Tamas Dalmay

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.

Butterfly genome reveals promiscuous exchange of mimicry adaptations among species

The evolutionary importance of hybridization and introgression has long been debated. Hybrids are usually rare and unfit, but even infrequent hybridization can aid adaptation by transferring beneficial traits between species. Here we use genomic tools to investigate introgression in Heliconius, a rapidly radiating genus of neotropical butterflies widely used in studies of ecology, behaviour, mimicry and speciation. We sequenced the genome of Heliconius melpomene and compared it with other taxa to investigate chromosomal evolution in Lepidoptera and gene flow among multiple Heliconius species and races. Among 12,669 predicted genes, biologically important expansions of families of chemosensory and Hox genes are particularly noteworthy. Chromosomal organization has remained broadly conserved since the Cretaceous period, when butterflies split from the Bombyx (silkmoth) lineage. Using genomic resequencing, we show hybrid exchange of genes between three co-mimics, Heliconius melpomene, Heliconius timareta and Heliconius elevatus, especially at two genomic regions that control mimicry pattern. We infer that closely related Heliconius species exchange protective colour-pattern genes promiscuously, implying that hybridization has an important role in adaptive radiation.

Deep sequencing of tomato short RNAs identifies microRNAs targeting genes involved in fruit ripening

In plants there are several classes of 21-24-nt short RNAs that regulate gene expression. The most conserved class is the microRNAs (miRNAs), although some miRNAs are found only in specific species. We used high-throughput pyrosequencing to identify conserved and nonconserved miRNAs and other short RNAs in tomato fruit and leaf. Several conserved miRNAs showed tissue-specific expression, which, combined with target gene validation results, suggests that miRNAs may play a role in fleshy fruit development. We also identified four new nonconserved miRNAs. One of the validated targets of a novel miRNA is a member of the CTR family involved in fruit ripening. However, 62 predicted targets showing near perfect complementarity to potential new miRNAs did not validate experimentally. This suggests that target prediction of plant short RNAs could have a high false-positive rate and must therefore be validated experimentally. We also found short RNAs from a Solanaceae-specific foldback transposon, which showed a miRNA/miRNA*-like distribution, suggesting that this element may function as a miRNA gene progenitor. The other Solanaceae-specific class of short RNA was derived from an endogenous pararetrovirus sequence inserted into the tomato chromosomes. This study opens a new avenue in the field of fleshy fruit biology by raising the possibility that fruit development and ripening may be under miRNA regulation.

Mutations in the seed region of human miR-96 are responsible for nonsyndromic progressive hearing loss

MicroRNAs (miRNAs) bind to complementary sites in their target mRNAs to mediate post-transcriptional repression, with the specificity of target recognition being crucially dependent on the miRNA seed region. Impaired miRNA target binding resulting from SNPs within mRNA target sites has been shown to lead to pathologies associated with dysregulated gene expression. However, no pathogenic mutations within the mature sequence of a miRNA have been reported so far. Here we show that point mutations in the seed region of miR-96, a miRNA expressed in hair cells of the inner ear, result in autosomal dominant, progressive hearing loss. This is the first study implicating a miRNA in a mendelian disorder. The identified mutations have a strong impact on miR-96 biogenesis and result in a significant reduction of mRNA targeting. We propose that these mutations alter the regulatory role of miR-96 in maintaining gene expression profiles in hair cells required for their normal function.

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.

The cartilage specific microRNA-140 targets histone deacetylase 4 in mouse cells.

MicroRNAs (miRNA) are short RNA molecules regulating the expression of specific mRNAs. We investigated the expression pattern and potential targets of mouse miR‐140 and found that miR‐140 is specifically expressed in cartilage tissues of mouse embryos during both long and flat bone development. MiR‐140 expression was detected in the limbs of E11.5 embryos in the primorida of future bones both in the fore and hindlimb and across autopod, zeugopod and stylopod. All digits of E14.5 fore‐ and hindlimbs showed accumulation of miR‐140, except the first digit of the hindlimb. MiR‐140 expression was also detected in the cartilagenous base of E17.5 skulls and in the sternum, the proximal rib heads and the developing vertebral column of E15.5 embryos. A potential target of miR‐140, histone deacetylase 4, was validated experimentally and the possible role of miR‐140 in long bone development is discussed.

