SINE RNA induces severe developmental defects in Arabidopsis thaliana and interacts with HYL1 (DRB1), a key member of the DCL1 complex

The proper temporal and spatial expression of genes during plant development is governed, in part, by the regulatory activities of various types of small RNAs produced by the different RNAi pathways. Here we report that transgenic Arabidopsis plants constitutively expressing the rapeseed SB1 SINE retroposon exhibit developmental defects resembling those observed in some RNAi mutants. We show that SB1 RNA interacts with HYL1 (DRB1), a double-stranded RNA-binding protein (dsRBP) that associates with the Dicer homologue DCL1 to produce microRNAs. RNase V1 protection assays mapped the binding site of HYL1 to a SB1 region that mimics the hairpin structure of microRNA precursors. We also show that HYL1, upon binding to RNA substrates, induces conformational changes that force single-stranded RNA regions to adopt a structured helix-like conformation. Xenopus laevis ADAR1, but not Arabidopsis DRB4, binds SB1 RNA in the same region as HYL1, suggesting that SINE RNAs bind only a subset of dsRBPs. Consistently, DCL4-DRB4-dependent miRNA accumulation was unchanged in SB1 transgenic Arabidopsis, whereas DCL1-HYL1-dependent miRNA and DCL1-HYL1-DCL4-DRB4-dependent tasiRNA accumulation was decreased. We propose that SINE RNA can modulate the activity of the RNAi pathways in plants and possibly in other eukaryotes.     

The interaction between DCL1 and HYL1 is important for efficient and precise processing of pri-miRNA in plant microRNA biogenesis

It has been reported that some double-stranded RNA (dsRNA) binding proteins interact with small RNA biogenesis-related RNase III enzymes. However, their biological significance is poorly understood. Here we examine the relationship between the Arabidopsis microRNA- (miRNA) producing enzyme DCL1 and the dsRNA binding protein HYL1. In the hyl1-2 mutant, the processing steps of miR163 biogenesis were partially impaired; increased accumulation of pri-miR163 and reduced accumulation of short pre-miR163 and mature miR163 as well as misplaced cleavages in the stem structure of pri-miR163 were detected. These misplaced cleavages were similar to those previously observed in the dcl1-9 mutant, in which the second double-stranded RNA binding domain of the protein was disrupted. An immunoprecipitation assay using Agrobacterium-mediated transient expression in Nicotiana benthamiana showed that HYL1 was able to form a complex with wild-type DCL1 protein, but not with the dcl1-9 mutant protein. We also examined miR164b and miR166a biogenesis in hyl1-2 and dcl1-9. Increased accumulation of pri-miRNAs and reduced accumulation of pre-miRNAs and mature miRNAs were detected. Misplaced cleavage on pri-miR164b was observed only in dcl1-9 but not in hyl1-2, whereas not on pri-miR166a in either mutant. These results indicate that HYL1 has a function in assisting efficient and precise cleavage of pri-miRNA through interaction with DCL1.     

Specific interactions between Dicer-like proteins and HYL1/DRB- family dsRNA-binding proteins in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Proteins that specifically bind double-stranded RNA (dsRNA) are involved in the regulation of cellular signaling events and gene expression, and are characterized by a conserved dsRNA-binding motif (dsRBM). Here we report the biochemical properties of nine such gene products, each containing one or two dsRBMs: four ArabidopsisDicer-like proteins (DCL1-4), ArabidopsisHYL1 and four of its homologs (DRB2, DRB4, DRB5 and OsDRB1). DCL1, DCL3, HYL1 and the four HYL1 homologs exhibit significant dsRNA-binding activity, indicating that these proteins are involved in RNA metabolism. The dsRBMs from dsRBM-containing proteins (dsRBPs) also function as a protein–protein interaction domain and homo- and heterodimerization are essential for biological functioning of these proteins. We show that DRB4 interacts specifically with DCL4, and HYL1 most strongly interacts with DCL1. These results indicate that each HYL1/DRB family protein interacts with one specific partner among the four Dicer-like proteins. Localization studies using GFP fusion proteins demonstrate that DCL1, DCL4, HYL1 and DRB4 localize in the nucleus, while DRB2 is present in the cytoplasm. Subcellular localizations of HYL1, DRB4, DCL1 and DCL4 further strengthen the notion that HYL1 and DCL1, and DRB4 and DCL4, exist as complexes. The presented data suggest that each member of the HYL1/DRB protein family may individually modulate Dicer function through heterodimerization with a Dicer-like protein in vivo.     

