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Small RNAs: essential regulators of gene expression and defenses against environmental stresses in plants

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Eukaryotic genomes produce thousands of diverse small RNAs (smRNAs), which play vital roles in regulating gene expression in all conditions, including in survival of biotic and abiotic environmental stresses. SmRNA pathways intersect with most of the pathways regulating different steps in the life of a messenger RNA (mRNA), starting from transcription and ending at mRNA decay. SmRNAs function in both nuclear and cytoplasmic compartments; the regulation of mRNA stability and translation in the cytoplasm and the epigenetic regulation of gene expression in the nucleus are the main and best‐known modes of smRNA action. However, recent evidence from animal systems indicates that smRNAs and RNA interference (RNAi) also participate in the regulation of alternative pre‐mRNA splicing, one of the most crucial steps in the fast, efficient global reprogramming of gene expression required for survival under stress. Emerging evidence from bioinformatics studies indicates that a specific class of plant smRNAs, induced by various abiotic stresses, the sutr‐siRNAs, has the potential to target regulatory regions within introns and thus may act in the regulation of splicing in response to stresses. This review summarizes the major types of plant smRNAs in the context of their mechanisms of action and also provides examples of their involvement in regulation of gene expression in response to environmental cues and developmental stresses. In addition, we describe current advances in our understanding of how smRNAs function in the regulation of pre‐mRNA splicing. WIREs RNA 2016, 7:356–381. doi: 10.1002/wrna.1340 This article is categorized under: RNA Processing > Splicing Regulation/Alternative Splicing Regulatory RNAs/RNAi/Riboswitches > RNAi: Mechanisms of Action Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs
SmRNAs in the regulation of gene expression in response to stresses. (a) Environmental stresses trigger rapid changes in gene expression. Adaption to environmental stresses requires rapid changes in gene expression on all levels in both nuclear and cytoplasmic compartments. These coordinated events result in global reprogramming and enhancement of stress tolerance. (b) Biogenesis and functions of smRNAs. SmRNAs are produced from dsRNA precursors. MiRNAs derive from the stems of hairpin‐forming miRNAs (top left), while siRNAs are produced from dsRNA precursors, which can be formed in various ways, most often by RNA‐directed RNA polymerases (RDRs) in plants (top right). Dicer proteins process the dsRNAs into mature smRNAs that are methylated at their 3′ end by the methyltransferase HEN1 to increase their stability and then incorporated into AGO effector complexes. The smRNA–AGO complexes regulate gene expression on multiple levels (bottom box). In the nucleus, smRNAs can mediate DNA methylation and histone modifications, participate in DNA double‐stranded break repairs, or regulate pre‐mRNA splicing. In the cytoplasm, smRNAs regulate mRNA decay and act in the regulation of translation. The products of smRNA‐mediated cleavage are generally subjected to the 3′→5′ exonucleolytic degradation by the exosome and 5′→3′ degradation by the exoribonuclease Xrn4 (the Arabidopsis homolog of yeast Xrn1).
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Regulation of pre‐mRNA splicing by smRNAs. (a) Schematics of splicing. Upper panel, the exon–intron architecture of eukaryotic genes. Exon sequences are boxed, constitutive exons are shown in blue, and alternative exons are in pink; black lines represent introns. The minimal essential core splicing cis‐elements, 5′ splice site (5′SS) with invariant GU, 3′ splice site (3′SS) with invariant AG dinucleotides, and branch point sequence (BP) are indicated and present in every intron. The polypyrimidine tract (PPT) is a pyrimidine‐rich sequence located between the BP and 3′SS. Spliceosome components are recruited to the pre‐mRNA molecule and splicing proceeds via stepwise interaction of spliceosomal snRNPs recognizing the core splicing cis‐elements on pre‐mRNAs through base‐pairing interactions. Lower panel, simplified schematics of the cis‐acting ‘splicing code.’ Multiple additional exonic and intronic cis‐regulatory elements, termed the ‘splicing code,’ define the correct splice sites and participate in regulation of alternative splicing. Intronic/exonic splicing enhancers, ISEs/ESEs, are marked in green and intronic/exonic splicing silencers, ISSs/ESSs, are marked in orange. Inclusion or skipping of alternative exon/s is regulated in a combinatorial manner by the relative strength of enhancers and silencers, which are bound by splicing trans‐acting factors. Green arrows indicate enhancing, orange arrows indicate silencing processes. (b) Model for the mechanism of Brachypodium sutr‐siRNAs action. 3′ UTRs of Brachypodium coding genes give rise (by unknown mechanisms) to mostly 24‐nt sutr‐siRNAs in response to various abiotic stresses (left panel). Sutr‐siRNAs have potential different groups of trans‐targets in the genome: 10% of sutr‐siRNAs are predicted to target 3′ UTRs of other genes (panel 1, top right), which would be consistent with these sutr‐siRNAs acting in translational regulation or mRNA stability of their targets. Over 90% of sutr‐siRNAs target intronic regions (panels 2 and 3, middle and bottom right). The indicated annotated/authentic 3′SS is the splice site used to produce full‐length protein. Over 30% of intron‐targeting sutr‐siRNAs target potential intron regulatory regions such as PPT and BP sequences. Sutr‐siRNAs target PPTs and BPs of splice sites, which could be cryptic or alternative, marked as an additional 3′SS (panel 3, bottom right). The choice of these additional/alternative splice sites would lead to introduction of a premature stop codon downstream of that splice site, resulting in either a alternative short splice isoform or, most likely, producing RNA substrate for nonsense‐mediated degradation, suggesting that sutr‐siRNAs might be involved in regulation of splicing by blocking cryptic cis‐elements from being recognized by U2 snRNP and other splicing proteins during stress conditions and thus promote the selection of the correct splice site.
