Small RNAs in Plants

Abstract

Small ribonucleic acids (smRNAs), 20–40 nucleotides in length, are the core components of basic eukaryotic regulatory processes collectively termed as RNA silencing. smRNAs afford sequence specificity to these processes, guiding effector proteins to deoxyribonucleic acid (DNA) or RNA targets through complementary base pairing. These effectors act either by cleaving cognate RNAs, blocking their productive translation or inducing the methylation of specific DNA targets. Plant smRNAs are produced from longer double‐stranded RNAs by a family of RNase III enzymes called DICER‐LIKE proteins and guide effector proteins of the ARGONAUTE family. Several endogenous smRNA classes are produced by distinct biosynthetic pathways and have critical functions in development, stress responses or genome stability. Moreover, the plant smRNA machinery contributes to defence against viruses. The proteins involved in these pathways are members of conserved protein families; thus, redundancy and compensation between family members create a complex network of smRNA regulation.

Key Concepts

  • Features of smRNAs: Plant smRNAs are 20–40‐nt long RNAs produced from longer double‐stranded (ds) RNA precursors. The smRNA duplex intermediates have 2‐nt overhangs at their 3′ ends. These products of DICER‐LIKE proteins also have hydroxyl groups at 3′ ends and phosphate groups at 5′ ends. The smRNA methyltransferase HEN1 subsequently modifies the 2′ hydroxyl group of the 3′ most distal ribose of each strand to make stable smRNA duplexes with 2′O‐methyl groups at 3′ ends.

  • Two smRNA categories in plants: Although all plant smRNAs have similar chemical attributes, they are grouped in two categories based on their origin. smRNAs produced from dsRNAs composed of two distinct RNA strands are called small interfering RNAs (siRNAs), whereas smRNAs produced from the foldback dsRNA regions of a single RNA strand are called microRNAs (miRNAs).

  • smRNAs guide effector proteins to cognate targets: smRNAs guide ARGONAUTE (AGO) effector proteins to cognate DNA or RNA targets. In plants, smRNAs are loaded into different AGO proteins depending, at least in part, on the identity of the first 5′ nucleotide of each smRNA. The 5′ nucleotide of the selected guide strand interacts with the MID domain of the protein and the 3′ nucleotide is incorporated into a binding pocket of the PAZ domain. As in animals, the passenger strand of plant smRNA duplexes is certainly removed by cleavage in the centre of the paired smRNA by the catalytic, RNase H activity located in the PIWI domain. The selected smRNA strand guides an AGO effector protein to cognate DNA or RNA targets through antiparallel complementary base pairing.

  • AGO effector proteins exert various functions: ARGONAUTE effector proteins act on nucleic acid targets in different ways: (i) by cleaving mRNAs, (ii) by blocking the productive translation of mRNAs or (iii) by inducing DNA methylation of specific targets.

  • Trans‐acting siRNAs are a class of plant‐specific siRNAs: Biogenesis of trans‐acting siRNAs (ta‐siRNAs), a class of endogenous, plant‐specific siRNAs, is initiated by smRNA‐guided cleavage or recognition of precursor transcripts at one or two distinct sites. Truncated RNA precursors serve as a template for the RNA‐dependent RNA polymerase RDR6, which synthesizes a complementary RNA strand. The resulting long dsRNA is processed by DCL4 in 21‐nt increments. Each 21‐nt siRNA regulates the expression of specific genes by guiding cleavage of complementary mRNAs. This can initiate production of additional ta‐siRNAs from the cleaved mRNA target; amplification cascade of ta‐siRNAs can regulate expression of genes with sequences unrelated to the initial smRNA trigger.

  • smRNAs versus viruses: The smRNA machinery of plants, which has dedicated functions in development, is also central to the co‐evolutionary race between plants and viruses. smRNA machinery contributes to plant defence against viruses by producing virus‐derived siRNAs that block virus proliferation. As a counter defence, viruses have evolved suppressor proteins that block smRNA machinery at specific steps.

Keywords: microRNA; DICER; siRNA; RNA silencing; ARGONAUTE

Figure 1.

A basic framework for biogenesis and function of plant smRNAs. Plant smRNAs are produced via smRNA duplex intermediates (c) (see Figure ) from long double‐stranded RNA (dsRNA) precursors by RNase III enzymes called DICER‐LIKE (DCL) proteins (a). smRNA origin defines two categories: siRNAs produced from dsRNAs made of two distinct strands and miRNAs produced from the foldback dsRNA region of a single RNA strand (RNA hairpin). DCL proteins certainly interact selectively with 2‐nt 3′ overhanged dsRNAs through the RNA‐binding domain PAZ; this places the DCL processing centre at a precise distance of approximately 20‐nt from the overhang. The two shifted catalytic residues (▴) cut a distinct strand of dsRNA to release smRNA duplex with 2‐nt overhangs at both 3′ ends (b). smRNA duplexes are subsequently 2′‐O‐methylated by HEN1 and loaded into AGO proteins for function. Strand selection occurs through interaction of the first 5′ nucleotide with a nucleotide‐specific binding pocket located in the MID domain. The 2‐nt 3′ overhang of the selected strand is then anchored in the PAZ domain (d). Plant smRNAs guide AGO proteins to cognate DNA or RNA targets through complementary base pairing (a). The ‘slicer’ RNase H activity of the PIWI domain (▴) of certain AGO proteins mediate the cleavage of the paired mRNA target.

