The cytoskeleton adaptor protein ankyrin-1 is upregulated by p53 following DNA damage and alters cell migration.

The integrity of the genome is maintained by a host of surveillance and repair mechanisms that are pivotal for cellular function. The tumour suppressor protein p53 is a major component of the DNA damage response pathway and plays a vital role in the maintenance of cell-cycle checkpoints. Here we show that a microRNA, miR-486, and its host gene ankyrin-1 (ANK1) are induced by p53 following DNA damage. Strikingly, the cytoskeleton adaptor protein ankyrin-1 was induced over 80-fold following DNA damage. ANK1 is upregulated in response to a variety of DNA damage agents in a range of cell types. We demonstrate that miR-486-5p is involved in controlling G1/S transition following DNA damage, whereas the induction of the ankyrin-1 protein alters the structure of the actin cytoskeleton and sustains limited cell migration during DNA damage. Importantly, we found that higher ANK1 expression correlates with decreased survival in cancer patients. Thus, these observations highlight ANK1 as an important effector downstream of the p53 pathway.

Microguards and micromessengers of the genome.

The regulation of gene expression is of fundamental importance to maintain organismal function and integrity and requires a multifaceted and highly ordered sequence of events. The cyclic nature of gene expression is known as 'transcription dynamics'. Disruption or perturbation of these dynamics can result in significant fitness costs arising from genome instability, accelerated ageing and disease. We review recent research that supports the idea that an important new role for small RNAs, particularly microRNAs (miRNAs), is in protecting the genome against short-term transcriptional fluctuations, in a process we term 'microguarding'. An additional emerging role for miRNAs is as 'micromessengers'-through alteration of gene expression in target cells to which they are trafficked within microvesicles. We describe the scant but emerging evidence that miRNAs can be moved between different cells, individuals and even species, to exert biologically significant responses. With these two new roles, miRNAs have the potential to protect against deleterious gene expression variation from perturbation and to themselves perturb the expression of genes in target cells. These interactions between cells will frequently be subject to conflicts of interest when they occur between unrelated cells that lack a coincidence of fitness interests. Hence, there is the potential for miRNAs to represent both a means to resolve conflicts of interest, as well as instigate them. We conclude by exploring this conflict hypothesis, by describing some of the initial evidence consistent with it and proposing new ideas for future research into this exciting topic.

Detecting new microRNAs in human osteoarthritic chondrocytes identifies miR-3085 as a human, chondrocyte-selective, microRNA.

OBJECTIVE: To use deep sequencing to identify novel microRNAs (miRNAs) in human osteoarthritic cartilage which have a functional role in chondrocyte phenotype or function. DESIGN: A small RNA library was prepared from human osteoarthritic primary chondrocytes using in-house adaptors and analysed by Illumina sequencing. Novel candidate miRNAs were validated by northern blot and qRT-PCR. Expression was measured in cartilage models. Targets of novel candidates were identified by microarray and computational analysis, validated using 3'-UTR-luciferase reporter plasmids. Protein levels were assessed by western blot and functional analysis by cell adhesion. RESULTS: We identified 990 known miRNAs and 1621 potential novel miRNAs in human osteoarthritic chondrocytes, 60 of the latter were expressed in all samples assayed. MicroRNA-140-3p was the most highly expressed microRNA in osteoarthritic cartilage. Sixteen novel candidate miRNAs were analysed further, of which six remained after northern blot analysis. Three novel miRNAs were regulated across models of chondrogenesis, chondrocyte differentiation or cartilage injury. One sequence (novel #11), annotated in rodents as microRNA-3085-3p, was preferentially expressed in cartilage, dependent on chondrocyte differentiation and, in man, is located in an intron of the cartilage-expressed gene CRTAC-1. This microRNA was shown to target the ITGA5 gene directly (which encodes integrin alpha5) and inhibited adhesion to fibronectin (dependent on alpha5beta1 integrin). CONCLUSION: Deep sequencing has uncovered many potential microRNA candidates expressed in human cartilage. At least three of these show potential functional interest in cartilage homeostasis and osteoarthritis (OA). Particularly, novel #11 (microRNA-3085-3p) which has been identified for the first time in man.

