiviroid, family Pospiviroidae), we have determined the termini of the ml (ϩ) RNA accumulating in vivo by rapid amplification of 5Ј and 3Ј cDNA ends (5Ј-and 3Ј-RACE) (25) and by in vitro ligation assays. The nature of these termini, 5Ј-phosphomono...

Changes in histone modifications are an attractive model through which environmental signals, such as diet, could be integrated in the cell for regulating its lifespan. However, evidence linking dietary interventions with specific alterations in histone modifications that subsequently affect lifespan remains elusive. We show here that deletion of histone N‐alpha‐terminal acetyltransferase Nat4 and loss of its associated H4 N‐terminal acetylation (N‐acH4) extend yeast replicative lifespan. Notably, nat4Δ‐induced longevity is epistatic to the effects of calorie restriction (CR). Consistent with this, (i) Nat4 expression is downregulated and the levels of N‐acH4 within chromatin are reduced upon CR, (ii) constitutive expression of Nat4 and maintenance of N‐acH4 levels reduces the extension of lifespan mediated by CR, and (iii) transcriptome analysis indicates that nat4Δ largely mimics the effects of CR, especially in the induction of stress‐response genes. We further show that nicotinamidase Pnc1, which is typically upregulated under CR, is required for nat4Δ‐mediated longevity. Collectively, these findings establish histone N‐acH4 as a regulator of cellular lifespan that links CR to increased stress resistance and longevity.

Histone modifications are key epigenetic regulators that control chromatin structure and gene transcription, thereby impacting on various important cellular phenotypes. Over the past decade, a growing number of studies have indicated that changes in various histone modifications have a significant influence on the aging process. Furthermore, it has been revealed that the abundance and localization of histone modifications are responsive to various environmental stimuli, such as diet, which can also affect gene expression and lifespan. This supports the notion that histone modifications can serve as a main cellular platform for signal integration. Hence, in this review we focus on the role of histone modifications during aging, report the data indicating that diet affects histone modification levels and explore the idea that histone modifications may function as an intersection through which diet regulates lifespan. A greater understanding of the epigenetic mechanisms that link environmental signals to longevity may provide new strategies for therapeutic intervention in age-related diseases and for promoting healthy aging.

The family Avsunviroidae comprises four viroid species with the ability to form hammerhead ribozymes that mediate self-cleavage of the multimeric plus and minus strands resulting from replication in the chloroplast through a symmetric rolling-circle mechanism. Research on these RNAs is restricted by their host range, which is limited to the plants wherein they were initially identified and some closely related species. Here we report cleavage and ligation in transplastomic Chlamydomonas reinhardtii expressing plus-and minus-strand dimeric transcripts of representative members of the family Avsunviroidae. Despite the absence of viroid RNA-RNA transcription, the C. reinhardtii-based system can be used to address intriguing questions about viroid RNA processing and, in particular, about the cellular factors involved in cleavage and ligation. (11,15,28). Although ASBVd, PLMVd, CChMVd, and ELVd do not contain the central conserved region characteristic of the family Pospiviroidae, they can form hammerhead ribozymes that mediate self-cleavage of multimeric plus and minus strands resulting from symmetric rolling-circle replication (3,8). The four members of the family Avsunviroidae have a restricted host range: they infect only the plants wherein they were discovered and related species (12, 13), a feature limiting the study of these intriguing RNAs. Although no viroid infects Arabidopsis thaliana, nuclear transformation of this model plant with dimeric cDNA constructs of members of the family Pospiviroidae has revealed its potential in investigation of viroid-host interactions (7). Here, we report that transplastomic Chlamydomonas reinhardtii expressing dimeric viroid transcripts can be used to explore viroid-host interactions in the family Avsunviroidae. Species of theTransformation of C. reinhardtii chloroplasts with dimeric viroid cDNAs. Transplastomic C. reinhardtii cell lines expressing different plus-strand viroid transcripts were obtained. These transcripts, which resemble replicative intermediates and are highly infectious when inoculated mechanically into their hosts (5), were from the genera Avsunviroid (ASBVd), Pelamoviroid (CChMVd), and Elaviroid (ELVd) of the family Avsunviroidae and from Citrus exocortis viroid (CEVd) (15) of the family Pospiviroidae. A cell line expressing a dimeric minus-strand ASBVd transcript was also included.Dimeric viroid cDNAs were inserted between the XbaI and EcoRI sites of the chloroplast transformation vector pCrcϩ157 (27), replacing the ␤-glucuronidase open reading frame. The recombinant plasmids, in which the insertions were preceded by the promoter, the 5Ј untranslated region, and the beginning of the open reading frame of the rbcL gene (from Ϫ70 to ϩ157) and followed by the 3Ј untranslated region of the psaB gene from C. reinhardtii (Fig. 1), were particle bombarded (1) into atpB-deficient C. reinhardtii strain CC-373 (ac-uc-2-21). Photoautotrophic transformants were selected, and for each construct, ten lines were grown in high-salt minimal medium at 32°C, with 12/12 h ...