Glossary

AGO4: ARGONAUTE 4, an essential component of the TGS machinery. It can load 24 nt siRNAs to recognize Pol V lncRNAs and trigger DNA methylation.

AS: alternative splicing, the regulated processing of mRNA that leads to the splicing of specific introns from a precursor mRNA and results in several mRNAs coding for multiple proteins, by combinations of alternative exons, from a single gene.

ceRNA: competing endogenous RNAs regulate other transcripts by being recognized by a common pool of miRNAs.

DCL3: DICER-like 3, an enzyme capable of cleaving RDR2-dependent dsRNAs into 24 nt siRNAs for TGS.

IR: inverted repeat, a genomic locus that is transcribed into a perfect or near-perfect dsRNA molecule.

lincRNA: long intergenic noncoding RNA.

lncNAT: long noncoding natural antisense transcript .

lncRNA: long noncoding RNA; ncRNAs longer than 200 nt.

miRNA: microRNA, a DICER-like 1(DCL1)-dependent 21 nt small RNA that, in complex with an AGO protein, induces cleavage or translational regulation of target mRNAs.

ncRNA: noncoding RNA, an RNA that does not encode a protein, but has other cellular functions.

Pol: DNA-dependent RNA polymerase.

PRC: polycomb repressive complex (1 or 2), a complex formed by polycomb group (PcG) proteins, with methyltransferase activity that catalyzes and maintains histone post-translational modifications.

RdDM: RNA-dependent DNA methylation, a plant-specific DNA methylation pathway involving lncRNAs and 24 nt siRNAs.

RDR2: RNA-dependent RNA polymerase 2; it can interact with Pol IV, transforming a nascent lncRNA into a dsRNA for TGS.

siRNA: small interfering RNA, the product of DICER-like cleavage of dsRNA. DCL1-, DCL2- and DCL4-dependent siRNAs (21 or 22 nt) trigger post-TGS, whereas DCL3-dependent siRNAs (24 nt) induce TGS.

SWI/SNF: an ATP-dependent nucleosome remodeling complex.

TF: transcription factor, a protein or complex that binds to specific DNA sequences to control the rate of transcription of a target gene.

TGS: transcriptional gene silencing, a mechanism depending on noncoding transcription and dsRNA production that controls the activity of transposable elements and expression of protein-coding genes through 24 nt siRNAs.

Battles and hijacks: noncoding transcription in plants

http://linkinghub.elsevier.com/retrieve/pii/S1360138515000552

90% of eukaryotic genomes are transcribed into RNAs, although only a small part corresponds to protein-coding mRNAs.

Genome-wide analyses revealed the existence of thousands of long noncoding RNAs (lncRNAs) in several plant species.

Plant lncRNAs are transcribed by the plant-specific RNA polymerases Pol IV and Pol V, leading to transcriptional gene silencing, as well as by Pol II.

function：gene expression, including chromatin remodeling, modulation of alternative splicing, fine-tuning of miRNA activity, and the control of mRNA translation or accumulation.

lncRNA 的分类

Antisense transcripts (lncNATs) initiate inside or 3′ to a protein-coding gene, are transcribed in its opposite direction, and overlap with at least one coding exon.

Intronic lncRNAs initiate inside an intron in either direction and terminate without overlapping with an exon.

Promoter lncRNAs are transcripts of the promoter region of a protein-coding gene.

Long intergenic ncRNAs (lincRNAs) are separate transcriptional units at a distance of at least 1 kb from protein-coding genes.

Protein-coding genes are represented with green exons and black introns, 5′ and 3′ untranslated regions. Red lines represent noncoding genes, which are shown without introns for clarity.Examples of each class are given with the corresponding reference.

Action of plant long noncoding (lnc) RNAs.

Plant lncRNAs can be transcribed by Pol II, Pol IV, or Pol V (black arrows).

RNA-dependent DNA methylation (RdDM, orange arrows) pathway begins when Pol IV transcribes a lncRNA transformed in a dsRNA by the action of the RNA-dependent RNA polymerase 2 (RDR2). These dsRNAs are processed by dicer-like 3 (DCL3) into 24nt siRNAs and are loaded in argonaute 4 (AGO4). Then, siRNA–AGO4 complexes are guided to their genomic targets by physical interaction with a** Pol V lncRNA**, inducing DNA methylation 文献.