MicroRNAs and cancer.

The Nobel Prize in Medicine and Physiology was awarded to the RNA interference (RNAi) field in 2006 because of the huge therapeutic potential this technique harbours. However, the RNAi technology is based on a natural mechanism that utilizes short noncoding RNA molecules (microRNAs) to regulate gene expression. This paper reviews our current knowledge about microRNAs focusing on their involvement in cancer and their potential as diagnostics and therapeutics.

MicroRNAs and the hallmarks of cancer.

It has become clear that particular microRNAs (miRNAs) function either as tumour suppressors or oncogenes, whose loss or overexpression, respectively, has diagnostic and prognostic significance. In several cases, miRNAs have been shown to affect target genes that are involved in the control of cell proliferation and apoptosis. However, malignant tumours display additional traits beyond the acquisition of enhanced growth potential and decreased cell death. Malignant disease is associated with altered tumour-host interactions leading to sustained angiogenesis and the ability to invade and metastasize. It is possible that miRNAs may act as master regulators of these aspects of tumour biology. Bioinformatic analysis of putative miRNA binding sites has indicated several novel potential gene targets of cancer-associated miRNAs that function in aspects of cell adhesion, neovascularization and tissue invasion. Among others, we speculate that miRNAs may find new roles in the regulation of E-cadherin, integrin alphavbeta3, hypoxia-inducible factor-1alpha, syndecan-1, lysyl oxidase, adamalysin metalloproteinase-17, tissue inhibitors of metalloproteinase-3, c-Met and CXCR-4 that underpin the tissue architectural changes associated with malignancy.

RNA polymerase IV directs silencing of endogenous DNA.

Plants encode subunits for a fourth RNA polymerase (Pol IV) in addition to the well-known DNA-dependent RNA polymerases I, II, and III. By mutation of the two largest subunits (NRPD1a and NRPD2), we show that Pol IV silences certain transposons and repetitive DNA in a short interfering RNA pathway involving RNA-dependent RNA polymerase 2 and Dicer-like 3. The existence of this distinct silencing polymerase may explain the paradoxical involvement of an RNA silencing pathway in maintenance of transcriptional silencing.

SDE3 encodes an RNA helicase required for post-transcriptional gene silencing in Arabidopsis.

Post-transcriptional gene silencing (PTGS) provides protection in plants against virus infection and can suppress expression of transgenes. Arabidopsis plants carrying mutations at the SDE3 locus are defective in PTGS mediated by a green fluorescent protein transgene. However, PTGS mediated by tobacco rattle virus (TRV) was not affected by sde3. From these results we conclude that SDE3, like the previously described RNA polymerase encoded by SDE1, acts at a stage in the mechanism that is circumvented when PTGS is mediated by TRV. The product of SDE3 is similar to RNA helicase-like proteins including GB110 in mouse and other proteins in Drosophila and humans. These proteins are similar to, but clearly distinct from Upf1p and SMG-2, which are required for nonsense-mediated mRNA decay in yeast and Caenorhabditis elegans and, in the case of SMG-2, for PTGS.

An RNA-dependent RNA polymerase gene in Arabidopsis is required for posttranscriptional gene silencing mediated by a transgene but not by a virus.

Posttranscriptional gene silencing is a defense mechanism in plants that is similar to quelling in fungi and RNA interference in animals. Here, we describe four genetic loci that are required for posttranscriptional gene silencing in Arabidopsis. One of these, SDE1, is a plant homolog of QDE-1 in Neurospora crassa that encodes an RNA-dependent RNA polymerase. The sde1 mutation was specific for posttranscriptional gene silencing induced by transgenes rather than by viruses. We propose that the role of SDE1 is to synthesize a double-stranded RNA initiator of posttranscriptional gene silencing. According to this idea, when a virus induces posttranscriptional gene silencing, the virus-encoded RNA polymerase would produce the double-stranded RNA and SDE1 would be redundant.