Identification of grapevine microRNAs and their targets using high throughput sequencing and degradome analysis

In plants, microRNAs (miRNAs) comprise one of three classes of small RNAs regulating gene expression at the post-transcriptional level. Many plant miRNAs are conserved, and play a role in development, abiotic stress responses or pathogen responses. However, some miRNAs have only been found in certain species. Here, we use deep-sequencing, computational and molecular methods to identify, profile, and describe conserved and non-conserved miRNAs in four grapevine (Vitis vinifera) tissues. A total of 24 conserved miRNA families were identified in all four tissues, and 26 known but non-conserved miRNAs were also found. In addition to known miRNAs, we also found 21 new grapevine-specific miRNAs together with their star strands. We have also shown that almost all of them originated from single genes. Furthermore, 21 other plausible miRNA candidates have been described. We have found that many known and new miRNAs showed tissue-specific expression. Finally, 112 target mRNAs of known and 44 target mRNAs of new grapevine-specific miRNAs were identified by genomic-scale high-throughput sequencing of miRNA cleaved mRNAs.

Sulphur starvation induces the expression of microRNA-395 and one of its target genes but in different cell types.

Plants play an important role in the global sulphur cycle because they assimilate sulphur from the environment and build it into methionine and cysteine. Several genes of the sulphur assimilation pathway are regulated by microRNA-395 (miR395) that is itself induced by a low-sulphur (-S) environment. Here, we show that the six Arabidopsis miR395 loci are induced differently. We find that MIR395 loci are expressed in the vascular system of roots and leaves and root tips. Induction of miR395 by a -S environment in both roots and leaves suggests that translocation of miR395 from leaves to roots through the phloem is not necessary for plants growing on -S soil/medium. We also demonstrate that induction of miR395 is controlled by SLIM1, a key transcription factor in the sulphur assimilation pathway. Unexpectedly, the mRNA level of a miR395 target gene, SULTR2;1, strongly increases during miR395 induction in roots. We show that the spatial expression pattern of MIR395 transcripts in the vascular system does not appear to overlap with the expression pattern previously reported for SULTR2;1 mRNA. These results illustrate that negative temporal correlation between the expression level of a miRNA and its target gene in a complex tissue cannot be a requirement for target gene validation.

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 αvβ3, hypoxia-inducible factor-1α, 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.

miR398 and miR408 are up-regulated in response to water deficit in Medicago truncatula

Plant microRNAs have been implicated in various abiotic stress responses. We identified several conserved microRNAs that showed differential expression in Medicago truncatula plants subjected to water deficit: miR169 is down-regulated only in the roots and miR398a/b and miR408 are strongly up-regulated in both shoots and roots. Down-regulation of miR169 in the roots did not correlate with accumulation of its target MtHAP2-1 transcripts, suggesting that its regulation may not occur at the mRNA level or may depend on other regulatory mechanisms, which do not involve this miRNA, in water-deficit conditions. The up-regulation of miR398a/b and miR408 and the clear down-regulation of their respective target genes, which encode the copper proteins COX5b (subunit 5b of mitochondrial cytochrome c oxidase) and plantacyanin, highlight the involvement of these miRNAs in response to water deprivation in M. truncatula. Also, miR398 up-regulation is inversely correlated with the down-regulation of copper superoxide dismutase, CSD1, during water deficit. The regulation of genes encoding copper proteins by miR398a/b and miR408 suggests a link between copper homeostasis and M. truncatula adaptation to progressive water deficit.