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PubMed2006

Arabidopsis endogenous small RNAs: highways and byways.

Several classes of endogenous small RNAs of 20-26 nucleotides, such as microRNAs and small interfering (si)RNAs, have been described in plants and animals. Arabidopsis currently seems to be an exception because a novel genetically distinguishable small RNA class, termed natural-antisense transcript derived siRNAs, was recently identified. Pathways for the biogenesis and function of each class are already well characterized, enabling their classification on the basis of both size and proteins required for their function. However, each pathway requires proteins belonging to conserved protein families, and recent genetic studies confirmed an expected partial redundancy between members of the Dicer-like (DCL) family. Moreover, several experimental results indicate redundancies among double-strand RNA binding proteins and ARGONAUTE proteins, which probably create a complex network of small RNA pathways.

Arabidopsis↗

PubMed2006

Silencing suppression by geminivirus proteins.

RNA silencing is an RNA-directed gene regulatory system that is present in a wide range of eukaryotes, and which functions as an antiviral defense in plants. Silencing pathways are complex and partially overlapping, but at least three basic classes can be distinguished: cytoplasmic RNA silencing (or post-transcriptional gene silencing; PTGS) mediated by small interfering RNAs (siRNAs), silencing mediated by microRNAs (miRNAs), and transcriptional gene silencing (TGS) mediated by siRNA-directed methylation of DNA and histone proteins. Recent advances in our understanding of different geminivirus silencing suppressors indicate that they can affect all three pathways, suggesting that multiple aspects of silencing impact geminivirus replication.

Capsid Proteins↗

PubMed2026

CRISPR-mediated intronic knock-in of pre-amiRNA enables targeted gene silencing.

This study introduces an intronic artificial microRNA (IamiRNA) strategy that combines CRISPR-Cas9-mediated knock-in with endogenous miRNA processing for targeted gene silencing in plants. By inserting amiRNA precursors into introns of endogenous genes, this approach enables effective, tissue-specific gene silencing without persistent transgene expression, offering a promising tool for functional genomics and crop improvement.

Introns↗

PubMed2005

The role of ARGONAUTE1 (AGO1) in meristem formation and identity.

The ARGONAUTE gene family is involved in the regulation of gene expression via the RNAi Silencing Complex (RISC). microRNA (miRNA) are 20-22bp RNAs that direct RISC to target genes. Several miRNA have been characterized in plants. Their roles include control of flowering time, floral organ identity, cell division patterns, and leaf polarity. ARGONAUTE1 (AGO1) is required for stem cell function and organ polarity, as is the closely related protein PINHEAD/ZWILLE (PNH/ZLL). Through phenotypic and double mutant analysis, we show that AGO1 regulates stem cell function via SHOOT MERISTEMLESS (STM). CUPSHAPED COTYLEDONS1 and 2 (CUC1 and CUC2) positively regulate STM and are targets of miRNA. The effect of AGO1 on leaf polarity is dependent, in part, on its role in meristem function revealed by interactions with ASYMMETRIC LEAVES1(AS1). AGO1 is required for full expression of LEAFY (LFY), APETALA1 (AP1) and AGAMOUS (AG). Flowering time is unaffected but floral meristem identity is partially restored in a curlyleaf (clf) background and this is not due to clf's affects on AG expression. CLF is over expressed in ago1, showing that the RNAi pathway regulates polycomb-type epigenetic modifiers.

Alleles↗

PubMed2004

Sm proteins, the constituents of the spliceosome, are components of nuage and mitochondrial cement in Xenopus oocytes.

