It has recently been shown that RNA 3′ end formation plays a more widespread role in controlling gene expression than previously thought. In order to examine the impact of regulated 3′ end formation genome-wide we applied direct RNA sequencing to A. thaliana. Here we show the authentic transcriptome in unprecedented detail and how 3′ end formation impacts genome organization. We reveal extreme heterogeneity in RNA 3′ ends, discover previously unrecognized non-coding RNAs and propose widespread re-annotation of the genome. We explain the origin of most poly(A)+ antisense RNAs and identify cis-elements that control 3′ end formation in different registers. These findings are essential to understand what the genome actually encodes, how it is organized and the impact of regulated 3′ end formation on these processes.

The spen family protein FPA is required for flowering time control and has been implicated in RNA silencing. The mechanism by which FPA carries out these functions is unknown. We report the identification of an activity for FPA in controlling mRNA 3' end formation. We show that FPA functions redundantly with FCA, another RNA binding protein that controls flowering and RNA silencing, to control the expression of alternatively polyadenylated antisense RNAs at the locus encoding the floral repressor FLC. In addition, we show that defective 3' end formation at an upstream RNA polymerase II-dependent gene explains the apparent derepression of the AtSN1 retroelement in fpa mutants. Transcript readthrough accounts for the absence of changes in DNA methylation and siRNA abundance at AtSN1 in fpa mutants, and this may explain other examples of epigenetic transitions not associated with chromatin modification.

Post-transcriptional gene silencing (PTGS) is INTRODUCTIONPost-transcriptional gene silencing (PTGS), first identified in plants, is now thought to be an ancient self-defense mechanism acting against molecular parasites (Waterhouse et al., 2001). Introduction of double-stranded RNA (dsRNA) into plant cells triggers PTGS, resulting in the degradation of dsRNA and cognate mRNAs (Schweizer et al., 2000). A similar mechanism appears to operate in a wide variety of organisms, including filamentous fungi, nematodes, Drosophila , mice, and cultured HeLa cells, and generally is referred to as RNA interference (RNAi) (Cogoni and Macino, 1999a;Fire, 1999;Grant, 1999;Sharp and Zamore, 2000;Elbashir et al., 2001a;Svoboda et al., 2000). Recently, homologous genes required for PTGS were identified from different organisms, demonstrating the conservation of the gene-silencing machinery Macino, 1999a, 1999b;Ketting et al., 1999;Tabara et al., 1999;Catalanotto et al., 2000;Dalmay et al., 2000Dalmay et al., , 2001Domeier et al., 2000;Fagard et al., 2000;Mourrain et al., 2000;Smardon et al., 2000;Wu-Scharf et al., 2000). The accumulation of 21-to 25-nucleotide RNAs corresponding to both sense and antisense strands of target RNA occurs during PTGS in plant and animal cells (Hamilton and Baulcombe, 1999;Hammond et al., 2000;Parrish et al., 2000). These 21-to 25-nucleotide RNAs are generated by an RNase III-like enzyme (DICER) as the initiation step of RNAi, providing the specificity of a second RNase complex (RISC) that targets the cognate single-stranded (ss) RNAs (Bernstein et al., 2001).In plants, PTGS has evolved as an antiviral system. PTGS is triggered efficiently by dsRNA intermediates of cytoplasmically replicating viruses. The RNA genome of the invading virus is targeted and eliminated specifically when this natural antiviral mechanism is activated (Waterhouse et al., 1998(Waterhouse et al., , 1999Baulcombe, 1999;Smith et al., 2000;. In higher plants, PTGS is not limited to the cells in which it is activated, because mobile signals produced by PTGS can spread and confer sequence-specific RNA degradation in distant tissues (Palauqui et al., 1997;Voinnet and Baulcombe, 1997).Consistent with the importance of PTGS as an antiviral response, many viruses encode gene-silencing suppressor proteins (Anandalakshmi et al., 1998;Beclin et al., 1998;Brigneti et al., 1998;Kasschau and Carrington, 1998;Voinnet et al., 1999Voinnet et al., , 2000. However, not all viruses are able to suppress PTGS, and some virus-infected plants recover after the development of the first systemic viral symptoms (e.g., 1 These authors contributed equally to this work. 2 To whom correspondence should be addressed. E-mail burgyan@ abc.hu; fax 36-28-430-416. Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.010366. 360The Plant Cell nepovirus-infected tobacco plants; Ratcliff et al., 1997). Upper leaves of recovered plants lack symptoms (or show attenuated symptoms), and the virus content in these leav...

Micro RNAs (miRNAs) represent a class of short, non-coding, endogenous RNAs which play important roles in post-transcriptional regulation of gene expression. While the diverse functions of miRNAs in model plants have been well studied, the impact of miRNAs in crop plant biology is poorly understood. Here we used high-throughput sequencing and bioinformatics analysis to analyze miRNAs in the tuber bearing crop potato (Solanum tuberosum). Small RNAs were analysed from leaf and stolon tissues. 28 conserved miRNA families were found and potato-specific miRNAs were identified and validated by RNA gel blot hybridization. The size, origin and predicted targets of conserved and potato specific miRNAs are described. The large number of miRNAs and complex population of small RNAs in potato suggest important roles for these non-coding RNAs in diverse physiological and metabolic pathways.

Key message Downy mildew resistance across days post-inoculation, experiments, and years in two interspecific grapevine F 1 families was investigated using linear mixed models and Bayesian networks, and five new QTL were identified. Abstract Breeding grapevines for downy mildew disease resistance has traditionally relied on qualitative gene resistance, which can be overcome by pathogen evolution. Analyzing two interspecific F 1 families, both having ancestry derived from Vitis vinifera and wild North American Vitis species, across 2 years and multiple experiments, we found multiple loci associated with downy mildew sporulation and hypersensitive response in both families using a single phenotype model. The loci explained between 7 and 17% of the variance for either phenotype, suggesting a complex genetic architecture for these traits in the two families studied. For two loci, we used RNA-Seq to detect differentially transcribed genes and found that the candidate genes at these loci were likely not NBS-LRR genes. Additionally, using a multiple phenotype Bayesian network analysis, we found effects between the leaf trichome density, hypersensitive response, and sporulation phenotypes. Moderate-high heritabilities were found for all three phenotypes, suggesting that selection for downy mildew resistance is an achievable goal by breeding for either physical-or non-physical-based resistance mechanisms, with the combination of the two possibly providing durable resistance.