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PubMed2006

The p53-induced Wig-1 protein binds double-stranded RNAs with structural characteristics of siRNAs and miRNAs.

Wig-1 is a p53-induced zinc finger protein. Here we show that human Wig-1 binds long (>or=23 bp) dsRNAs with 5'-overhangs. The first zinc finger domain is necessary but not sufficient for this dsRNA-binding in vitro. Wig-1 also binds dsRNA in living cells via zinc fingers 1 and 2. Both zinc fingers 1 and 2 are important for Wig-1-mediated growth suppression. Moreover, Wig-1 binds 21 bp dsRNAs with 3'-protruding ends. These findings demonstrate that human Wig-1 can bind different types of dsRNAs, including dsRNAs resembling small interfering RNAs (siRNAs) and microRNAs (miRNAs), and indicate that dsRNA binding has a role in Wig-1-mediated regulation of cell growth.

Binding Sites↗

PubMed2006

Analysis of short RNAs in the malaria parasite and its red blood cell host.

RNA interference (RNAi) is an RNA degradation process that involves short, double-stranded RNAs (dsRNA) as sequence specificity factors. The natural function of the RNAi machinery is to generate endogenous short double-stranded RNAs to regulate gene expression. It has been shown that treatment of Plasmodium falciparum, the etiologic agent of malaria, with dsRNA induces degradation of the corresponding microRNA (miRNA), yet typical RNAi-associated genes have not been identifiable in the parasite genome. To clarify this discrepancy we set out to clone short RNAs from P. falciparum-infected red blood cells and from purified parasites. We did not find any short RNA that was not a rRNA or tRNA fragment. Indeed, only known human miRNAs were isolated in parasite preparations indicating that very few if any short RNAs exist in P. falciparum. This suggests a different mechanism than classical RNAi in observations of dsRNA-mediated degradation. Of the human miRNAs identified, the human miRNA mir-451 accumulates at a very high level in both infected and healthy red blood cells. Interestingly, mir-451 was not detectable in a series of immortalised cell lines representing progenitor stages of all major blood lineages, suggesting that mir-451 may play a role in the differentiation of erythroid cells.

Animals↗

PubMed2005

Asymmetry of intronic pre-miRNA structures in functional RISC assembly.

The two oligonucleotide strands of a siRNA duplex are functionally asymmetric in assembling the RNAi effector, RNA-induced gene silencing complex (RISC). Based on this asymmetric RISC assembly model in vitro, formation of a microRNA (miRNA) and complementary miRNA (miRNA*) duplex was proposed to be an essential step for the assembly of miRNA-associated RISC (miRISC). We observed here that a strong structural bias exists in the selection of a mature miRNA strand for RISC assembly in zebrafish using an intronic miRNA-like vector to target EGFP mRNA for regulation. The position of the stemloop in a precursor miRNA (pre-miRNA) was involved in the determination of miRNA-miRNA* asymmetry of the pre-miRNA stemarm, leading to different miRNA maturation during miRISC assembly. These findings suggest that the miRISC assembly is likely different from the RISC assembly model of siRNA in zebrafish, providing the first in vivo evidence for asymmetric miRISC assembly.

Animals↗

PubMed2006

Identification and characterization of lin-28 homolog B (LIN28B) in human hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Several studies have identified signature gene sets that may be useful as potential diagnostic tools by global microarray analysis. Here we report the cloning and characterization of a novel gene, lin-28 homolog B (LIN28B), which is overexpressed in hepatocellular carcinoma. The heterochronic gene lin-28 is a key regulator of developmental timing in the nematode Caenorhabditis elegans. Similar with lin-28 proteins, LIN28B conserves a cold shock domain and a pair of CCHC zinc finger domains. Phylogenetic analysis suggests that they might arise as a result of duplication from an ancestral gene. Overexpression of LIN28B was noted in most HCC cell lines and clinical samples. By western blot analysis using a polyclonal antibody against LIN28B, a short LIN28B isoform was also identified in non-tumor liver tissue and fetal liver. Although predominantly localized in the cytoplasm, we found that LIN28B protein shows cell cycle-dependent nuclear translocation in Huh7 cells. Induced expression of exogenous LIN28B in a tet-off cell line promoted cancer cell proliferation. Interestingly, the segment of the unusually long 3'UTR of LIN28B contains complementary sites to let-7 microRNA of mammals. And our studies provided indirect evidence that LIN28B is a possibly natural target for let-7 mediated regulation. These findings strongly implicate a critical role of LIN28B during development and tumorigenesis and suggest a possible novel mechanism.

