N6-methyladenosine (m6A) is the most prevalent internal modification of eukaryotic mRNA. Very little is known of the function of m6A in the immune system or its role in host–pathogen interactions. Here we investigated the topology, dynamics, and bidirectional influences of the viral–host RNA methylomes during HIV-1 infection of human CD4 T cells. We show that viral infection triggers a massive increase in m6A in both host and viral mRNAs. In HIV-1 mRNA, we identified 14 methylation peaks in coding and noncoding regions, splicing junctions, and splicing regulatory sequences. We also identified a set of 56 human gene transcripts that were uniquely methylated in HIV-1-infected T cells and were enriched for functions in viral gene expression. The functional relevance of m6A for viral replication was demonstrated by silencing of the m6A writer or the eraser enzymes, which decreased or increased HIV-1 replication, respectively. Furthermore, methylation of two conserved adenosines in the stem loop II region of HIV-1 Rev Response Element (RRE) RNA enhanced binding of HIV-1 Rev protein to the RRE in vivo and influenced nuclear export of RNA. Our results identify a new mechanism for the control of HIV-1 replication and its interaction with the host immune system.

Summary Argonaute (AGO) proteins are critical components of RNA silencing pathways that bind small RNAs and mediate gene silencing at their target sites. We found that Arabidopsis AGO2 is highly induced by the bacterial pathogen Pseudomonas syringae pv. tomato (Pst). Further genetic analysis demonstrated that AGO2 functions in antibacterial immunity. One abundant species of AGO2-bound small RNAs is miR393b*, which targets a Golgi-localized SNARE gene MEMB12. Pst infection down-regulates MEMB12 in a miR393b*-dependent manner. Loss-of-function of MEMB12 but not SYP61, another intracellular SNARE, leads to increased exocytosis of an antimicrobial pathogenesis-related protein PR1. Overexpression of miR393b* resembles memb12 mutant in resistance responses. Thus, AGO2 functions in antibacterial immunity by binding miR393b* to modulate exocytosis of antimicrobial PR proteins via MEMB12. Since miR393 also contributes to antibacterial responses, miR393*/miR393 represent an example of a miRNA*/miRNA pair that functions in immunity through two distinct AGOs - miR393* through AGO2 while miR393 through AGO1.

Ethylene (C 2 H 4 ) is a unique plant-signaling molecule that regulates numerous developmental processes. The key enzyme in the two-step biosynthetic pathway of ethylene is 1-aminocyclopropane-1-carboxylate synthase (ACS), which catalyzes the conversion of S-adenosylmethionine (AdoMet) to ACC, the precursor of ethylene. To understand the function of this important enzyme, we analyzed the entire family of nine ACS isoforms (ACS1, ACS2, ACS4-9, and ACS11) encoded in the Arabidopsis genome. Our analysis reveals that members of this protein family share an essential function, because individual ACS genes are not essential for Arabidopsis viability, whereas elimination of the entire gene family results in embryonic lethality. Phenotypic characterization of single and multiple mutants unmasks unique but overlapping functions of the various ACS members in plant developmental events, including multiple growth characteristics, flowering time, response to gravity, disease resistance, and ethylene production. Ethylene acts as a repressor of flowering by regulating the transcription of the FLOWERING LOCUS C. Each single and high order mutant has a characteristic molecular phenotype with unique and overlapping gene expression patterns. The expression of several genes involved in light perception and signaling is altered in the high order mutants. These results, together with the in planta ACS interaction map, suggest that ethylene-mediated processes are orchestrated by a combinatorial interplay among ACS isoforms that determines the relative ratio of homo-and heterodimers (active or inactive) in a spatial and temporal manner. These subunit isoforms comprise a combinatorial code that is a central regulator of ethylene production during plant development. The lethality of the null ACS mutant contrasts with the viability of null mutations in key components of the ethylene signaling apparatus, strongly supporting the view that ACC, the precursor of ethylene, is a primary regulator of plant growth and development.

