Regulation role of small RNA

The regulation role of miRNA in animals

The miRNAs regulate the gene expression by targeting the 3’-UTR of mRNAs, resulting in degradation of mRNAs and repression of translation. miRNAs are reported strongly associated with various pathways such as cancer pathway and cholesterol metabolic pathway.

The well-known example in cancer pathway is that miRNA-34 (miR-34) family, miR-34 a, b and c, is down-regulated in various cancers. Figure S9 demonstrates the regulation role of miR-34 family in cancer pathway [138]. p53 is the key factor which regulate the abundance of miR-34 in cancer pathway.

miR-34 family targets the set of mRNAs which support tumor formation such as E2F3, Bcl2, Notch, HMGA2, CDK4, CDK6 and Cyclin E2. E2F3 is an important transcription factor which initiated the production of proteins that affect cell-cycle checkpoint, DNA repair and replication. miR-34 family inhibits cell proliferation and activates cell death pathways by down-regulating the expression of E2F3. Bcl2 which usually over-expressed in cancer cells is the important gene in tumor formation. It protects tumor cells processed into apoptosis pathway. Reduction of Bcl2 expression level by miR-34 family targeting induces the apoptosis of cancer cells. The other miR-34 target genes such as Notch, HMGA2, CDK4, CDK6 and Cyclin E2 are involved self-renewal and survival of cancer stem cells. Therefore, the miR-34 family can inhibit tumor formation. According to the function of these miR-34 target genes, miR-34 family is a tumor suppressor in cancer pathway.

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Recent studies reported miRNAs involve in cholesterol metabolic pathway.

miR-33 a and b are the intragenic miRNAs [139-141]. Their host genes are SREBP (sterol regulatory element-binding protein) family. SREBP family is the transcription factor which is the key protein in cholesterol biosynthesis. miR-33 is co-expressed with its host gene (SREBP) and targets ABCA1 (adenosine triphosphate-binding cassette transporter A1). ABCA1 is the important regulator in HDL (high-density lipoprotein) synthesis and reversing cholesterol transport.

Figure S10 demonstrates the regulation role of miR-33 in cholesterol metabolic pathway. Cholesterol accumulation induces the expression of ABCA1 and inhibits the expression of SREBP. ABCA1 medicates cholesterol efflux to apolipoprotein A1 (apoA-1) and HDL. The expression of ABCA1 is down-regulated by miR-33.

Therefore, the role of miR-33 in cholesterol metabolic pathway is regulating the expression level of HDL with SREBP (the host gene of miR-33) by targeting ABCA1.

The regulation role of small RNAs in plants

In plants, many genes silencing are through small RNA such as miRNAs and siRNAs. miRNAs regulate the expression level of genes by targeting the coding region of the mRNA. siRNAs regulate the gene expression through multiple ways such as directly targeting the coding region of mRNA or transcriptional gene silencing by triggering DNA methylation and histone modifications. Table S1 lists the reported miRNAs which are involved in root development in Arabidopsis and Rice [142].

The miRNAs involve in auxin signing pathway are miR160, miR164, miR167, miR390 and miR393. miR160 targets ARF10, ARF16 and ARF17. The function of ARF10 and ARF16 is for root cap development. Overexpressing miR160 causes

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serious root cap defect in rice (the targeting relationships do not be validated yet). The function of ARF17 is reducing primary root length and decreasing root branching in Arabidopsis. miR164 is the key regulator in lateral root development.

miR164 targets NAC1 which transduces auxin signal for the emergence of lateral root. So, miR164 down-regulates auxin signal in lateral root development.

miR167 targets ARF6 and ARF8 which are positive regulator of shoot-borne root emergence. miR390 regulate the expression of ARF2, ARF3 and ARF4 which involve in auxin signaling pathway by indirect targeting. miR390 target TAS3 which is the non-coding transcript and produce tasiRNAs. These tasiRNAs direct target ARF2, ARF3 and ARF4. miR390 regulates the auxin signal concentration and lateral root development by TAS3-ARF2/ARF3/ARF4 pathway.

miR393 targets TIR1 (transport inhibitor response 1), AFB2 (auxin signaling F-Box proteins 2) and AFB3. So, miR393 regulates auxin signaling. Moreover, NAC1, the target gene of miR164, is the downstream of TIR1. NAC1 transduce auxin signal to promote lateral root development. Therefore, miR164 and miR393 may participate together in lateral root development.

Vegetative and reproductive processes of plant root require mineral elements in soil such as macronutrients, micronutrients and heavy metals.

Several miRNA families involve in nutrition response or metabolism which affect plant growth. The first miRNA family is miR395 which is the key regulatore in sulphate metabolism by targeting SULRT2;1 (low-affinity sulphate transporter), APS1 and APS2 (ATP sulphurylases). The second miRNA family is miR398 which affects copperand zinc homeostasis by targeting CSD1 and CSD2. CSD1 and CSD2 are copper/zinc superoxide dismutase genes. Heavy metals such as copper and zinc are required for root growth. Therefore, miRNA398 has great influence in

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plant root development and growth behavior of plants. The final miRNA family is miR399. miR399 involves in phosphate starvation response in Arabidopsis. The concentration of phosphate is one of important factor in root system architecture and growth habit. miR399 regulates phosphate homeostasis by targeting PHO2.

External abiotic stress such as drought affects plant root growth. miR169 is induced by drought stress in Rice. But the target genes of miR169 are not identified.

miRNAs in plants also involve in other pathway excluding root development [133]. For example, miR156 targets over 50% members of SPL family. SPL 3, 4 and 5 promote the change of vegetative phase and floral transition. The length of plastochron is regulated by SPL9 and SPL15. miR172 targets AP2 (APETALA2) which regulates floral transitions. miR159 targets MYB33, MYB65 and MYB101.

These MYB genes induce Gibberellin-responsive (GA) genes in the aleurone layer.

Floral pattering and leaf morhpgenesis are regulated through miR164 targeting CUC1 (CUPSHAPED COTYLDON1) and CUC2 (CUPSHAPED COTYLDON2).

miR165/166 targets RHB and REV which promote abaxial identity of lateral organs in both Arabidopsis and maize. miR319 targets several TCP genes (a large family of proteins containing “TCP domain”). TCP genes regulate cell divisons during leaf morphogenesis.

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