MicroRNAs and siRNAs in plants

In plants, small RNAs (19-27 nt) can be classified into two classes, microRNAs (miRNAs) and short interfering RNAs (siRNAs), play an important role in gene regulation. siRNAs contain trans-acting siRNAs (tasiRNAs), natural antisense transcript siRNAs (nat-siRNAs) and heterochromatic siRNAs (hc-siRNAs). Figure S4 demonstrates that the biogenesis and relationship of miRNAs and each type of sRNA in plants.

The biogenesis of miRNAs in plants

The biogenesis of miRNAs in plants has some difference with animals. In plants, the length of stem-loops generally is longer than that of animal stem-loops. DCL1 (DICER-LIKE1) has the functions of Drosha and Dicer. The miRNA/miRNA*

duplex is produced through DCL1 interacting with HYL1 (HYPONASTIC LEVAES1) to cleave the miRNA precursor in nucleus [95-98]. The mature miRNA is methylated by HEN1 (HEN1 is also in nucleus) [94]. Methylation protects small RNAs from degradation and polyuridylation. Exporting miRNA duplexs or mature miRNAs to the cytoplasm is completed by HASTY [99] (the function is similar with Exportin-5) (Figure S5).

Like animals, plant miRNAs require Argonaute protein for forming RNA-induced silencing complex (RISC) [100]. However, the degree of complementarity between miRNAs and their targets is different in plants. Unlike animals, nearly perfect complementarity between miRNAs and their targets is required in plants [100-103]. Most plants miRNAs only have four or less mismatches with their targets. These mismatches usually locate in the 3’ region of miRNAs [104]. The degree of complemnetarity in the region of miRNA/mRNA duplex at position 3-11 affects the efficiency of cleavage in mRNAs [105]. The

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affection of miRNAs and their targets is also different between animals and plants. In animals, most miRNAs guide translational repression of their target genes. In plants, most miRNAs lead to mRNA cleavage by targeting the coding region of mRNAs. The previous studies demonstrated that about two-thirds of plant miRNAs regulate the expression of transcription factors during plant development. In additional to their great affection in development [106-107], they also play important roles in plant responses to biotic and abiotic stresses and nutrient homeostasis [106, 108-112].

The biogenesis of tasiRNAs

Trans-acting siRNAs (tasiRNAs) which has similar function to miRNAs down-regulate the expression of genes are 21 nt regulatory siRNAs. TasiRNAs are generated from specific miRNAs cutting TAS primary RNAs process. TAS genes transcribe long primary RNAs which do not generate protein products. Its’

function is serving as the precursors of the tasiRNA. In processing tasiRNAs, miRNAs guide the cleavage of tasiRNA primary transcripts which are converted into the structure of dsRNA by SGS3 and RDR6 binding to one of the two single-stranded TAS cleavage sequences. Then, DCL4 (DICER-LIKE4) with DRB4 cuts the dsRNAs and produces 21 nt tasiRNA-mRNA duplexes (Figure S6) [113-115]. Like miRNAs, tasiRNAs are methylated by HEN1 for avoiding degradation and polyuridylation. Different tasiRNA families regulate the gene expression by forming RISC with different AGO family protein (Figure S4). AGO1 involves with TAS2 tasiRNAs and AGO7 involves with TAS3 tasiRNAs. The high degree of complementarity between tasiRNAs and mRNA is needed to guide mRNA cleavage. The different members of same gene family are targeted by either miRNAs or tasiRNAs. For example, miR161 or TAS2 tasiRNA target the

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members of PPR family and miR160, miRN167 or TAS3 tasiRNA target the members of ARF family, respectively. According to previous studies report, tasiRNAs are found only in plants [116].

The biogenesis of nat-siRNAs

Natural antisense transcript siRNAs (nat-siRNAs), 24 or 21 nt siRNAs, are generated from natural antisense transcripts (NATs). NATs are formed by two coding or non-coding RNAs that have complementary regions. There are two classes of NATs, cis-NATs and trans-NATs [50, 117-125]. The transcripts in the same genomic locus but in different strands form cis-NATs. For example, the genomic locus of SRO5 is at chr5: 25097998-2509997 [+]. The genomic locus of P5CDH is at chr5: 25099003-25103298 [-]. The overlapping region is at chr5:

25099003-2509997. The cis-NATs can be categorized into three groups, convergent (overlapping in the 3’ ends of two transcripts), divergent (overlapping in the 5’ end of two transcripts) and enclosed (one transcript can completely overlap another transcript) according to the conditions of overlapping. Trans-NATs are formed by the overlapping regions of two transcripts from different genomic locus.

The 24 nt nat-siRNAs are derived from the complementary region by the interaction of DCL2 (DICER-LIKE2), NRPD1a, RDR6 and SGS3. These 24 nt nat-siRNAs guides the cleavage of the constitutive transcript and establishes a phase for the sequential production of 21 nt nat-siRNAs by DCL1 (Figure 7).

Unlike tasiRNAs, the function of nat-siRNAs is not for targeting other mRNAs. It can lead to post-transcriptional gene silencing by hybridizing to the cis-strand of mRNA. The member of AGO family involves in this mechanism does not be understood clearly. The well known example is SRO5 and P5CDH in Arabidopsis.

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SRO5 and P5CDH have the function of regulating salt tolerance [126]. SRO5 and P5CDH have the overlapping region in their 3’end of transcripts. P5CDH is expressed constitutively. When SRO5 is induced in response to salt stress, the 24 nt nat-siRNAs are derived from the overlapping region of these two transcripts by DCL2, RDR6 and SGS3 interaction. These siRNAs direct the cleavage of P5CDH transcripts (Figure S7). Meanwhile, the dsRNAs are formed by RDR6, SGS3 and SDE4. 21 nt nat-siRNAs are produced from the cleavage of dsRNAs by DCL1. Then, the mRNA of P5CDH are degraded through these 21 nt nat-siRNAs targeting P5CDH.

The biogenesis of trans- nat-siRNAs does not be understood clearly.

However, previous studies suggests that trans- NAT-siRNAs are involved in alternative splicing, post-transcriptional gene silencing [127-129]. There are some studies using the computational methods to screen whole Arabidopsis transcripts to find trans-NATs.

The biogenesis of hc-siRNAs

Heterochromatic siRNAs (hc-siRNAs), 24 nt siRNAs, are produced from transposable or repetitive elements [130-132]. Single strand non-coding transcripts are transcribed from heterochromatic locs by Pol IV and CLASSY1 (Figure S8). RDR2 involves in the formation of dsRNAs through using transcripts as templates. Then, the dsRNAs are processed into 24 nt siRNAs by DCL3. Methylation by HEN1 joins the process for protecting degradation and polyuridylation. The siRNAs are loaded into AGO4-RISC complex. DNA methylation and heterochomatic histone modifications are affected by PoI V and AGO4 interaction and the interaction in DRD1 (potential chromatin remodeling protein), DMS3 (structural maintenance of chromosomes hinge-domian protien)

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and Pol V [133-137]. In Arabidopsis, the siRNAs derived from a transposable element inserting in the intron of FLC (FLOWERING LOCUS C) genes lead to the reduction of FLC and early flower. FWA gene is silenced through the siRNAs, generated from two tandem repeats in the promoter region of FWA, triggering DNA methylation. The siRNAs derived from the tandem repeats in the promoter region of SDC, a gene ecncoding the F-box protein, silenced SDC by triggering DNA methylation.

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