Pol V transcripts can also recruit the SWI/SNF chromatin-remodeling complex to target gene promoter regions 文献, whereas** Pol II lncRNAs** can recruit** PRC** components for chromatin remodeling 文献 (light-blue arrows).

Pol II lncRNAs forming hairpin structures (dark blue arrows) can be precursors of 24 nt siRNAs by DCL3 cleavage, triggering TGS, or of si/miRNAs by DCL1, DCL2, or DCL4 processing, triggering post-TGS.

However, Pol II lncRNAs may also kidnap miRNAs by target mimicry 文献 and 文献, allowing normal translation of its target mRNA (yellow arrows).

LncNATs (green arrows) can trigger degradation of the transcript pair 文献 and文献 or promote the translation of the overlapping mRNA through recruitment to polysomes 文献.

In addition, Pol II lncRNAs can act as molecular transporters for nucleo cytoplasmic trafficking of RNA-binding proteins (RBP) 文献, or interact with splicing factors (SFs, purple arrows). These SFs can be hijacked by lncRNAs, consequently affecting alternative splicing of target mRNAs

lncRAN 发挥作用实例

The repression of FLC under vernalization is mediated by the transcription of two lncRNAs. COOLAIR, a lncNAT encompassing the whole FLC locus, and COLDAIR, a sense lncRNA encoded in the first intron of FLC.

参考文献： Ietswaart, R. et al. (2012) Flowering time control: another window to the connection between antisense RNA and chromatin. Trends Genet. 28, 445–453

In Arabidopsis, the expression of a heat stress transcription factor (HSFB2a) is counteracted by a lncNAT, asHSFB2a, influencing vegetative and gameto-phytic development

The production of regulatory siRNAs derived from NAT pairs was first described for dsRNAs produced by two partially-overlapping mRNAs in response to salt stress

lncRNAs in the arena of DNA methylation

RNA-directed DNA methylation (RdDM) is a model proposed to explain a mechanism controlling genome activity and involving Pol IV- and Pol V-dependent lncRNA. Transposable elements, repetitive regions and transgenes give rise to 24 nt siRNAs that target homologous genomic regions to trigger DNA methylation and post-translational histone modifications, thus promoting heterochromatin formation and transcriptional repression.

The Landscape of long noncoding RNA classificatio

http://linkinghub.elsevier.com/retrieve/pii/S0168952515000542

Schematic diagram illustrating various classes of ncRNAs.

Three hypothetical loci are shown. Protein coding exons are shown as green (locus 1) or yellow boxes (locus 3). Locus 2 signifies a pseudogene of locus 1.

Regulatory regions of locus 1 are shown in purple (promoter) and magenta (enhancer). Repeats are denoted by brown boxes. Lines with arrows represent ncRNAs.

The role depicted here for CARs and ciRNAs in stabilising a chromatin loop is hypothetical.

Abbreviations: CAR, chromatin-associated RNA; ceRNA, competing endogenous RNA; ciRNA, chromatin-interlinking RNA (grey) or circular intronic RNA (green); eRNA, enhancer-associated RNA; ecircRNA, exonic circular RNA; lincRNA, long intervening non-coding RNA; ncRNA, noncoding RNA; ncRNA-a, activating ncRNAs; PALR, promoter-associated long RNA; PIN, partially intronic RNA; TIN, totally intronic RNA; TSSa-RNA, transcription-start-site-associated RNA; T-UCR, transcribed ultraconserved regions; uaRNA, 3′ UTR-derived RNAs; vlincRNA, very long intergenic ncRNA.