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A model for diRNA‐mediated DSB repair in Arabidopsis. In the vicinity of DSB sites, ssRNAs are generated, presumably by RNA Pol IV. SsRNAs are then converted into dsRNAs by RDR or through bidirectional transcription. DCL2/DCL3/DCL4 processes the dsRNAs into mature diRNAs, which are subsequently incorporated into AGO2. AGO2–diRNA complexes localize to the DSB site through interaction with scaffold transcripts made by Pol V. The AGO2–diRNA complexes may activate the DNA damage response (DDR) by recruiting DDR components (1), and may modify local chromatin by recruiting chromatin‐modifying components (2) or enable repair of the DSB by recruiting repair proteins (3). Adapted from Ref .
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Biogenesis and action of nat‐siRNAs and long siRNAs in Arabidopsis. (a) Nat‐siRNA processing from the SRO5‐P5CDH gene pair and nat‐siRNA function in a regulatory loop in response to salt stress. Nat‐siRNAs can originate both from cisNAT and transNAT transcript pairs. Salt stress leads to accumulation of reactive oxygen species (ROS), which can cause oxidation of DNA, proteins, and lipids, and induce the expression of SRO5. This triggers a series of nat‐siRNA processing steps, resulting in the downregulation of P5CDH. The primary SRO5 and P5CDH transcripts form dsRNA in their overlapping region, leading to production of 24‐nt nat‐siRNAs by DCL2. The initial cleavage of the P5CDH mRNA by 24‐nt nat‐siRNAs causes phased generation of 21‐nt nat‐siRNAs by a DCL1‐dependent mechanism and additional cleavage of the P5CDH transcript. RDR6, SGS3, and Pol IV may contribute to the formation of both 24‐ and 21‐nt SRO5‐P5CDH nat‐siRNAs. The 24‐nt and 21‐nt nat‐siRNAs mediate the downregulation of P5CSH mRNAs, leading to proline accumulation, which contributes to salt tolerance. However, it also causes accumulation of the proline catabolic intermediate P5C and thus accumulation of ROS; ROS can harm cells, but also act as a signal that activates stress responses. SRO5 may counteract the accumulation of ROS, thus fine‐tuning ROS levels and the resulting stress response. Adapted from Ref . (b) The biogenesis and mechanism of action of AtlsiRNA‐1. LsiRNAs are 30–40 nt in length. AtlsiRNA‐1 originates from the SRRLK‐AtRAP NAT pair in response to infection by bacterial pathogens carrying the avrRpt2 effector and forms a complex with AGO7. AtlsiRNA‐1 biogenesis requires DCL1, HYL1, HEN1, and HST1; DCL4, Pol IV, and RDR6 may also function in secondary amplification of AtlsiRNA‐1. AtlsiRNAs may destabilize mRNAs via promoting decapping and subsequent XRN4‐mediated 5′→3′ degradation in cis.
