In plants, microRNAs (miRNAs) comprise one of two classes of small RNAs that function primarily as negative regulators at the posttranscriptional level. Several MIRNA genes in the plant kingdom are ancient, with conservation extending between angiosperms and the mosses, whereas many others are more recently evolved. Here, we use deep sequencing and computational methods to identify, profile and analyze non-conserved MIRNA genes in Arabidopsis thaliana. 48 non-conserved MIRNA families, nearly all of which were represented by single genes, were identified. Sequence similarity analyses of miRNA precursor foldback arms revealed evidence for recent evolutionary origin of 16 MIRNA loci through inverted duplication events from protein-coding gene sequences. Interestingly, these recently evolved MIRNA genes have taken distinct paths. Whereas some non-conserved miRNAs interact with and regulate target transcripts from gene families that donated parental sequences, others have drifted to the point of non-interaction with parental gene family transcripts. Some young MIRNA loci clearly originated from one gene family but form miRNAs that target transcripts in another family. We suggest that MIRNA genes are undergoing relatively frequent birth and death, with only a subset being stabilized by integration into regulatory networks.

Eukaryotes contain a diversified set of small RNA-guided pathways that control genes, repeated sequences, and viruses at the transcriptional and posttranscriptional levels. Genome-wide profiles and analyses of small RNAs, particularly the large class of 24-nucleotide (nt) short interfering RNAs (siRNAs), were done for wild-type Arabidopsis thaliana and silencing pathway mutants with defects in three RNA-dependent RNA polymerase (RDR) and four Dicer-like (DCL) genes. The profiling involved direct analysis using a multiplexed, parallel-sequencing strategy. Small RNA-generating loci, especially those producing predominantly 24-nt siRNAs, were found to be highly correlated with repetitive elements across the genome. These were found to be largely RDR2- and DCL3-dependent, although alternative DCL activities were detected on a widespread level in the absence of DCL3. In contrast, no evidence for RDR2-alternative activities was detected. Analysis of RDR2- and DCL3-dependent small RNA accumulation patterns in and around protein-coding genes revealed that upstream gene regulatory sequences systematically lack siRNA-generating activities. Further, expression profiling suggested that relatively few genes, proximal to abundant 24-nt siRNAs, are regulated directly by RDR2- and DCL3-dependent silencing. We conclude that the widespread accumulation patterns for RDR2- and DCL3-dependent siRNAs throughout the Arabidopsis genome largely reflect mechanisms to silence highly repeated sequences.

Posttranscriptional RNA silencing of many endogenous transcripts, viruses, and transgenes involves the RNA-DEPENDENT RNA POLYMERASE6/DICER-LIKE4 (RDR6/DCL4)-dependent short interfering RNA (siRNA) biogenesis pathway. Arabidopsis thaliana contains several families of trans-acting siRNAs (tasiRNAs) that form in 21-nucleotide phased arrays through the RDR6/DCL4-dependent pathway and that negatively regulate target transcripts. Using deep sequencing technology and computational approaches, the phasing patterns of known tasiRNAs and tasiRNA-like loci from across the Arabidopsis genome were analyzed in wild-type plants and silencing-defective mutants. Several gene transcripts were found to be routed through the RDR6/DCL4-dependent pathway after initial targeting by one or multiple miRNAs or tasiRNAs, the most conspicuous example of which was an expanding clade of genes encoding pentatricopeptide repeat (PPR) proteins. Interestingly, phylogenetic analysis using Populus trichocarpa revealed evidence for small RNA-mediated regulatory mechanisms within a similarly expanded group of PPR genes. We suggest that posttranscriptional silencing mechanisms operate on an evolutionary scale to buffer the effects of rapidly expanding gene families.

We propose a new statistical test for assessing differential gene expression using RNA sequencing (RNA-Seq) data. Commonly used probability distributions, such as binomial or Poisson, cannot appropriately model the count variability in RNA-Seq data due to overdispersion. The small sample size that is typical in this type of data also prevents the uncritical use of tools derived from large-sample asymptotic theory. The test we propose is based on the NBP parameterization of the negative binomial distribution. It extends an exact test proposed by Robinson and Smyth (2007, 2008). In one version of Robinson and Smyth’s test, a constant dispersion parameter is used to model the count variability between biological replicates. We introduce an additional parameter to allow the dispersion parameter to depend on the mean. Our parametric method complements nonparametric regression approaches for modeling the dispersion parameter. We apply the test we propose to an Arabidopsis data set and a range of simulated data sets. The results show that the test is simple, powerful and reasonably robust against departures from model assumptions.

The ascomycete fungus Tolypocladium inflatum, a pathogen of beetle larvae, is best known as the producer of the immunosuppressant drug cyclosporin. The draft genome of T. inflatum strain NRRL 8044 (ATCC 34921), the isolate from which cyclosporin was first isolated, is presented along with comparative analyses of the biosynthesis of cyclosporin and other secondary metabolites in T. inflatum and related taxa. Phylogenomic analyses reveal previously undetected and complex patterns of homology between the nonribosomal peptide synthetase (NRPS) that encodes for cyclosporin synthetase (simA) and those of other secondary metabolites with activities against insects (e.g., beauvericin, destruxins, etc.), and demonstrate the roles of module duplication and gene fusion in diversification of NRPSs. The secondary metabolite gene cluster responsible for cyclosporin biosynthesis is described. In addition to genes necessary for cyclosporin biosynthesis, it harbors a gene for a cyclophilin, which is a member of a family of immunophilins known to bind cyclosporin. Comparative analyses support a lineage specific origin of the cyclosporin gene cluster rather than horizontal gene transfer from bacteria or other fungi. RNA-Seq transcriptome analyses in a cyclosporin-inducing medium delineate the boundaries of the cyclosporin cluster and reveal high levels of expression of the gene cluster cyclophilin. In medium containing insect hemolymph, weaker but significant upregulation of several genes within the cyclosporin cluster, including the highly expressed cyclophilin gene, was observed. T. inflatum also represents the first reference draft genome of Ophiocordycipitaceae, a third family of insect pathogenic fungi within the fungal order Hypocreales, and supports parallel and qualitatively distinct radiations of insect pathogens. The T. inflatum genome provides additional insight into the evolution and biosynthesis of cyclosporin and lays a foundation for further investigations of the role of secondary metabolite gene clusters and their metabolites in fungal biology.