A conserved feature of germ cells in many animal species is the presence of perinuclear electron-dense material called the "nuage" that is believed to be a precursor of germinal (or polar or P) granules. In Xenopus oogenesis the nuage is first observed near the nuclear envelope and subsequently in close contact with mitochondria, at which stage it is called the mitochondrial cement. In this study, we found that, in Xenopus pre-stage I and stage I oocytes, nuage and mitochondrial cement contain the spliceosomal Sm proteins, Xcat2 mRNA, and DEAD-box RNA helicase XVLG1. Other components of Cajal bodies or splicing machinery such as coilin, SMN protein, and snRNAs are absent from the nuage and mitochondrial cement. We suggest that Xenopus Sm proteins have adapted to a role independent of pre-mRNA splicing and that instead of binding to their traditional spliceosomal partner such as snRNA, they bind mRNAs that are the components of germinal granules (i.e., Xcat2 mRNA) and facilitate the transport of these mRNAs from the nucleus to the nuage that is a precursor of germinal granules. In addition, the presence of Vasa-like DEAD-box helicase in Xenopus nuage suggests involvement of nuage in the microRNA and/or RNAi pathway, similar to the role of nuage in Drosophila.

Active Transport, Cell Nucleus↗

PubMed2026

A muskrat tissue atlas of small non-coding RNAs and their regulatory roles in muskrat musk secretion.

Muskrat musk, secreted by the male muskrat scent gland during the secretion period, is a valuable natural product with promising pharmacological activities. Its synthesis is regulated not only by coding genes but also by small non-coding RNAs (sncRNAs). However, a comprehensive tissue atlas of sncRNAs in muskrats has been lacking. To address this issue, we systematically profiled the expression levels of 6 sncRNA categories (piRNA [PIWI-interacting RNA], miRNA [microRNA], snoRNA [small nucleolar RNA], snRNA [small nuclear RNA], tRNA [transfer RNA], and other RNAs) across 13 tissues of the muskrat via deep sequencing. We identified 23,957 sncRNAs (∼ 65%) exhibiting tissue-specific expression patterns throughout the body. The results show that miRNA is the main contributor to tissue specificity. Eleven tissues showed a significantly higher number of miRNA-5p arm expressions compared to the 3p arm, although the brain was an exception. Quantitative results imply that miR-477-3p/5p and miR-794-3p are potentially involved in the regulation of muskrat musk synthesis and secretion in the muskrat scent glands. Their target genes were enriched in pathways related to energy supply, lipid metabolism, and cyclic morphological changes of the scent gland. Furthermore, we constructed a set of co-expressed miRNAs based on the hormone-dominated "brain-testis-scent gland" axis and the energy metabolism-dominated "brain-liver-scent gland" axis. These results provide the most comprehensive description to date of tissue-specific and ubiquitous sncRNAs in individual muskrat tissues. We anticipate that these data will enhance the understanding of the molecular regulation underlying muskrat musk synthesis and secretion.

Animals↗

PubMed2003

Asymmetry in the assembly of the RNAi enzyme complex.

A key step in RNA interference (RNAi) is assembly of the RISC, the protein-siRNA complex that mediates target RNA cleavage. Here, we show that the two strands of an siRNA duplex are not equally eligible for assembly into RISC. Rather, both the absolute and relative stabilities of the base pairs at the 5' ends of the two siRNA strands determine the degree to which each strand participates in the RNAi pathway. siRNA duplexes can be functionally asymmetric, with only one of the two strands able to trigger RNAi. Asymmetry is the hallmark of a related class of small, single-stranded, noncoding RNAs, microRNAs (miRNAs). We suggest that single-stranded miRNAs are initially generated as siRNA-like duplexes whose structures predestine one strand to enter the RISC and the other strand to be destroyed. Thus, the common step of RISC assembly is an unexpected source of asymmetry for both siRNA function and miRNA biogenesis.

Adenosine Diphosphate↗

PubMed2003

Functional siRNAs and miRNAs exhibit strand bias.