Homodimerization of HYL1 ensures the correct selection of cleavage sites in primary miRNA

MicroRNA (miRNA) plays an important role in the control of gene expression. HYPONASTIC LEAVES1 (HYL1) is a double-stranded RNA-binding protein that forms a complex with DICER-LIKE1 (DCL1) and SERRATE (SE) to process primary miRNA (pri-miRNA) into mature miRNA. Although HYL1 has been shown to partner with DCL1 to enhance miRNA accuracy, the mechanism by which HYL1 selects the DCL1-targeted cleavage sites in pri-miRNA has remained unknown. By mutagenesis of HYL1 and analysis of in vivo pri-miRNA processing, we investigated the role of HYL1 in pri-miRNA cleavage. HYL1 forms homodimers in which the residues Gly¹⁴⁷ and Leu¹⁶⁵ in the dsRBD2 domain are shown to be critical. Disruption of HYL1 homodimerization causes incorrect cleavage at sites in pri-miRNA without interrupting the interaction of HYL1 with DCL1 and accumulation of pri-miRNAs in HYL1/pri-miRNA complexes, leading to a reduction in the efficiency and accuracy of miRNAs that results in strong mutant phenotypes of the plants. HYL1 homodimers may function as a molecular anchor for DCL1 to cleave at a distance from the ssRNA–dsRNA junction in pri-miRNA. These results suggest that HYL1 ensures the correct selection of pri-miRNA cleavage sites through homodimerization and thus contributes to gene silencing and plant development.     

Gene structures and processing of Arabidopsis thaliana HYL1-dependent pri-miRNAs

Arabidopsis thaliana HYL1 is a nuclear double-stranded RNA-binding protein involved in the maturation of pri-miRNAs. A quantitative real-time PCR platform for parallel quantification of 176 pri-miRNAs was used to reveal strong accumulation of 57 miRNA precursors in the hyl1 mutant that completely lacks HYL1 protein. This approach enabled us for the first time to pinpoint particular members of MIRNA family genes that require HYL1 activity for efficient maturation of their precursors. Moreover, the accumulation of miRNA precursors in the hyl1 mutant gave us the opportunity to carry out 3′ and 5′ RACE experiments which revealed that some of these precursors are of unexpected length. The alignment of HYL1-dependent miRNA precursors to A. thaliana genomic sequences indicated the presence of introns in 12 out of 20 genes studied. Some of the characterized intron-containing pri-miRNAs undergo alternative splicing such as exon skipping or usage of alternative 5′ splice sites suggesting that this process plays a role in the regulation of miRNA biogenesis. In the hyl1 mutant intron-containing pri-miRNAs accumulate alongside spliced pri-miRNAs suggesting the recruitment of HYL1 into the miRNA precursor maturation pathway before their splicing occurs.     

A role for the Dicer helicase domain in the processing of thermodynamically unstable hairpin RNAs

In humans a single species of the RNAseIII enzyme Dicer processes both microRNA precursors into miRNAs and long double-stranded RNAs into small interfering RNAs (siRNAs). An interesting but poorly understood domain of the mammalian Dicer protein is the N-terminal helicase-like domain that possesses a signature DExH motif. Cummins et al. created a human Dicer mutant cell line by inserting an AAV targeting cassette into the helicase domain of both Dicer alleles in HCT116 cells generating an in-frame 43-amino-acid insertion immediately adjacent to the DExH box. This insertion creates a Dicer mutant protein with defects in the processing of most, but not all, endogenous pre-miRNAs into mature miRNA. Using both biochemical and computational approaches, we provide evidence that the Dicer helicase mutant is sensitive to the thermodynamic properties of the stems in microRNAs and short-hairpin RNAs, with thermodynamically unstable stems resulting in poor processing and a reduction in the levels of functional mi/siRNAs. Paradoxically, this mutant exhibits enhanced processing efficiency and concomitant RNA interference when thermodynamically stable, long-hairpin RNAs are used. These results suggest an important function for the Dicer helicase domain in the processing of thermodynamically unstable hairpin structures.     

Molecular mechanisms that funnel RNA precursors into endogenous small-interfering RNA and microRNA biogenesis pathways in Drosophila