Genome-wide view of natural antisense transcripts in Arabidopsis thaliana

这篇文献通讯作者是浙江大学陈铭教授，其实验室主要研究NAT和lncRNA。多篇文章虽然分数不高，但是分析方法可以学习。

http://dnaresearch.oxfordjournals.org/cgi/doi/10.1093/dnares/dsv008

Natural antisense transcripts (NATs) are endogenous transcripts that can form double-stranded RNA structures. Many protein-coding genes (PCs) and non-protein-coding genes (NPCs) tend to form cis-NATs and trans-NATs, respectively. In this work, we identified 4,080 cis-NATs and 2,491 trans-NATs genome-widely in Arabidopsis. Of these, 5,385 NAT-siRNAs were detected from the small RNA sequencing data. NAT-siRNAs are typically 21nt, and are processed by Dicer-like 1 (DCL1)/DCL2 and RDR6 and function in epigenetically activated situations, or 24nt, suggesting these are processed by DCL3 and RDR2 and function in environment stress. NAT-siRNAs are significantly derived from PC/PC pairs of trans-NATs and NPC/NPC pairs of cis-NATs. Furthermore, NAT pair genes typically have similar pattern of epigenetic status. Cis-NATs tend to be marked by euchromatic modifications, whereas trans-NATs tend to be marked by heterochromatic modifications.

NAT的分类：

Cis-NATs （顺式）are transcribed from the same genomic locus as their sense transcripts but from the opposite DNA strand, and display perfect sequence complementarity.

trans-NATs （反式）are transcribed from** separate genomic loci** and typically form partial complementarity.

我们的研究已顺式为主

cis****-NATs can be categorized into four subtypes:

• head-to-head (divergent),

• tail-to-tail (convergent),****

• fully containing overlap (enclosed)

• nearby (nearby head-to-head and nearby tail-to-tail) .

focus on the role of plants NATs in post-transcription regulation.

NATs are involved in epigenetic regulation, including DNA methylation and histone modification.

Different histone modifications have distinct distribution and distinct functions.

• H3K4me3 and H3K36me3 are known as euchromatic marks and are often abundant in highly expressed genes. H3K4me3 accumulate in the promoters and 5′ genic regions, while H3K36me3 accumulate across the transcribed region.

• H3K9me2 and H3K27me3 are major silencing mechanisms in plants. H3K9me2 is enriched in both the promoter and gene body, which has been found in a limited number of repressed genes in Arabidopsis, while H3K27me3 prefers to mark across transcribed regions which is associated with tissue-specific and developmentally regulated genes.

• Methylation can occur at any cytosine in plants: CG, CHG and CHH (where H = A, C or T). Genes are usually methylated within the promoters (so-called** ‘promoter-methylated**’), or within the transcribed regions away from the 3′ and 5′ end (so-called ‘body-methylated’).

• Promoter-methylation is usually linked to transcriptional silencing, whereas body-methylation prefers exons and likely plays an effect on exon definition during splicing.

• Growing evidence shows that NATs are involved in epigenetic regulation, including DNA methylation and histone modification.

NAT genes and NAT-siRNAs in chromosomes.

From outermost to innermost, the first track demonstrates Arabidopsis chromosomes, the second and third tracks show the NAT genes on Watson and Crick strands, respectively, the fourth and fifth tracks show the distributions of all smRNAs and NAT-siRNAs, innermost links demonstrate the NAT pairs’ loci in chromosomes.

Statistics of genes and candidate NATs in A. thaliana

Long Non-coding RNAs and Their Biological Roles in Plants

http://www.sciencedirect.com/science/article/pii/S167202291500042X

long npcRNAs or ncRNAs (lnpcRNAs or lncRNAs) represent diverse classes of transcripts longer than 200 nucleotides

lncRNA : RNA transcripts that contain >200 nt but lack protein- coding potential.

lncRNAs are transcribed by RNA polymerase II or III, and additionally, by polymerase IV/V in plants

They are processed by splicing or non-splicing, polyadenylation or non-polyadenylation, and can be located in the nucleus or cytoplasm. Functional analyses of lncRNAs have shown that they are potent cis- and trans- regulators of gene transcription, and act as scaffolds for chromatin-modifying complexes. As potent regulatory compo- nents involved in gene regulation from various aspects, lncRNAs can exert their effects during tissue development and in response to external stimuli.