Both microRNAs (miRNA) and small interfering RNAs (siRNA) share a common set of cellular proteins (Dicer and the RNA-induced silencing complex [RISC]) to elicit RNA interference. In the following work, a statistical analysis of the internal stability of published miRNA sequences in the context of miRNA precursor hairpins revealed enhanced flexibility of miRNA precursors, especially at the 5'-anti-sense (AS) terminal base pair. The same trend was observed in siRNA, with functional duplexes displaying a lower internal stability (Delta0.5 kcal/mol) at the 5'-AS end than nonfunctional duplexes. Average internal stability of siRNA molecules retrieved from plant cells after introduction of long RNA sequences also shows this characteristic thermodynamic signature. Together, these results suggest that the thermodynamic properties of siRNA play a critical role in determining the molecule's function and longevity, possibly biasing the steps involved in duplex unwinding and strand retention by RISC.

Animals↗

PubMed2004

Dicers at RISC; the mechanism of RNAi.

The pathway of RNA interference starts when Dicer cuts dsRNA into small interfering RNAs (siRNAs) that subsequently target homologous mRNAs for destruction. microRNA processing from stem loop precursors similarly requires Dicer activity. Two papers in this issue of Cell now demonstrate that Dicer is also essential for the execution phase of RNAi and explore the distinct requirements for Dicers in the siRNA and miRNA pathways.

Animals↗

PubMed2003

Posttranscriptional gene silencing is not compromised in the Arabidopsis CARPEL FACTORY (DICER-LIKE1) mutant, a homolog of Dicer-1 from Drosophila.

Posttranscriptional silencing (PTGS) in plants, nematodes, Drosophila, and perhaps all eukaryotes operates by sequence-specific degradation or translational inhibition of the target mRNA. These processes are mediated by duplexed RNA. In Drosophila and nematodes, double-stranded (ds)RNA or self-complementary RNA is processed into fragments of approximately 21 nt by Dicer-1. These small interfering RNAs (siRNAs) serve as guides to target degradation of homologous single-stranded (ss)RNA. In some cases, the approximately 21 nt guide fragments derived from endogenous, imperfectly self-complementary RNAs cause translational inhibition of their target mRNAs, with which they have substantial, but not perfect sequence complementarity. These small temporal RNAs (stRNAs) belong to a class of noncoding microRNAs (miRNAs), 20-24 nt in length, that are found in flies, plants, nematodes, and mammals. In nematodes, the Dicer-1 enzyme catalyzes the production of both siRNA and stRNA. Mutation of the Arabidopsis Dicer-1 homolog, CARPEL FACTORY (CAF), blocks miRNA production. Here, we report that the same caf mutant does not block either PTGS or siRNA production induced by self-complementary hairpin RNA. This suggests either that this mutation only impairs miRNA formation or, more interestingly, that plants have two distinct dicer-like enzymes, one for miRNA and another for siRNAi production.

Animals↗

PubMed2003

Arabidopsis HEN1: a genetic link between endogenous miRNA controlling development and siRNA controlling transgene silencing and virus resistance.

In animals, double-stranded short interfering RNA (siRNA) and single-stranded microRNA (miRNA) regulate gene expression by targeting homologous mRNA for cleavage or by interfering with their translation, respectively. siRNAs are processed from injected or transgene-derived, long, perfect double-stranded RNA (dsRNA), while miRNAs are processed from short, imperfect dsRNA precursors transcribed from endogenous intergenic regions. In plants, both siRNAs and miRNAs activate cleavage of homologous RNA targets, but little is known about the genes controlling their production or action. The SGS2/SDE1 protein contributes to produce transgene siRNA, while DCL1 and HEN1 contribute to endogenous miRNA accumulation. Here, we show that: i) SGS2, SGS3, AGO1, and HEN1 contribute to produce transgene siRNA involved in sense posttranscriptional gene silencing (S-PTGS); ii) HEN1, but not SGS2, SGS3, or AGO1, contributes to the accumulation of the endogenous miR171 miRNA and to the cleavage of Scarecrow target mRNA by miR171; iii) SGS2, SGS3, AGO1, and HEN1 contribute to resistance against cucumber mosaic virus, but not to siRNA and IR-PTGS triggered by hairpin transgenes directly producing perfect dsRNA; and iv) the actions of HEN1 in miRNA/development and siRNA/S-PTGS can be uncoupled by single-point mutations at different positions in the protein.