Potato virus X amplicons in arabidopsis mediate genetic and epigenetic gene silencing.

Amplicon transgenes from potato virus X (PVX) are based on a modified version of the viral genome and are efficient activators of post-transcriptional gene silencing (PTGS). To determine whether PVX amplicons activate PTGS in Arabidopsis, we used constructs based on the genome of PVX carrying a green fluorescent protein (GFP) reporter gene. Our analysis of the transgene phenotype exploited previous observations indicating that PTGS is associated with short 25-nucleotide RNA species, transgene methylation, and homology-dependent virus resistance. We also used the ability of turnip mosaic virus to suppress gene silencing as a means of dissecting stages of the mechanism. The results showed that a PVX:GFP amplicon induces weak PTGS and that this PTGS was enhanced in the presence of a GFP reporter gene. Our interpretation of these data is that the PTGS induced by the amplicon was genetically determined and equivalent to the initiation stage of the PTGS mechanism. The PTGS induced by the combined amplicon and reporter gene was equivalent to the maintenance stage and was associated with an epigenetic conversion of the transgene. The distinction between genetic and epigenetic PTGS explains the well-characterized effects of transgene dosage on PTGS that have been previously interpreted in terms of RNA expression thresholds.

Secondary structure-dependent evolution of Cymbidium ringspot virus defective interfering RNA.

Mutational analysis of defective interfering (DI) RNAs of Cymbidium ringspot virus (CymRSV) was used to study the mechanism of DI RNA evolution. It was shown that a highly base-paired structure in the 3' region of the longer DI RNA directed the formation of smaller DI RNA molecules. Mutations which increased the stability of the computer-predicted, highly structured 3' region of the longest DI RNA of CymRSV significantly enhanced the generation and accumulation of the smaller derivatives. Sequence analysis of smaller progeny molecules revealed that the highly base-paired region was deleted from the precursor DI RNA. Moreover, sites of recombination were found in other regions of the DI RNA progenies due to transposition of the highly base-paired structure. It is likely that the deletion event was structure- and not sequence-specific, and operated when a foreign sequence containing a 37-nt-long base-paired stem was inserted at the appropriate position of DI RNA.

Complete nucleotide sequence of tobacco necrosis virus strain DH and genes required for RNA replication and virus movement.

The complete genome sequence of tobacco necrosis virus strain D (Hungarian isolate, TNV-DH) was determined. The genome (3762 nt) has an organization identical to that reported for TNV-D. Highly infectious synthetic transcripts from a full-length TNV-DH cDNA clone were prepared, the first infectious necrovirus transcript reported. This clone was used for reverse genetic studies to map the viral genes required for replication and movement. Protoplast inoculation with delta 22 and delta 82 mutants revealed that both the 22 kDa and 82 kDa gene products are required for RNA replication. Although the products of three small central genes (p7(1), p7a and p7b) were not essential for RNA replication in protoplasts, mutations in these ORFs prevented infection of plants. In contrast, viral RNA accumulation and cell-to-cell movement were observed in the inoculated, but not the systemically infected, leaves of Nicotiana benthamiana challenged with RNA lacking the intact coat protein (CP) gene. These results strongly suggest that p7(1), p7a, p7b and CP are involved in TNV-DH cell-to-cell and long-distance movement, respectively.

Localization of cis-acting sequences essential for cymbidium ringspot tombusvirus defective interfering RNA replication.

The smallest defective interfering RNA (DI-2) of cymbidium ringspot tombusvirus (CyRSV) was used to identify the cis-acting sequences necessary for its replication by making a series of deletions throughout the 404 nt long molecule and testing the biological activity of mutants. Deletion or substitution of the conserved sequence blocks (A, B and C) always yielded inactive molecules. The deletion of only a few nucleotides could be tolerated beyond the natural deletion sites in blocks A and B. However, either half of block C1 (34 nt) and the first 25 nt of C2 (102 nt) could be deleted without loss of infectivity. It was also demonstrated that either one of the two halves of block C1 was specifically required for replication. We suggest that the last 77 nt of the viral genome and either half of block C1 represent the complementary strand promoter sequence recognized by the viral replicase.