About 6480 lncRNAs were identified from 200 Arabidopsis thaliana transcriptomic data sets, with either organ-specific or stress-induced expression profiles

文献 Genome-Wide Analysis Uncovers Regulation of Long Intergenic Noncoding RNAs in Arabidopsis

Using a strand-specific RNA sequencing approach

文献 Long noncoding RNAs responsive to Fusarium oxysporum （镰刀菌） infection in Arabidopsis thaliana

和病菌相关 http://onlinelibrary.wiley.com/doi/10.1111/nph.12537/full

DNA demethylases target promoter transposable elements to positively regulate stress responsive genes in Arabidopsis https://genomebiology.biomedcentral.com/articles/10.1186/s13059-014-0458-3

In plants, the presence of poly(A) lncRNAs was revealed in seedlings of A. thaliana under different stress conditions using RNA-seq . Compared to poly(A)+ lncRNAs, poly(A) lncRNAs are shorter, have lower expression, and are more specific in response to stresses.

文献 Characterization of stress-responsive lncRNAs in Arabidopsis thaliana by integrating expression, epigenetic and structural features http://onlinelibrary.wiley.com/doi/10.1111/tpj.12679/full

在 rice 中的情况
In addition, Zhang et al. performed strand-specific RNA sequencing of rice anthers, pistils, seeds, and shoots. In combination with the analysis of other available rice RNA-seq datasets, they systematically identified 2224 lncRNAs from rice and showed that rice lncRNAs were highly tissue-specific or stage-specific. In total 2292 putative cis-NATs were shown to be expressed, among which 503 cis-NATs were expressed under specific conditions

文献 Genome-wide screening and functional analysis identify a large number of long noncoding RNAs involved in the sexual reproduction of rice

http://genomebiology.biomedcentral.com/articles/10.1186/s13059-014-0512-1

plant lncRNA function

Plant lncRNAs as precursors of miRNAs and other sRNAs

Some lncRNAs are primary transcripts of small regula-tory RNAs such as miRNAs and siRNAs. plant-specific RNA polymerase IV/V (Pol IV/Pol V)- dependent siRNAs and secondary endogenous siRNAs.Biogenesis pathway of the Pol IV/Pol V-dependent siRNAs also produces a plant-specific class of lncRNAs called the Pol IV/V-dependent lncRNAs, which are required for RNA- directed DNA methylation (RdDM) .

Plant lncRNAs as miRNA target mimics

在动物中是competing endogenous RNAs (ceRNAs)

Target mimicry effects can be induced by both endogenous and engineered artificial miRNA TMs

Plant lncRNAs and vernalization

Vernalization is the best-studied regulatory process in plants that is known to involve lncRNAs, primarily in the regulation of FLOWERING LOCUS C (FLC) gene (acts as a repressor to inhibit flowering under cold temperature).FLC gene is located at a complex locus. Recent studies have shown that at least two types of lncRNAs are present in this locus. A group of long antisense RNAs, called COLD INDUCED LONG ANTISENSE INTRAGENIC RNAs (COOLAIR) are transcribed in antisense orientation in relation to FLC and, whereas another lncRNA COLD ASSISTED INTRONIC NONCODING RNA (COLDAIR), is transcribed from the intron of FLC gene in the sense orientation. Both lncRNAs can help recruit PHD-PRC2 complex to enable histone modifications of FLC via epigenetic regulation.

Plant lncRNAs and photomorphogenesis

They focused on the roles of lncRNAs in response to light and identified 626 concordant and 766 discordant NAT pairs in A. thaliana, with many light-responsive lncNATs related to histone modifications.

Plant lncRNAs and phosphate homeostasis Phosphate

Plant lncRNAs and alternative splicing

Bardou et al. reported the involvement of lncRNA in alternative splicing in Arabidopsis. They found that an lncRNA acts as an alternative splicing competitor (ASCO). The ASCO-lncRNA and the nuclear speckle RNA-binding protein (NSR) could form an alternative splicing regulatory module. http://www.sciencedirect.com/science/article/pii/S1534580714004067

Plant lncRNAs and modulation of chromatin loop dynamics

the dual APOLO transcription could control the chromatin loop dynamics to regulate the promoter activity of the neighbor PID gene,

Summary of the lncRNAs reported in plants

Note: COLDAIR, cold assisted intronic noncoding RNA; COOLAIR, cold induced long antisense intragenic RNAs; LDMAR, long day-specific male-fertility-associated RNA; HID1, hidden treasure 1; IPS1, induced by phosphate starvation 1; PHO1;2, PHOSPHATE1;2; PI1, phosphate-limitation inducible gene 1; OsPI1,Oryza sativa phosphate-limitation inducible gene 1; TPS11, tomato phosphate starvation-induced gene; asHSFB2a, natural long non-coding antisense RNA of heat stress transcription factor B; CesA6 lncNAT, natural antisense of CesA6 cellulose synthase gene; SHO, an enzyme responsible for the synthesis of plant cytokinins; ENOD40, early nodulin 40; ASCO, alternative splicing competitor; APOLO, auxin-regulated promoter loop.