Arabidopsis↗

PubMed2003

miRNAs: micro managers of programmed cell death.

Genomes encode numerous small RNAs, but the function of these molecules has been elusive. Recent studies show that two distinct microRNAs regulate programmed cell death, and provide new mechanisms for the regulation of animal development.

Animals↗

PubMed2013

Application of mutated miR-206 target sites enables skeletal muscle-specific silencing of transgene expression of cardiotropic AAV9 vectors.

Insertion of completely complementary microRNA (miR) target sites (miRTS) into a transgene has been shown to be a valuable approach to specifically repress transgene expression in non-targeted tissues. miR-122TS have been successfully used to silence transgene expression in the liver following systemic application of cardiotropic adeno-associated virus (AAV) 9 vectors. For miR-206-mediated skeletal muscle-specific silencing of miR-206TS-bearing AAV9 vectors, however, we found this approach failed due to the expression of another member (miR-1) of the same miR family in heart tissue, the intended target. We introduced single-nucleotide substitutions into the miR-206TS and searched for those which prevented miR-1-mediated cardiac repression. Several mutated miR-206TS (m206TS), in particular m206TS-3G, were resistant to miR-1, but remained fully sensitive to miR-206. All these variants had mismatches in the seed region of the miR/m206TS duplex in common. Furthermore, we found that some m206TS, containing mismatches within the seed region or within the 3' portion of the miR-206, even enhanced the miR-206- mediated transgene repression. In vivo expression of m206TS-3G- and miR-122TS-containing transgene of systemically applied AAV9 vectors was strongly repressed in both skeletal muscle and the liver but remained high in the heart. Thus, site-directed mutagenesis of miRTS provides a new strategy to differentiate transgene de-targeting of related miRs.

Base Pairing↗

PubMed2005

Systematic discovery of regulatory motifs in human promoters and 3' UTRs by comparison of several mammals.

Comprehensive identification of all functional elements encoded in the human genome is a fundamental need in biomedical research. Here, we present a comparative analysis of the human, mouse, rat and dog genomes to create a systematic catalogue of common regulatory motifs in promoters and 3' untranslated regions (3' UTRs). The promoter analysis yields 174 candidate motifs, including most previously known transcription-factor binding sites and 105 new motifs. The 3'-UTR analysis yields 106 motifs likely to be involved in post-transcriptional regulation. Nearly one-half are associated with microRNAs (miRNAs), leading to the discovery of many new miRNA genes and their likely target genes. Our results suggest that previous estimates of the number of human miRNA genes were low, and that miRNAs regulate at least 20% of human genes. The overall results provide a systematic view of gene regulation in the human, which will be refined as additional mammalian genomes become available.

3' Untranslated Regions↗

PubMed2005

SERRATE coordinates shoot meristem function and leaf axial patterning in Arabidopsis.

Leaves of flowering plants are determinate organs produced by pluripotent structures termed shoot apical meristems. Once specified, leaves differentiate an adaxial (upper) side specialized for light capture, and an abaxial (lower) side specialized for gas exchange. A functional relationship between meristem activity and the differentiation of adaxial leaf fate has been recognized for over fifty years, but the molecular basis of this interaction is unclear. In Arabidopsis thaliana, activity of the class I KNOX (KNOTTED1-like homeobox) genes SHOOTMERISTEMLESS (STM) and BREVIPEDICELLUS (BP) is required for meristem function but excluded from leaves, whereas members of the HD-Zip III (class III homeodomain leucine zipper) protein family function to promote both meristem activity and adaxial leaf fate. Here we show that the zinc-finger protein SERRATE acts in a microRNA (miRNA) gene-silencing pathway to regulate expression of the HD-Zip III gene PHABULOSA (PHB) while also limiting the competence of shoot tissue to respond to KNOX expression. Thus, SERRATE acts to coordinately regulate meristem activity and leaf axial patterning.