In Drosophila, three types of endogenous small RNAs—microRNAs (miRNAs), PIWI-interacting RNAs (piRNAs), and endogenous small-interfering RNAs (endo-siRNAs or esiRNAs)—function as triggers in RNA silencing. Although piRNAs are produced independently of Dicer, miRNA and esiRNA biogenesis pathways require Dicer1 and Dicer2, respectively. Recent studies have shown that among the four isoforms of Loquacious (Loqs), Loqs-PB and Loqs-PD are involved in miRNA and esiRNA processing pathways, respectively. However, how these Loqs isoforms function in their respective small RNA biogenesis pathways remains elusive. Here, we show that Loqs-PD associates specifically with Dicer2 through its C-terminal domain. The Dicer2–Loqs-PD complex contains R2D2, another known Dicer2 partner, and excises both exogenous siRNAs and esiRNAs from their corresponding precursors in vitro. However, Loqs-PD, but not R2D2, enhanced Dicer2 activity. The Dicer2–Loqs-PD complex processes esiRNA precursor hairpins with long stems, which results in the production of AGO2-associated small RNAs. Interestingly, however, small RNAs derived from terminal hairpins of esiRNA precursors are loaded onto AGO1; thus, they are classified as a new subset of miRNAs. These results suggest that the precursor RNA structure determines the biogenesis mechanism of esiRNAs and miRNAs, thereby implicating hairpin structures with long stems as intermediates in the evolution of Drosophila miRNA.     

Functional mapping of the plant small RNA methyltransferase: HEN1 physically interacts with HYL1 and DICER-LIKE 1 proteins

Methylation of 3′-terminal nucleotides of miRNA/miRNA* is part of miRNAs biogenesis in plants but is not found in animals. In Arabidopsis thaliana this reaction is carried out by a multidomain AdoMet-dependent 2′-O-methyltransferase HEN1. Using deletion and structure-guided mutational analysis, we show that the double-stranded RNA-binding domains R¹ and R² of HEN1 make significant but uneven contributions to substrate RNA binding, and map residues in each domain responsible for this function. Using GST pull-down assays and yeast two-hybrid analysis we demonstrate direct HEN1 interactions, mediated by its FK506-binding protein-like domain and R² domain, with the microRNA biogenesis protein HYL1. Furthermore, we find that HEN1 forms a complex with DICER-LIKE 1 (DCL1) ribonuclease, another key protein involved in miRNA biogenesis machinery. In contrast, no direct interaction is detectable between HEN1 and SERRATE. On the basis of these findings, we propose a mechanism of plant miRNA maturation which involves binding of the HEN1 methyltransferase to the DCL1•HYL1•miRNA complex excluding the SERRATE protein.     

Structural and mechanistic insight into stem‐loop RNA processing by yeast Pichia stipitis Dicer

Dicer is a member of the ribonuclease III enzyme family and processes double‐stranded RNA into small functional RNAs. The variation in the domain architecture of Dicer among different species whilst preserving its biological dicing function is intriguing. Here, we describe the structure and function of a novel catalytically active RNase III protein, a non‐canonical Dicer (PsDCR1), found in budding yeast Pichia stipitis. The structure of the catalytically active region (the catalytic RNase III domain and double‐stranded RNA‐binding domain 1 [dsRBD1]) of DCR1 showed that RNaseIII domain is structurally similar to yeast RNase III (Rnt1p) but uniquely presents dsRBD1 in a diagonal orientation, forming a catalytic core made of homodimer and large RNA‐binding surface. The second dsRNA binding domain at C‐terminus, which is absent in Rnt1, enhances the RNA cleavage activity. Although the cleavage pattern of PsDCR1 anchors an apical loop similar to Rnt1, the cleavage activity depended on the sequence motif at the lower stem, not the apical loop, of hairpin RNA. Through RNA sequencing and RNA mutations, we showed that RNA cleavage by PsDCR1 is determined by the stem‐loop structure of the RNA substrate, suggesting the possibility that stem‐loop RNA‐guided gene silencing pathway exists in budding yeast.     

The Role of Human Dicer-dsRBD in Processing Small Regulatory RNAs

One of the most exciting recent developments in RNA biology has been the discovery of small non-coding RNAs that affect gene expression through the RNA interference (RNAi) mechanism. Two major classes of RNAs involved in RNAi are small interfering RNA (siRNA) and microRNA (miRNA). Dicer, an RNase III enzyme, plays a central role in the RNAi pathway by cleaving precursors of both of these classes of RNAs to form mature siRNAs and miRNAs, which are then loaded into the RNA-induced silencing complex (RISC). miRNA and siRNA precursors are quite structurally distinct; miRNA precursors are short, imperfect hairpins while siRNA precursors are long, perfect duplexes. Nonetheless, Dicer is able to process both. Dicer, like the majority of RNase III enzymes, contains a dsRNA binding domain (dsRBD), but the data are sparse on the exact role this domain plays in the mechanism of Dicer binding and cleavage. To further explore the role of human Dicer-dsRBD in the RNAi pathway, we determined its binding affinity to various RNAs modeling both miRNA and siRNA precursors. Our study shows that Dicer-dsRBD is an avid binder of dsRNA, but its binding is only minimally influenced by a single-stranded – double-stranded junction caused by large terminal loops observed in miRNA precursors. Thus, the Dicer-dsRBD contributes directly to substrate binding but not to the mechanism of differentiating between pre-miRNA and pre-siRNA. In addition, NMR spin relaxation and MD simulations provide an overview of the role that dynamics contribute to the binding mechanism. We compare this current study with our previous studies of the dsRBDs from Drosha and DGCR8 to give a dynamic profile of dsRBDs in their apo-state and a mechanistic view of dsRNA binding by dsRBDs in general.     