Plant lncRNA database

Summary of databases depositing plant lncRNAs

Note:TAIR, The Arabidopsis Information Resource; PlantNATsDB, Plant Natural Antisense Transcripts DataBase; lncRNAdb, A reference database for lncRNAs;NONCODE, An integrated knowledge database of ncRNAs; PLncDB, A plant lncRNA database; PNRD, A plant ncRNA database.

文献

http://nar.oxfordjournals.org/content/43/D1/D982

http://nar.oxfordjournals.org/content/44/D1/D203.long

Long noncoding RNA transcriptome of plants

http://doi.wiley.com/10.1111/pbi.12336

蔡南海实验室

lncRNA的另一种分类

polyadenylated or nonpolyadenylated transcripts with low protein-coding potentia

Nonpolyadenylated lncRNAs

These non-polyadenylated transcripts which are 50–300nt in length have low protein-coding potential and do not show sequence similarity to any known ncRNAs. They are named intermediate-sized ncRNAs (im-ncRNAs)

文献 Genome-wide identification of long noncoding natural antisense transcripts and their responses to light in Arabidopsis http://genome.cshlp.org/cgi/doi/10.1101/gr.165555.113

In addition, hundreds of >200nt non-polyadenylated ncRNAs are induced by** specific abiotic stresses** in Arabidopsis

文献 Characterization of stress-responsive lncRNAs in Arabidopsis thaliana by integrating expression, epigenetic and structural features http://onlinelibrary.wiley.com/doi/10.1111/nph.12537/abstract;jsessionid=41AE7D471510D45F0BDB1B6742541643.f02t03

Polyadenylated lncRNAs

In addition, various types of unstable lncRNAs can also be transcribed from the genomic regions around transcription start sites, enhancer regions, intron splicing sites and/or transcription termination sites

Based on their genomic origins, these lncRNAs can be generally classified into** three large groups**: (i) long intergenic ncRNAs (lincRNAs), (ii) intronic ncRNAs (incRNAs) derived from introns and (iii) natural antisense transcripts (NATs) transcribed from complementary DNA strand of their associated genes.

lncRNA确实含有短的保守序列

Some lncRNA genes may be associated with short conserved elements. Around 11–22% genomic regions encoding lincRNAs of mouse and rice** contain short conserved elements.**

http://www.nature.com/nature/journal/v458/n7235/full/nature07672.html?cookies=accepted

This proportion is similar to or lower than that of the short conserved elements encoded by intronic regions of protein-coding genes http://www.nature.com/ng/journal/v45/n8/full/ng.2684.html

In general, lincRNA, incRNA and NATs expression are highly tissue specific and many are responsive to biotic and/or abiotic stresses. In addition, some** plant pseudogenes or repeats** with transcriptional activities produce RNAs that can also be regarded as a type of lncRNAs

LncRNA biogenesis

Around 40–50% of lncRNA genes contain introns. Introns for some lincRNAs may have regulatory functions in transcription (Chung et al., 2006; Rose, 2002), RNA nuclear export (Akua and Shaul, 2013; Valencia et al., 2008) and suppression of RNA silencing pathway (Christie

Regulation of the transcription machinery by lncRNAs

One mode of action of plant lncRNA is to trigger the formation of a stable RNA–DNA triplex so as to control TF binding specificity on promoter region

LncRNA regulation on histone modification

LncRNA and small RNA pathways

lncRNA和TE以及重复序列有大量重合

A considerable number of lncRNA genes (~49% lincRNAs in Arabidopsis)** overlap with transposable elements or repeats**, producing the so-called repeat-containing lncRNAs (RC-lncRNA)

http://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1003470

recently developed bioinformatics tools have predicted quite a number of endogenous miRNA target mimics in plants. Putative target mimics for 20 conserved miRNAs have been reported and some of them have been shown to be functional by transgenic experiments http://www.plantphysiol.org/content/161/4/1875.full

LncRNA-mediated post-translational regulation

Being flexible, lncRNAs are ideal candidates to specifically interact with RNA-binding proteins (RBPs) to regulate protein–protein interaction,protein modification, subunit assembly of protein complex and/or protein subcellular location.