Alleles↗

PubMed2006

Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways.

RNA interference (RNAi) is a universal and evolutionarily conserved phenomenon of post-transcriptional gene silencing by means of sequence-specific mRNA degradation, triggered by small double-stranded RNAs. Because this mechanism can be efficiently induced in vivo by expressing target-complementary short hairpin RNA (shRNA) from non-viral and viral vectors, RNAi is attractive for functional genomics and human therapeutics. Here we systematically investigate the long-term effects of sustained high-level shRNA expression in livers of adult mice. Robust shRNA expression in all the hepatocytes after intravenous infusion was achieved with an optimized shRNA delivery vector based on duplex-DNA-containing adeno-associated virus type 8 (AAV8). An evaluation of 49 distinct AAV/shRNA vectors, unique in length and sequence and directed against six targets, showed that 36 resulted in dose-dependent liver injury, with 23 ultimately causing death. Morbidity was associated with the downregulation of liver-derived microRNAs (miRNAs), indicating possible competition of the latter with shRNAs for limiting cellular factors required for the processing of various small RNAs. In vitro and in vivo shRNA transfection studies implied that one such factor, shared by the shRNA/miRNA pathways and readily saturated, is the nuclear karyopherin exportin-5. Our findings have fundamental consequences for future RNAi-based strategies in animals and humans, because controlling intracellular shRNA expression levels will be imperative. However, the risk of oversaturating endogenous small RNA pathways can be minimized by optimizing shRNA dose and sequence, as exemplified here by our report of persistent and therapeutic RNAi against human hepatitis B virus in vivo.

Animals↗

PubMed2006

A germline-specific class of small RNAs binds mammalian Piwi proteins.

Small RNAs associate with Argonaute proteins and serve as sequence-specific guides to regulate messenger RNA stability, protein synthesis, chromatin organization and genome structure. In animals, Argonaute proteins segregate into two subfamilies. The Argonaute subfamily acts in RNA interference and in microRNA-mediated gene regulation using 21-22-nucleotide RNAs as guides. The Piwi subfamily is involved in germline-specific events such as germline stem cell maintenance and meiosis. However, neither the biochemical function of Piwi proteins nor the nature of their small RNA guides is known. Here we show that MIWI, a murine Piwi protein, binds a previously uncharacterized class of approximately 29-30-nucleotide RNAs that are highly abundant in testes. We have therefore named these Piwi-interacting RNAs (piRNAs). piRNAs show distinctive localization patterns in the genome, being predominantly grouped into 20-90-kilobase clusters, wherein long stretches of small RNAs are derived from only one strand. Similar piRNAs are also found in human and rat, with major clusters occurring in syntenic locations. Although their function must still be resolved, the abundance of piRNAs in germline cells and the male sterility of Miwi mutants suggest a role in gametogenesis.

Animals↗

PubMed2005

Second-generation shRNA libraries covering the mouse and human genomes.

Loss-of-function phenotypes often hold the key to understanding the connections and biological functions of biochemical pathways. We and others previously constructed libraries of short hairpin RNAs that allow systematic analysis of RNA interference-induced phenotypes in mammalian cells. Here we report the construction and validation of second-generation short hairpin RNA expression libraries designed using an increased knowledge of RNA interference biochemistry. These constructs include silencing triggers designed to mimic a natural microRNA primary transcript, and each target sequence was selected on the basis of thermodynamic criteria for optimal small RNA performance. Biochemical and phenotypic assays indicate that the new libraries are substantially improved over first-generation reagents. We generated large-scale-arrayed, sequence-verified libraries comprising more than 140,000 second-generation short hairpin RNA expression plasmids, covering a substantial fraction of all predicted genes in the human and mouse genomes. These libraries are available to the scientific community.

Animals↗