Small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are essential regulatory molecules of many cellular processes. Arabidopsis has at least three classes of endogenous siRNAs-chromatin-associated siRNAs, trans-acting siRNAs (tasiRNAs), and natural antisense transcript (NAT)-associated siRNAs (nat-siRNAs)-all 20-25 nucleotides (nt) in length. Here, we identified a novel class of small RNAs, long siRNAs (lsiRNAs), which are 30-40 nt and share many common features with known siRNAs. The lsiRNAs identified so far are induced by pathogen infection or under specific growth conditions. One of the lsiRNAs, AtlsiRNA-1, is generated from SRRLK/AtRAP NAT pair and specifically induced by the bacterium Pseudomonas syringae carrying effector avrRpt2. Recently, 25-to 31-nt PIWI-interacting RNAs (piRNAs) and repeat-associated siRNAs (rasiRNAs) were identified in animal germline cells. In contrast to the biogenesis of piRNAs/rasiRNAs, which is dicer independent and requires PIWI subfamily proteins, generation of AtlsiRNA-1 requires DCL1, DCL4, and the ARGONAUTE subfamily protein AGO7. It also depends on HYL1, HEN1, HST1, RDR6, and Pol IV. Induction of AtlsiRNA-1 silences AtRAP, which encodes a RAP-domain protein involved in disease resistance. Our further analysis implies that AtlsiRNA-1 may destabilize target mRNA through decapping and XRN4-mediated 5-to-3 degradation.[Keywords: Long siRNAs (lsiRNAs); DCL1; AGO7; decapping; bacteria-induced] Supplemental material is available at http://www.genesdev.org. Small RNA-mediated gene silencing plays important roles in many cellular processes, including development, genome maintenance and integrity, and adaptive responses to biotic and abiotic stress in most eukaryotes. Small RNAs, usually 20-25 nucleotides (nt) in length, can be grouped into two classes on the basis of their origins: microRNAs (miRNAs) and small interfering RNAs (siRNAs). They guide heterochromatin formation, mRNA degradation, translational inhibition, and DNA elimination (Baulcombe 2004;Zamore and Haley 2005;Vazquez 2006). Rapid and phenomenal progress has been achieved in unraveling the components and mechanisms involved in the biogenesis and function of various small RNAs. In plants, small RNAs are highly diverse. In general, miRNAs in Arabidopsis are processed from singlestranded (ss) hairpin RNA precursors by an RNase IIItype enzyme Dicer-like (DCL) 1. A recent study shows that a couple of newly identified miRNAs are processed by DCL4 (Rajagopalan et al. 2006). The precursors of these miRNAs tend to have more complementarity within the foldback structure than that in most previously identified DCL1-dependent miRNAs. miRNAs are primarily associated with ARGONAUTE 1 (AGO1) to guide mRNA cleavage or translational inhibition. Endogenous siRNAs are usually processed from long doublestranded RNAs (dsRNAs). The generation of trans-acting siRNAs (tasiRNAs) is initiated by miRNAs, and requires DCL4 and RNA-dependent RNA polymerase (RDR) 6 for their biogenesis. Both miRNAs and tasiRNAs are 21-to 2...

BackgroundMany eukaryotic genomes encode cis-natural antisense transcripts (cis-NATs). Sense and antisense transcripts may form double-stranded RNAs that are processed by the RNA interference machinery into small interfering RNAs (siRNAs). A few so-called nat-siRNAs have been reported in plants, mammals, Drosophila, and yeasts. However, many questions remain regarding the features and biogenesis of nat-siRNAs.ResultsThrough deep sequencing, we identified more than 17,000 unique siRNAs corresponding to cis-NATs from biotic and abiotic stress-challenged Arabidopsis thaliana and 56,000 from abiotic stress-treated rice. These siRNAs were enriched in the overlapping regions of NATs and exhibited either site-specific or distributed patterns, often with strand bias. Out of 1,439 and 767 cis-NAT pairs identified in Arabidopsis and rice, respectively, 84 and 119 could generate at least 10 siRNAs per million reads from the overlapping regions. Among them, 16 cis-NAT pairs from Arabidopsis and 34 from rice gave rise to nat-siRNAs exclusively in the overlap regions. Genetic analysis showed that the overlapping double-stranded RNAs could be processed by Dicer-like 1 (DCL1) and/or DCL3. The DCL3-dependent nat-siRNAs were also dependent on RNA-dependent RNA polymerase 2 (RDR2) and plant-specific RNA polymerase IV (PolIV), whereas only a fraction of DCL1-dependent nat-siRNAs was RDR- and PolIV-dependent. Furthermore, the levels of some nat-siRNAs were regulated by specific biotic or abiotic stress conditions in Arabidopsis and rice.ConclusionsOur results suggest that nat-siRNAs display distinct distribution patterns and are generated by DCL1 and/or DCL3. Our analysis further supported the existence of nat-siRNAs in plants and advanced our understanding of their characteristics.