It is reasonable to hypothesize that ABA-triggered complex assembly and relocalization of RBP proteins may be mediated by lncRNAs; however, detailed mechanisms of these processes and their biological significance require clarification.

lncRNA database papers

Characterization of stress-responsive lncRNAs in Arabidopsis thaliana by integrating expression, epigenetic and structural features

http://doi.wiley.com/10.1111/tpj.12679

Genome-Wide Analysis Uncovers Regulation of Long Intergenic Noncoding RNAs in Arabidopsis
http://www.plantcell.org/cgi/doi/10.1105/tpc.112.102855

Long noncoding RNAs responsive to Fusarium oxysporum infection in Arabidopsis thaliana
http://onlinelibrary.wiley.com/doi/10.1111/nph.12537/full

Genome-wide view of natural antisense transcripts in Arabidopsis thaliana
http://dnaresearch.oxfordjournals.org/cgi/doi/10.1093/dnares/dsv008

Genome-wide identification of long noncoding natural antisense transcripts and their responses to light in Arabidopsis
http://genome.cshlp.org/cgi/doi/10.1101/gr.165555.113

NONCODE 2016: An informative and valuable data source of long non-coding RNAs
http://nar.oxfordjournals.org/lookup/doi/10.1093/nar/gkv1252

stress and lncRNA

http://doi.wiley.com/10.1111/tpj.12679

The framework to identify and characterize lncRNAs in Arabidopsis.

The RNA sequencing reads are first assembled into transcripts. Four filter steps are carried out to remove noises. A total of 995 novel lncRNA transcripts is identified. An integrative model combines expression patterns, epigenetic signatures, sequence and structural features, and classifies the lncRNAs into three confidence levels. Characterization of lncRNAs focus on three aspects: poly(A)+ and poly(A)− classification, stress association by expression pattern, and stress association by sequence and structural motifs. Hundreds of stress-associated poly(A)+ and poly(A)− lncRNAs are identified within different levels.

disease and lncRNA

http://doi.wiley.com/10.1111/nph.12537

• THE role of lncRNAs in disease resistance, we used a strand-specific RNA-sequencing approach to identify lncRNAs responsive to Fusarium oxysporum infection in Arabidopsis thaliana.
• Several noncoding natural antisense transcripts responsive to F. oxysporum infection were found in genes implicated in disease defense.
• the majority of the novel transcriptionally active regions (TARs) were adjacent to annotated genes and could be an extension of the annotated transcripts, 159 novel intergenic TARs, including 20 F. oxysporum-responsive lncTARs, were identified.
• Promoter analysis suggests that some of the F. oxysporum-induced lncTARs are direct targets of transcription factor(s) responsive to pathogen attack.

More and more evidence implies that a significant proportion of these ‘dark matter’ transcripts are noncoding RNAs (ncRNAs) with an important role in a wide range of biological processes, including responding to biotic or abiotic stresses.

Based on their length, ncRNAs are arbitrarily grouped into short (< 200 base pairs (bp)) and long ncRNAs (lncRNAs; > 200 bp). Based on their genomic location and orientation, ncRNAs are classified into intergenic, intronic, promoter- and terminator-associated ncRNAs, and natural antisense ncRNAs.

In Arabidopsis thaliana, a large number of novel transcriptionally active regions (TARs), including TARs induced under various abiotic stress conditions, and natural antisense transcripts (NATs), have been identifie. A number of these TARs and NATs are potential candidate lncRNAs with a biological function.

Fusarium oxysporum is a soilborne plant fungal pathogen causing vascular wilt disease through roots in a wide range of plants, including A. thaliana and economically important crops such as cotton and tomato.

We identified a number of F. oxysporum-responsive lncRNAs, including long noncoding NATs (lncNATs) and lincRNAs, and demon-strated that several of these lncRNAs may play an important role in antifungal immunity.

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