Cytoplasmic HYL1 modulates miRNA-mediated translational repression

MicroRNAs (miRNAs) control various biological processes by repressing target mRNAs. In plants, miRNAs mediate target gene repression via both mRNA cleavage and translational repression. However, the mechanism underlying this translational repression is poorly understood. Here, we found that Arabidopsis thaliana HYPONASTIC LEAVES1 (HYL1), a core component of the miRNA processing machinery, regulates miRNA-mediated mRNA translation but not miRNA biogenesis when it localized in the cytoplasm. Cytoplasmic HYL1 localizes to the endoplasmic reticulum and associates with ARGONAUTE1 (AGO1) and ALTERED MERISTEM PROGRAM1. In the cytoplasm, HYL1 monitors the distribution of AGO1 onto polysomes, binds to the mRNAs of target genes, represses their translation, and partially rescues the phenotype of the hyl1 null mutant. This study uncovered another function of HYL1 and provides insight into the mechanism of plant gene regulation.

The nuclear miRNA biogenesis factor HYL1 also localizes to the cytoplasm to modulate miRNA-mediated translational repression.     

Genetic analyses reveal a requirement for Dicer1 in the mouse urogenital tract

Despite the increasing interest in other classes of small RNAs, microRNAs (miRNAs) remain the most widely investigated and have been shown to play a role in a number of different processes in mammals. Many studies investigating miRNA function focus on the processing enzyme Dicer1, which is an RNAseIII protein essential for the biogenesis of active miRNAs through its cleavage of precursor RNA molecules. General deletion of Dicer1 in the mouse confirms that miRNAs are essential for development because embryos lacking Dicer1 fail to reach the end of gastrulation. Here we investigate the role of Dicer1 in urogenital tract development. We utilised a conditional allele of the Dicer1 gene and two Cre-expressing lines, driven by HoxB7 and Amhr2, to investigate the effect of Dicer1 deletion on both male and female reproductive tract development. Data presented here highlight an essential role for Dicer1 in the correct morphogenesis and function of the female reproductive tract and confirm recent findings that suggest Dicer1 is required for female fertility. In addition, HoxB7:Cre-mediated deletion in ureteric bud derivatives leads to a spectrum of anomalies in both males and females, including hydronephrotic kidneys and kidney parenchymal cysts. Male reproductive tract development, however, remains largely unaffected in the absence of Dicer1. Thus, Dicer1 is required for development of the female reproductive tract and also normal kidney morphogenesis.     

Stop the dicing in hematopoiesis: What have we learned?

MicroRNAs (miRNAs) belong to an abundant class of highly conserved small (22nt) non-coding RNAs. MiRNA profiling studies indicate that their expression is highly cell type-dependent. DICER1 is an essential RNase III endoribonuclease for miRNA processing. Hematopoietic cell type- and developmental stage-specific Dicer1 deletion models show that miRNAs are essential regulators of cellular survival, differentiation and function. For instance, miRNA deficiency in hematopoietic stem cells and progenitors of different origins results in decreased cell survival, dramatic developmental aberrations or dysfunctions in mice. We recently found that homozygous Dicer1 deletion in myeloid-committed progenitors results in an aberrant expression of stem cell genes and induces a regained self-renewal capacity. Moreover, Dicer1 deletion causes a block in macrophage development and myeloid dysplasia, a cellular condition that may be considered as a preleukemic state. However, Dicer1-null cells do not develop leukemia in mice, indicating that depletion of miRNAs is not enough for tumorigenesis. Surprisingly, we found that heterozygous Dicer1 deletion in myeloid-committed progenitors, but not Dicer1 knockout, collaborates with p53 deletion in leukemic progression and results in various types of leukemia. Our data indicate that Dicer1 is a haploinsufficient tumorsuppressor in hematopoietic neoplasms, which is consistent with the observed downregulation of miRNA expression in human leukemia samples. Here, we review the various hematopoietic specific Dicer1 deletion mouse models and the phenotypes observed within the different hematopoietic lineages and cell developmental stages. Finally, we discuss the role for DICER1 in mouse and human malignant hematopoiesis.