Generation of defective interfering RNA dimers of cymbidium ringspot tombusvirus.

Inoculation of Nicotiana clevelandii and N. benthamiana plants with in vitro transcripts of both genomic and defective interfering (DI) RNAs of cymbidium ringspot tombusvirus resulted in a rapid accumulation of new DI-like RNA species which were demonstrated by cloning and sequencing to be head-to-tail dimers of unit length DI RNAs. The junction regions of dimers were represented by sequences derived precisely from the 5' and 3' termini of DI RNAs. Only infection with DI RNAs of smaller size (DI-2 and DI-3, 402 and 482 nt, respectively) produced detectable amount of dimers; in contrast, infection with the largest DI RNA (DI-13, 679 nt) was unable to accumulate dimers during viral infection. Analysis of mutant DI RNAs containing deletions or insertions revealed that the size of the monomer molecule is a major factor in the accumulation of dimers. Monomeric DI RNAs were formed in both plants and protoplasts inoculated with in vitro-transcribed dimers. No heterodimers were found in plants inoculated simultaneously with DI-2 and DI-3 RNA molecules, which may indicate that replicase is not released from the template during synthesis of dimer molecules. However, the occurrence of a recombinant DI RNA dimer molecule derived from the two DI RNAs suggests that simultaneous infection of the same cells with two DI RNAs did indeed take place and that absence of heterodimers did not depend on compartmentalization.

Replication of cymbidium ringspot virus satellite RNA mutants.

Cymbidium ringspot tombusvirus (CyRSV) supports the replication of an RNA molecule known to be a satellite (sat-RNA). Sequence analysis of wild-type sat-RNA showed that the 3' terminus is heterogeneous and terminates with one, two, or three C residues. Transcripts from mutant clones which lacked up to eight nucleotides at the 3' end were biologically active and yielded progeny RNA that had the 3' end restored as in wild-type RNA. Deletions or substitutions at the 5' end produced nonviable molecules. Studies with mutants containing deletions in internal regions showed that replication was affected essentially by the location of deletions; viable mutants were much less encapsidated than the wild type, showing that size also may be important in the encapsidation and survival of CyRSV sat-RNA.

The nature of multimeric forms of cymbidium ringspot tombusvirus satellite RNA.

The multimeric forms of cymbidium ringspot tombusvirus (CyRSV) satellite (sat) RNA were analysed. Attempts to amplify the putative junction region of oligomers using the polymerase chain reaction (PCR) failed, indicating the absence of such structures. SatRNA-related species having the size double than the unit length were shown to be double-stranded monomers and not single-stranded dimers. Similarly, satRNA species of a size four times the unit length were shown to be constituted by aggregates of double-stranded monomers. The absence of single-stranded CyRSV satRNA oligomers indicates that the formation of multimers is not a step in the replication of this RNA molecule.

Expression of homologous and heterologous viral coat protein-encoding genes using recombinant DI RNA from cymbidium ringspot tombusvirus.

Defective interfering (DI) RNA of cymbidium ringspot tombusvirus (CyRSV) was tested as a potential RNA vector. The coat protein-encoding gene (CP) of the same virus or of the unrelated tomato aspermy cucumovirus (TAV) was inserted in a biologically active clone of CyRSV DI-3 RNA. Both homologous and heterologous CP genes were inserted into DI-3 cDNA clone in two different positions. The CyRSV CP was expressed only in the leaves which were inoculated with DI-3CPWt plus CP-less mutant helper virus (delta ABgII). Chimaeric DI RNA carrying a heterologous CP gene (DI-3HCPtav) was able to replicate and express the inserted TAV CP in the presence of a wild-type (wt) helper genome. Both constructs, which were stable and active for gene expression, carried the inserted CP genes in the same position, between the A and B blocks of DI-3 RNA. Other constructs in which the CP were cloned between the B and C blocks of DI-3 RNA were not able to direct the translation of the encoded CP. The expression level of CP derived from recombinant DI RNA was lower relative to expression of CP from wt virus infection.