3' Untranslated Regions↗

PubMed2026

Alternative End Joining Dependency Imposed by miR-21-5p Defines Radiation Resistance and a Targetable Vulnerability in Oral Squamous Cell Carcinoma.

PURPOSE: Clinical control of oral squamous cell carcinoma (OSCC) is constrained by heterogeneous radiosensitivity driven by divergent DNA damage response programs. The architecture and functional contribution of alternative end joining (Alt-EJ), an error-prone DNA double-strand break (DSB) repair pathway frequently upregulated in cancer, to radiation resistance remains poorly defined. METHODS AND MATERIALS: We profiled microRNAs in radioresistant OSCC clones and performed multiomic integration across an institutional OSCC cohort, an external OSCC cohort from the Gene Expression Omnibus, The Cancer Genome Atlas pan-cancer tumors, and cell lines characterized by Sanger Genomics of Drug Sensitivity in Cancer to infer DNA damage response characteristics, genomic scar features, drug sensitivity, and radiation therapy outcomes. DSB repair capacity and pathway usage were validated using functional assays, including Alt-EJ reporters and droplet digital PCR quantification of microhomology-mediated repair events. Core Alt-EJ effectors such as PARP1 and POLQ were perturbed genetically and pharmacologically. Therapeutic efficacy of PARP or POLQ inhibition with or without irradiation was tested in a syngeneic OSCC model, followed by bulk tumor transcriptomics to assess pathway engagement. RESULTS: Upregulation of miR-21-5p was not only selectively detected in radioresistant OSCC, but also modulated radiosensitivity in vitro and in vivo, and was associated with inferior postradiation therapy survival. A calibrated miR-21-5p target-gene signature tracked Alt-EJ activity across patient and mouse tumors and cancer cell lines, correlated with microhomology-mediated indels and broader genomic scarring, and predicted sensitivity to clinically available PARP inhibitors. Functionally, enforced miR-21-5p expression increased Alt-EJ usage and accelerated DSB repair, whereas inhibition or depletion of key Alt-EJ effectors reduced repair efficiency and restored radiosensitivity. In vivo, Alt-EJ targeting with PARP or POLQ inhibitor abrogated miR-21-5p-driven radiation resistance; transcriptomic profiling supported suppression of Alt-EJ programs as the operative mechanism. CONCLUSIONS: These findings establish a mechanistic link between miR-21-5p activity and Alt-EJ dependence, provide a clinically deployable signature to identify Alt-EJ-dependent OSCC, and support rational combinations of Alt-EJ targeting agents with radiation therapy to overcome treatment failure and advance precision radiation oncology.

MicroRNAs↗

PubMed2006

Differentiation of 2'-O- and 3'-O-methylated ribonucleosides by tandem mass spectrometry.

Recent studies revealed that the 3'-terminal nucleotides in plant microRNAs were methylated on the ribose at the 2' or 3' hydroxyl groups. Here we examined the fragmentation of the electrospray-produced [M + H]+ and [M - H]- ions of 2'- and 3'-O-methylated ribonucleosides. It turned out that the predominant fragmentation pathway for the [M + H]+ ions of ribose-methylated nucleosides was the neutral loss of the methylated ribose, which made it impossible to distinguish 2'-O-methylation from 3'-O-methylation by positive-ion MS/MS. However, characteristic fragment ions, resulting from the cleavage through the ribose rings, were produced for the [M - H]- ions of each pair of ribose-methylated nucleosides. In this respect, the neutral loss of a 90-Da fragment (C3H6O3) was observed for 2'-O-methylated cytidine, guanosine and adenosine, but not for their 3'-O-methylated counterparts. On the other hand, the neutral loss of a 60-Da fragment (C2H4O2) was found for 3'-O-methyluridine, but not for 2'-O-methyluridine.