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Biogenesis of miRNAs, lmiRNAs, and tasiRNAs in Arabidopsis. (a) Schematics of miRNA and lmiRNA biogenesis. Most mature Arabidopsis miRNAs are 21 nt and produced by DCL1 from primary miRNA transcripts containing imperfect, self‐complementary fold‐back regions. However, DCL3 can process some miRNA hairpins, resulting in 24‐nt lmiRNAs (left). Some miRNA transcripts can also give rise to both canonical miRNAs and lmiRNAs. The methyltransferase HEN1 also 2′‐O‐methylates the 3′ nucleotides of mature miRNA and lmiRNA duplexes to enhance their stability before they are loaded into AGO complexes. The miRNA duplex assembles with AGO1 into RISC, which then base pairs with mRNAs to induce miRNA‐directed silencing through mRNA degradation or affects on translation. In contrast, lmiRNAs function with AGO4 and may strictly function in directing DNA methylation in cis and in trans, similar to the function of het‐siRNAs. (b) Biogenesis and action of tasiRNAs in Arabidopsis. The productions of secondary phased 21‐nt tasiRNAs are initiated by miRNA‐mediated cleavage of TAS (TAS1–4) transcripts, which are transcribed by RNA Pol II. In the one‐hit model, AGO1‐loaded 22‐nt miR173 and miR828 target a single site on TAS1, TAS2, and TAS4 transcripts. In the two‐hit model, AGO7‐loaded 21‐nt miR390 targets TAS3 transcripts, which have two target sites. Cleaved transcripts are then converted into dsRNA by SGS3 and RDR6, and processed by DCLs before loading into AGO complexes. TasiRNAs can function in PTGS in trans and in TGS pathways in cis. Specific classes of 21‐nt tasiRNAs are preferentially processed by DCL4 (with the redundant activities of DCL1/2/3) and loaded into AGO1 to direct target mRNA cleavage in trans post‐transcriptionally. Other classes are processed predominantly by DCL1 (with DCL2/3/4 acting redundantly) and loaded into AGO4/6 complexes to recruit other RdDM effectors to mediate DNA methylation of TAS loci in cis.
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SmRNAs in transcriptional gene silencing (TGS). (a) The RNA‐dependent DNA methylation pathway (RdDM) and het‐siRNAs. Pol IV transcripts serve as precursors for het‐siRNAs, while Pol V‐produced lncRNA scaffolds act as targets recognized by siRNAs. Pol IV is recruited to its genomic loci by SHH1 (H3K9me reader) and the SNF2 domain‐containing chromatin remodeler CLSY1 facilitates Pol IV transcription. RDR2 converts single‐stranded Pol IV transcripts into dsRNA and these dsRNAs are further processed by DCL3 into mature 24‐nt het‐siRNAs. Mature het‐siRNAs are stabilized by methylation at the 3′ end by HEN1 and exported to the cytoplasm and loaded onto AGO4. AGO4–siRNA complexes are reimported into the nucleus to guide the targeting of nascent Pol V scaffold transcripts by sequence complementarity. Pol V transcription is facilitated by the DDR complex and SUVH2 and/or SUVH9 (H3K9 methyltransferase) aid Pol V recruitment to its genomic loci. The IDN2–IDP complex bound to Pol V scaffold RNAs interacts with SWI/SNF complex, which adjusts nucleosome positioning. The interaction of AGO4 and KTF1 (a putative transcription elongation factor) aids in recruiting AGO4–siRNA complexes to Pol V transcripts; the AGO4–siRNA complex pairs with Pol V transcripts and, along with RDM1 (RNA‐DIRECTED DNA METHYLATION 1), recruits DRM2 (cytosine‐5‐methyltransferase), which catalyzes de novo cytosine methylation to silence the locus. There is crosstalk between the DNA and H3K9 methylation pathways. H3K9 methylation by KYP (SUVH9), SUVH5, and SUVH6 amplifies silencing mediated by DNA methylation (extensively reviewed in Ref ). Together this results in transcriptional silencing at the genomic loci that are transcribed by Pol IV and Pol V, particularly TEs and other repetitive DNA. Adapted from Refs , , . (b) EasiRNAs. EasiRNAs are functionally equivalent to animal‐specific piRNAs. Developmental stresses during the reprogramming of the plant germ line trigger easiRNA production. Unlike animals, plant germ cells arise from somatic stem cells. The Arabidopsis male gametophyte (pollen grain) is binucleate, with one large vegetative cell enclosing a smaller cell that eventually gives rise to two sperm cells. The vegetative nucleus (VN) performs only supportive functions and does not contribute DNA to the next generation. Reprogramming in the germ line coincides with loss of chromatin remodelers in the VN. In the VN, DDM1 expression is repressed, leading to reversible chromatin decondensation and DNA demethylation of transposons, which reactivates TE transcription and triggers easiRNA production via a pathway requiring RDR6, DCL4/DCL2, and AGO1/AGO2. Over 50 known endogenous miRNAs, which target TEs post‐transcriptionally, can also trigger production of easiRNAs. The 21‐nt easiRNAs from the VN can move to induce PTGS in the nuclei of the sperm cells. They may also inhibit epigenetic modification to affect TEs. The easiRNAs can also form via an miRNA‐independent pathway, but the details of this pathway remain to be elucidated. Adapted from Refs , , .
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