Consequences of gene transfer between distantly related tombusviruses.

Hybrid cDNA clones were constructed by fusing the coat protein-encoding gene and/or the 3'-terminal region (including the 22- and 19-kDa protein-encoding genes) derived from a clone of artichoke mottled crinkle tombusvirus to the 5'-terminal region of a full-length clone of cymbidium ringspot tombusvirus. In vitro transcripts from recombinant clones were infectious when inoculated into Nicotiana clevelandii plants. Inoculated plants showed symptoms different from those induced by parent viruses. In particular, systemic invasion depended very much, although not exclusively, on the type of protein that coated progeny viral RNA, suggesting a role of the capsid protein in the long-distance movement of tombusvirus infections.

Functional analysis of cymbidium ringspot virus genome.

Genomic RNA of cymbidium ringspot tombusvirus (CyRSV) contains five large open reading frames (ORFs). The two 5' proximal ORFs encode proteins of 33 and 92 kDa and the three 3' proximal ORFs encode proteins of 41, 22, and 19 kDa. In addition, a small reading frame encoding a protein of 4 kDa was recently observed by computer analysis of the 3' nontranslated region of CyRSV and other tombusvirus RNAs (ORF 6). The function of putative gene products was investigated with the use of several mutants. Mutants in ORFs 1 and 2 were not viable indicating that both 33- and 92-kDa proteins are required for replication. Deletion of a large portion of the coat protein gene, encoding a 41-kDa protein, did not prevent replication of viral RNA and cell-to-cell movement, but interfered severely with long-distance translocation. No virus particles or viral RNA could be detected in inoculated or upper leaves of plants inoculated with transcripts obtained from mutants not expressing the 22-kDa protein. However, replication and encapsidation occurred normally in inoculated protoplasts indicating that this gene product is a transport protein. Conversely, no role could be assigned to putative gene products of ORF5 (19-kDa protein) and ORF6. It was also shown that factors other than gene expression can influence the replication of RNA mutants, probably due to instability of RNA molecules.

Defective interfering RNA-mediated resistance against cymbidium ringspot tombusvirus in transgenic plants.

Defective interfering (DI) RNA of cymbidium ringspot tombusvirus was cloned downstream from the bacteriophage T7 RNA polymerase promoter. In vitro synthesized RNA was biologically active when coinoculated with parental genomic RNA onto Nicotiana benthamiana plants and prevented the occurrence of apical necrosis. N. benthamiana plants were transformed with the DI RNA sequences in both the positive and negative orientations relative to the cauliflower mosaic virus 35S promoter. Integration of DI RNA sequences in the plant genome was verified using PCR amplification of DNA extracts and Northern blot analysis of RNA extracts. DI RNA-related transcripts were detected in uninfected transgenic plants, but inoculation with the parental virus induced replication of the DI RNA only in transgenic plants expressing DI RNA in the positive orientation. Transgenic plants in which DI RNA accumulated were protected from apical necrosis and death.

Repair in vivo of altered 3' terminus of cymbidium ringspot tombusvirus RNA.

Progeny RNA of CyRSV RNA transcripts ending with -GGG, instead of -CCC as in wild-type RNA, was analyzed and shown to revert to wild-type. A new full-length cDNA clone of CyRSV was prepared, bearing the correct 3' terminus, from which transcripts could be synthesized having very high infectivity. Mutants were prepared which lacked the three terminal Cs; transcripts were infectious and most cDNA clones recovered from progeny RNA had regained the wild-type sequence. Transcripts ending in -GGCCAn were also infectious, and the 3' sequence of most cDNA clones recovered after inoculation was the same as the inoculum sequence. Deletion of G at position -4 completely abolished infectivity. The occurrence of a repair mechanism at the 3' end of CyRSV similar to telomerase activity on chromosomes is suggested.