Methylation↗

PubMed2004

Endogenous trans-acting siRNAs regulate the accumulation of Arabidopsis mRNAs.

Here we describe a set of endogenous short interfering RNAs (siRNAs) in Arabidopsis, some of which direct the cleavage of endogenous mRNAs. These siRNAs correspond to both sense and antisense strands of a noncoding RNA (At2g27400) that apparently is converted to double-stranded RNA and then processed in 21 nt increments. These siRNAs differ from previously described regulatory small RNAs in two respects. First, they require components of the cosuppression pathway (RDR6 and SGS3) and also components of the microRNA (miRNA) pathway (AGO1, DCL1, HEN1, and HYL1) but not components needed for heterochromatic siRNAs (DCL3 and RDR2), another class of endogenous plant siRNAs. Second, these siRNAs repress the expression of genes that have little overall resemblance to the genes from which they originate, a characteristic previously reported only for miRNAs. The identification of this silencing pathway provides yet another dimension to posttranscriptional mRNA regulation in plants.

Arabidopsis↗

PubMed2005

Biochemical specialization within Arabidopsis RNA silencing pathways.

In plants, the RNA silencing machinery responds to numerous inputs, including viral infection, microRNAs, and endogenous siRNAs that may act both in trans and in cis. Additionally, the full spectrum of silencing outcomes has been demonstrated in plants, ranging from mRNA degradation to repression at the level of protein synthesis to chromatin remodeling. Genetic studies in Arabidopsis have indicated that individual response pathways are functionally compartmentalized. However, to date, no biochemical systems have been available to investigate the roles of specific proteins within silencing pathways or the effects of selected mutations on the biochemical activity of those components. Here, we describe the generation of Arabidopsis extracts that reproduce many aspects of RNA silencing reactions in vitro. We find that specific members of the Dicer and Argonaute families have distinct biochemical activities, which provides insight into their roles within RNA silencing pathways in Arabidopsis.

Adenosine Triphosphate↗

PubMed2005

The developmental timing regulator AIN-1 interacts with miRISCs and may target the argonaute protein ALG-1 to cytoplasmic P bodies in C. elegans.

In metazoans, microRNAs (miRNAs) carry out various regulatory functions through association with multiprotein miRNA-induced silencing complexes (miRISCs) that contain Dicer and Argonaute proteins. How miRNAs regulate the expression of their mRNA targets remains a major research question. We have identified the C. elegans ain-1 gene through a genetic suppressor screen and shown that it functions with the heterochronic genetic pathway that regulates developmental timing. Biochemical analysis indicates that AIN-1 interacts with protein complexes containing an Argonaute protein, Dicer, and miRNAs. AIN-1 shares homology with the candidate human neurological disease protein GW182, shown to localize in cytoplasmic processing bodies that are sites of mRNA degradation and storage. A functional AIN-1::GFP also localizes at the likely worm processing bodies. When coexpressed from transgenes, AIN-1 targets ALG-1 to the foci. These results suggest a model where AIN-1 regulates a subset of miRISCs by localization to the processing bodies, facilitating degradation or translational inhibition of mRNA targets.

Animals↗

PubMed2006

Short RNAs repress translation after initiation in mammalian cells.

