Loss of Function of OsDCL1 Affects MicroRNA Accumulation and Causes Developmental Defects in Rice

Abstract

MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are two types of noncoding RNAs involved in developmental regulation, genome maintenance, and defense in eukaryotes. The activity of Dicer or Dicer-like (DCL) proteins is required for the maturation of miRNAs and siRNAs. In this study, we cloned and sequenced 66 candidate rice (Oryza sativa) miRNAs out of 1,650 small RNA sequences (19 to approximately 25 nt), and they could be further grouped into 21 families, 12 of which are newly identified and three of which, OsmiR528, OsmiR529, and OsmiR530, have been confirmed by northern blot. To study the function of rice DCL proteins (OsDCLs) in the biogenesis of miRNAs and siRNAs, we searched genome databases and identified four OsDCLs. An RNA interference approach was applied to knock down two OsDCLs, OsDCL1 and OsDCL4, respectively. Strong loss of function of OsDCL1IR transformants that expressed inverted repeats of OsDCL1 resulted in developmental arrest at the seedling stage, and weak loss of function of OsDCL1IR transformants caused pleiotropic developmental defects. Moreover, all miRNAs tested were greatly reduced in OsDCL1IR but not OsDCL4IR transformants, indicating that OsDCL1 plays a critical role in miRNA processing in rice. In contrast, the production of siRNA from transgenic inverted repeats and endogenous CentO regions were not affected in either OsDCL1IR or OsDCL4IR transformants, suggesting that the production of miRNAs and siRNAs is via distinct OsDCLs.

Duplication and expression analysis of multicopy miRNA gene family members in Arabidopsis and rice

“…Indeed, the vast majority of the predicted or verified miRNA targets encode members of large families of transcription factors that regulate development [15]. Mutants that lack miRNA pathway proteins, such as DICER-LIKE 1 (DCL1), ARGONAUTE 1 (AGO1) and HUA ENHANCER 1 (HEN1), exhibit morphological defects in Arabidopsis and rice, suggesting an important role for miRNA in plant development [8,14,[16][17][18][19][20]. Recently, more and more plant miRNAs have been reported for their specialized developmental functions, such as miR159, miR-JAW, miR160, miR162, miR165/166, miR168, and miR172.…”

Fine tuning of auxin signaling by miRNAs

“…This is evident from the study of loss of function mutants of enzymes involved in miRNA biogenesis. dcl1 mutant shows reduced expression level of mature miRNAs and exhibits many developmental abnormalities including immature embryos, altered leaf shape and morphology, delayed floral transition, and female sterility (Park et al, 2002;Reinhart et al, 2002;Liu et al, 2005;Kurihara et al, 2006). Similarly, hst mutant also exhibits pleiotropic abnormalities, such as abnormal leaf and flower morphology, accelerated phase transition and reducing fertility (Bollman et al, 2003).…”

The Cornucopia of Small RNAs in Plant Genomes

“…Initially, many miRNAs in rice were sequenced through the traditional Sanger sequencing method, most of which turned out to be the high-abundance miRNAs [16,33] Fig. 3 The plant miRNA biogenesis pathway and cellular function.…”

“…Information about rice DCLs is limited in comparison to Arabidopsis. However, studies by Liu et al [15,16] utilized knock-down and loss-of-function dcl1 and dcl4 RNAi mutants to demonstrate a role for OsDCL1 and OsDCL4 in small RNA biogenesis and plant development. The loss of function of OsDCL1 led to shoot and root abnormalities, such as rolled leaves and reduced root elongation.…”

“…Functional redundancies and competition among DCL2, DCL3, and DCL4 in small RNA biogenesis have been reported [12,13]. Similarly, in rice, OsDCL1 and OsDCL4 also function in the biogenesis of miRNAs and siRNAs, respectively [14][15][16].…”