Figure 2.

Characteristic features of plant smRNAs. Representative intermediate smRNA duplex with 2‐nt overhangs, hydroxyl groups and HEN1‐dependent 2′‐O‐methyl groups at both 3′ ends and phosphate groups at both 5′ ends (expanded region shows a detailed molecular view).

Figure 3.

Schematic representation of the ra‐siRNA pathway. Self‐sustained loop that maintains ra‐siRNA‐guided methylation of DNA repeats and methylated DNA regions. Noncoding transcripts are produced from certain methylated regions by Pol IV and are used by RDR2 to form dsRNAs that are then processed by DCL3 into 24‐nt ra‐siRNAs. An amplification loop using as a template, the RDR2‐generated dsRNAs, is indicated by the upward arrow. ra‐siRNAs guide AGO4 proteins to DNA targets where they recruit additional proteins to maintain DNA and histone methylation (★).

Figure 4.

Pathways for endogenous smRNA that mediate posttranscriptional regulations. Opened bars represent genes with their transcription initiation indicated by arrows. Dotted arrow of NAT gene 2 indicates it is stress‐inducible. Thin black lines represent transcripts of genes encoding smRNAs and blue lines represent target mRNAs transcribed from independent genes (not represented). The miRNA (a) and trans‐acting siRNA (b) precursors are noncoding RNAs, whereas nat‐siRNA precursors derive from cis‐antisense overlapping coding transcripts (c). All three precursors are transcribed by Pol II. In the nucleus, hairpin‐folded miRNA precursors (a) are processed in two steps by the DCL1/HYL1 proteins to produce an miRNA duplex with 2‐nt 3′ overhangs. This duplex will be 2′‐O‐methylated at the 3′ ends by the dsRNA methylase HEN1. The miRNA duplexes are then exported from the nucleus and one strand is selected to guide the AGO1 protein to complementary mRNAs, which are then cleaved in the centre of the paired region. Alternatively, miRNA can guide translation inhibition of these mRNAs by recruiting additional proteins. Certain miRNAs guide the cleavage of TAS precursors through AGO1 or AGO7 (b). The resulting fragment is used by RDR6 for production of a long dsRNA, which is then cleaved in a phase every 21‐nt by DCL4 and its partner DRB4. ta‐siRNAs are then 2′‐O‐methylated at their 3′ ends by HEN1 and guide AGO1 protein to their mRNA targets for cleavage. In the proposed nat‐siRNA pathway (c), the double‐stranded region resulting from pairing of cis‐antisense transcripts is thought to be processed by DCL2 to generate a unique 24‐nt siRNA that guides cleavage of transcripts arising from the constitutively expressed gene. In principle, RDR6 would synthesize a complementary strand to the resulting fragments. The resulting dsRNA would then be processed like TAS duplexes in a phase by DCL1. Each resulting 21‐nt nat‐siRNA potentially reinforces cleavage of mRNAs transcribed from the constitutively expressed gene.

Figure 5.

Schematic representation of possible source of virus‐derived siRNAs. Virus‐derived siRNAs, which provide protection against viruses and against subsequent infections by related viruses, form by the action of DCLs on diverse dsRNA substrates. Viral dsRNAs can form by conversion of ssRNA templates to dsRNAs by viral or cellular RNA‐dependent RNA polymerases (RdRP or RDR, respectively), by bidirectional transcription of DNA viruses, or by intramolecular hairpin folding of dedicated viral RNAs. Virus‐derived siRNAs are subsequently loaded into AGO proteins to block virus proliferation.

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Further Reading

Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116: 281–297.

Brodersen P and Voinnet O (2006) The diversity of RNA silencing pathways in plants. Trends in Genetics 22: 268–280.

Collins RE and Cheng X (2005) Structural domains in RNAi. FEBS Letters 579: 5841–5849.

Ding SW and Voinnet O (2007) Antiviral immunity directed by small RNAs. Cell 130: 413–426.

Hannon GJ (2006) RNA interference (RNAi) and microRNAs. Encyclopedia of Life Sciences. doi: 10.1002/9780470015902.a0006256.

Mallory AC and Vaucheret H (2006) Functions of microRNAs and related small RNAs in plants. Nature Genetics 38: S31–S36.

Meins F Jr., Si‐Ammour A and Blevins T (2006) RNA silencing systems and their relevance to plant development. Annual Review of Cell and Developmental Biology 21: 297–318.

Meyer P (2006) Gene silencing in plants. Encyclopedia of Life Sciences. doi: 10.1002/9780470015902.a0002022.pub2.

Ramachandran V and Chen X (2008) Small RNA metabolism in Arabidopsis. Trends in Plant Science 13: 368–374.

Vazquez F (2006) Arabidopsis endogenous small RNAs: highways and byways. Trends in Plant Science 11: 460–468.

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Vazquez, Franck(Mar 2009) Small RNAs in Plants. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0020123]