Gardening the genome: DNA methylation in Arabidopsis thaliana.

DNA methylation has two essential roles in plants and animals - defending the genome against transposons and regulating gene expression. Recent experiments in Arabidopsis thaliana have begun to address crucial questions about how DNA methylation is established and maintained. One cardinal insight has been the discovery that DNA methylation can be guided by small RNAs produced through RNA-interference pathways. Plants and mammals use a similar suite of DNA methyltransferases to propagate DNA methylation, but plants have also developed a glycosylase-based mechanism for removing DNA methylation, and there are hints that similar processes function in other organisms.

Quantitative RNA-seq analysis of the campylobacter jejuni transcriptome

Campylobacter jejuni is the most common bacterial cause of foodborne disease in the developed world. Its general physiology and biochemistry, as well as the mechanisms enabling it to colonize and cause disease in various hosts, are not well understood, and new approaches are required to understand its basic biology. High-throughput sequencing technologies provide unprecedented opportunities for functional genomic research. Recent studies have shown that direct Illumina sequencing of cDNA (RNA-seq) is a useful technique for the quantitative and qualitative examination of transcriptomes. In this study we report RNA-seq analyses of the transcriptomes of C. jejuni (NCTC11168) and its rpoN mutant. This has allowed the identification of hitherto unknown transcriptional units, and further defines the regulon that is dependent on rpoN for expression. The analysis of the NCTC11168 transcriptome was supplemented by additional proteomic analysis using liquid chromatography-MS. The transcriptomic and proteomic datasets represent an important resource for the Campylobacter research community. © 2011 SGM.

RNA processing and Arabidopsis flowering time control.

Plants control their flowering time in order to ensure that they reproduce under favourable conditions. The components involved in this complex process have been identified using a molecular genetic approach in Arabidopsis and classified into genetically separable pathways. The autonomous pathway controls the level of mRNA encoding a floral repressor, FLC, and comprises three RNA-binding proteins, FCA, FPA and FLK. FCA interacts with the 3'-end RNA-processing factor FY to autoregulate its own expression post-transcriptionally and to control FLC. Other components of the autonomous pathway, FVE and FLD, regulate FLC epigenetically. This combination of epigenetic and post-transcriptional control gives precision to the control of FLC expression and flowering time.

Dissecting Arabidopsis thaliana DICER function in small RNA processing, gene silencing and DNA methylation patterning.

Small RNAs have several important biological functions. MicroRNAs (miRNAs) and trans-acting small interfering RNAs (tasiRNAs) regulate mRNA stability and translation, and siRNAs cause post-transcriptional gene silencing of transposons, viruses and transgenes and are important in both the establishment and maintenance of cytosine DNA methylation. Here, we study the role of the four Arabidopsis thaliana DICER-LIKE genes (DCL1-DCL4) in these processes. Sequencing of small RNAs from a dcl2 dcl3 dcl4 triple mutant showed markedly reduced tasiRNA and siRNA production and indicated that DCL1, in addition to its role as the major enzyme for processing miRNAs, has a previously unknown role in the production of small RNAs from endogenous inverted repeats. DCL2, DCL3 and DCL4 showed functional redundancy in siRNA and tasiRNA production and in the establishment and maintenance of DNA methylation. Our studies also suggest that asymmetric DNA methylation can be maintained by pathways that do not require siRNAs.

MicroRNAs and other small RNAs enriched in the Arabidopsis RNA-dependent RNA polymerase-2 mutant.

The Arabidopsis genome contains a highly complex and abundant population of small RNAs, and many of the endogenous siRNAs are dependent on RNA-Dependent RNA Polymerase 2 (RDR2) for their biogenesis. By analyzing an rdr2 loss-of-function mutant using two different parallel sequencing technologies, MPSS and 454, we characterized the complement of miRNAs expressed in Arabidopsis inflorescence to considerable depth. Nearly all known miRNAs were enriched in this mutant and we identified 13 new miRNAs, all of which were relatively low abundance and constitute new families. Trans-acting siRNAs (ta-siRNAs) were even more highly enriched. Computational and gel blot analyses suggested that the minimal number of miRNAs in Arabidopsis is approximately 155. The size profile of small RNAs in rdr2 reflected enrichment of 21-nt miRNAs and other classes of siRNAs like ta-siRNAs, and a significant reduction in 24-nt heterochromatic siRNAs. Other classes of small RNAs were found to be RDR2-independent, particularly those derived from long inverted repeats and a subset of tandem repeats. The small RNA populations in other Arabidopsis small RNA biogenesis mutants were also examined; a dcl2/3/4 triple mutant showed a similar pattern to rdr2, whereas dcl1-7 and rdr6 showed reductions in miRNAs and ta-siRNAs consistent with their activities in the biogenesis of these types of small RNAs. Deep sequencing of mutants provides a genetic approach for the dissection and characterization of diverse small RNA populations and the identification of low abundance miRNAs.