MicroRNAs (miRNAs) are predicted to regulate 30% of mammalian protein-encoding genes by interactions with their 3' untranslated regions (UTRs). We use partially complementary siRNAs to investigate the mechanism by which miRNAs mediate translational repression in human cells. Repressed mRNAs are associated with polyribosomes that are engaged in translation elongation, as shown by puromycin sensitivity. The inhibition appears to be postinitiation because translation driven by the cap-independent processes of HCV IRES and CrPV IRES is repressed by short RNAs. Further, metabolic labeling suggests that silencing occurs before completion of the nascent polypeptide chain. In addition, silencing by short RNAs causes a decrease in translational readthrough at a stop codon, and ribosomes on repressed mRNAs dissociate more rapidly after a block of initiation of translation than those on control mRNAs. These results suggest that repression by short RNAs, and thus probably miRNAs, is primarily due to ribosome drop off during elongation of translation.

Animals↗

PubMed2006

AGO1 homeostasis entails coexpression of MIR168 and AGO1 and preferential stabilization of miR168 by AGO1.

Arabidopsis ARGONAUTE1 (AGO1) encodes the RNA slicer enzyme of the microRNA (miRNA) pathway and is regulated by miR168-programmed, AGO1-catalyzed mRNA cleavage. Here, we describe two additional regulatory processes required for AGO1 homeostasis: transcriptional coregulation of MIR168 and AGO1 genes, and posttranscriptional stabilization of miR168 by AGO1. Disrupting any of these regulatory processes by using mutations or transgenes disturbs a proper functioning of the miRNA pathway. In contrast, minor perturbation leads to fine-tuned posttranscriptional adjustment of miR168 and AGO1 levels, thereby maintaining a proper balance of other miRNAs, which, together with AGO1, control the mRNA levels of miRNA targets. We suggest that miR168 stabilization occurs at the level of silencing-complex assembly and that modulating the efficiency of assembling miRNA-programmed silencing complexes will also be important in other contexts.

Arabidopsis↗

PubMed2006

Recapitulation of short RNA-directed translational gene silencing in vitro.

microRNAs (miRNAs) are a large class of endogenous short RNAs that repress gene expression. Many miRNAs are conserved throughout evolution, and dysregulation of miRNA pathways has been correlated with an increasing number of human diseases. In animals, miRNAs typically bind to the 3' untranslated region (3'UTR) of target mRNAs with imperfect sequence complementarity and repress translation. Despite their importance in regulating biological processes in numerous organisms, the mechanisms of miRNA function are largely unknown. Here, we report in vitro reactions for miRNA-directed translational gene silencing. These reactions faithfully recapitulate known in vivo hallmarks of mammalian miRNA function, including a requirement for a 5' phosphate and perfect complementarity to the mRNA target in the 5' seed region. Translational gene silencing by miRNAs in vitro requires target mRNAs to possess a 7-methyl G cap and a polyA tail, whereas increasing polyA tail length alone can increase miRNA silencing activity.

Animals↗

PubMed2005

Genetic control of shoot organ boundaries.

The initiation of plant lateral organs from the shoot meristem is associated with the formation of boundaries that separate the primordia from surrounding tissue. A distinctive set of cells is present along the boundary, and these 'boundary cells' display characteristic patterns of cell division, morphology and gene expression. A certain class of the NAC transcription factors is important for growth suppression at the boundary, and auxin and microRNAs participate in boundary formation by regulating NAC gene expression. Other factors regulate different aspects of boundary functions, such as the establishment of the border of different cell identities, the initiation of axillary meristems, or the proper development of organs and tissues in adjacent regions.

Gene Expression Regulation, Plant↗

PubMed2006

Advanced techniques in placental biology -- workshop report.

Major advances in placental biology have been realized as new technologies have been developed and existing methods have been refined in many areas of biological research. Classical anatomy and whole-organ physiology tools once used to analyze placental structure and function have been supplanted by more sophisticated techniques adapted from molecular biology, proteomics, and computational biology and bioinformatics. In addition, significant refinements in morphological study of the placenta and its constituent cell types have improved our ability to assess form and function in highly integrated manner. To offer an overview of modern technologies used by investigators to study the placenta, this workshop: Advanced techniques in placental biology, assembled experts who discussed fundamental principles and real time examples of four separate methodologies. Y. Sadovsky presented the principles of microRNA function as an endogenous mechanism of gene regulation. J. Robinson demonstrated the utility of correlative microscopy in which light-level and transmission electron microscopy are combined to provide cellular and subcellular views of placental cells. A. Croy provided a lecture on the use of microdissection techniques which are invaluable for isolating very small subsets of cell types for molecular analysis. Finally, G. Rice presented an overview methods on profiling of complex protein mixtures within tissue and/or fluid samples that, when refined, will offer databases that will underpin a systems approach to modern trophoblast biology.

Animals↗

PubMed2005

Post-transcriptional gene silencing by siRNAs and miRNAs.

Recent years have seen a rapid increase in our understanding of how double-stranded RNA (dsRNA) and 21- to 25-nucleotide small RNAs, microRNAs (miRNAs) and small interfering RNAs (siRNAs), control gene expression in eukaryotes. This RNA-mediated regulation generally results in sequence-specific inhibition of gene expression; this can occur at levels as different as chromatin modification and silencing, translational repression and mRNA degradation. Many details of the biogenesis and function of miRNAs and siRNAs, and of the effector complexes with which they associate have been elucidated. The first structural information on protein components of the RNA interference (RNAi) and miRNA machineries is emerging, and provides some insight into the mechanism of RNA-silencing reactions.

Animals↗

PubMed2005

Mechanisms of translational control by the 3' UTR in development and differentiation.

Translational control plays a major role in early development, differentiation and the cell cycle. In this review, we focus on the four main mechanisms of translational control by 3' untranslated regions: 1. Cytoplasmic polyadenylation and deadenylation; 2. Recruitment of 4E binding proteins; 3. Regulation of ribosomal subunit binding; 4. Post-initiation repression by microRNAs. Proteins with conserved functions in translational control during development include cytoplasmic polyadenylation element binding proteins (CPEB/Orb), Pumilio, Bruno, Fragile X mental retardation protein and RNA helicases. The translational regulation of the mRNAs encoding cyclin B1, Oskar, Nanos, Male specific lethal 2 (Msl-2), lipoxygenase and Lin-14 is discussed.

3' Untranslated Regions↗

PubMed2005

siRNA and miRNA: an insight into RISCs.

Two classes of short RNA molecule, small interfering RNA (siRNA) and microRNA (miRNA), have been identified as sequence-specific posttranscriptional regulators of gene expression. siRNA and miRNA are incorporated into related RNA-induced silencing complexes (RISCs), termed siRISC and miRISC, respectively. The current model argues that siRISC and miRISC are functionally interchangeable and target specific mRNAs for cleavage or translational repression, depending on the extent of sequence complementarity between the small RNA and its target. Emerging evidence indicates, however, that siRISC and miRISC are distinct complexes that regulate mRNA stability and translation. The assembly of RISCs can be traced from the biogenesis of the small RNA molecules and the recruitment of these RNAs by the RISC loading complex (RLC) to the transition of the RLC into the active RISC. Target recognition by the RISC can then take place through different interacting modes.

Animals↗

PubMed2005

Small non-coding RNAs as magic bullets.

RNA interference (RNAi) - inhibition of gene expression by small, non-coding RNAs [small interfering RNAs (siRNAs) or microRNAs (miRNAs)] - has changed our view of regulation of expression dramatically. The application of siRNAs for both functional analysis of genes and medication raises several questions. These include the design of the double-stranded oligonucleotides, their preparation and introduction into cells or animals either as chemically synthesized entities or as transcripts from a suitable vector. Delivery of the oligonucleotides, choice of vector, chemical modification to stabilize against nucleases and avoidance of side effects (e.g. stimulation of interferons) are major challenges. Work to identify the multiple targets of miRNAs is still in its infancy, and a clear distinction between siRNAs and miRNAs is difficult in some instances. Moreover, transcriptional silencing by RNAi is poorly understood; it is evident that the siRNA machinery is involved but the details await clarification. Given the multitude of interactions of the small non-coding RNAs revealed so far, we should be prepared to encounter, as yet, undiscovered interactions and mechanisms.

Animals↗