Physcomitrella patens DCL3 Is Required for 22–24 nt siRNA Accumulation, Suppression of Retrotransposon-Derived Transcripts, and Normal Development

Endogenous 24 nt short interfering RNAs (siRNAs), derived mostly from intergenic and repetitive genomic regions, constitute a major class of endogenous small RNAs in flowering plants. Accumulation of Arabidopsis thaliana 24 nt siRNAs requires the Dicer family member DCL3, and clear homologs of DCL3 exist in both flowering and non-flowering plants. However, the absence of a conspicuous 24 nt peak in the total RNA populations of several non-flowering plants has raised the question of whether this class of siRNAs might, in contrast to the ancient 21 nt microRNAs (miRNAs) and 21–22 nt trans-acting siRNAs (tasiRNAs), be an angiosperm-specific innovation. Analysis of non-miRNA, non-tasiRNA hotspots of small RNA production within the genome of the moss Physcomitrella patens revealed multiple loci that consistently produced a mixture of 21–24 nt siRNAs with a peak at 23 nt. These Pp23SR loci were significantly enriched in transposon content, depleted in overlap with annotated genes, and typified by dense concentrations of the 5-methyl cytosine (5 mC) DNA modification. Deep sequencing of small RNAs from two independent Ppdcl3 mutants showed that the P. patens DCL3 homolog is required for the accumulation of 22–24 nt siRNAs, but not 21 nt siRNAs, at Pp23SR loci. The 21 nt component of Pp23SR-derived siRNAs was also unaffected by a mutation in the RNA-dependent RNA polymerase mutant Pprdr6. Transcriptome-wide, Ppdcl3 mutants failed to accumulate 22–24 nt small RNAs from repetitive regions while transcripts from two abundant families of long terminal repeat (LTR) retrotransposon-associated reverse transcriptases were up-regulated. Ppdcl3 mutants also displayed an acceleration of leafy gametophore production, suggesting that repetitive siRNAs may play a role in the development of P. patens. We conclude that intergenic/repeat-derived siRNAs are indeed a broadly conserved, distinct class of small regulatory RNAs within land plants

Arabidopsis lyrata Small RNAs: Transient MIRNA and Small Interfering RNA Loci within the Arabidopsis Genus[W][OA]

This study exploits the recent production of a draft nuclear genome sequence for Arabidopsis lyrata to examine evolution of plant MIRNA and p4-siRNA loci between two congenic Brassicaceae species. The results indicate that many MIRNAs and most p4-siRNA hot spots are rapidly changing and evolutionarily transient within the Arabidopsis genus

Improved Placement of Multi-mapping Small RNAs

High-throughput sequencing of small RNAs (sRNA-seq) is a popular method used to discover and annotate microRNAs (miRNAs), endogenous short interfering RNAs (siRNAs), and Piwi-associated RNAs (piRNAs). One of the key steps in sRNA-seq data analysis is alignment to a reference genome. sRNA-seq libraries often have a high proportion of reads that align to multiple genomic locations, which makes determining their true origins difficult. Commonly used sRNA-seq alignment methods result in either very low precision (choosing an alignment at random), or sensitivity (ignoring multi-mapping reads). Here, we describe and test an sRNA-seq alignment strategy that uses local genomic context to guide decisions on proper placements of multi-mapped sRNA-seq reads. Tests using simulated sRNA-seq data demonstrated that this local-weighting method outperforms other alignment strategies using three different plant genomes. Experimental analyses with real sRNA-seq data also indicate superior performance of local-weighting methods for both plant miRNAs and heterochromatic siRNAs. The local-weighting methods we have developed are implemented as part of the sRNA-seq analysis program ShortStack, which is freely available under a general public license. Improved genome alignments of sRNA-seq data should increase the quality of downstream analyses and genome annotation efforts

The CLASSY family controls tissue-specific DNA methylation patterns in Arabidopsis.

DNA methylation shapes the epigenetic landscape of the genome, plays critical roles in regulating gene expression, and ensures transposon silencing. As is evidenced by the numerous defects associated with aberrant DNA methylation landscapes, establishing proper tissue-specific methylation patterns is critical. Yet, how such differences arise remains a largely open question in both plants and animals. Here we demonstrate that CLASSY1-4 (CLSY1-4), four locus-specific regulators of DNA methylation, also control tissue-specific methylation patterns, with the most striking pattern observed in ovules where CLSY3 and CLSY4 control DNA methylation at loci with a highly conserved DNA motif. On a more global scale, we demonstrate that specific clsy mutants are sufficient to shift the epigenetic landscape between tissues. Together, these findings reveal substantial epigenetic diversity between tissues and assign these changes to specific CLSY proteins, elucidating how locus-specific targeting combined with tissue-specific expression enables the CLSYs to generate epigenetic diversity during plant development

Expression and cellular localization of ZIP1 transporter under zinc deficiency in wild emmer wheat

Zinc deficiency is a common problem leading to severe decreases in grain yield and has detrimental effects on nutritional quality in cereals. Wild emmer wheat, Triticum turgidum ssp. dicoccoides, exhibits a potential genetic resource for wheat improvement due to its compatibility with modern wheat. In this study, Zn deficiency response of wild progenitors and modern wheat were examined using molecular and physiological approaches with plants grown under various Zn concentrations. The results revealed wide variation in response to Zn deficiency between wild emmer accessions. Among the wild emmer accessions studied, accession MM 5/4 was found to be most tolerant and accession 19-36 was the most sensitive to Zn deficiency. To better understand Zn transport mechanisms in wild emmer wheat, we analyzed the expression patterns of a ZRT/IRT-like gene, Zrt-, Irt-like protein (ZIP)1, in the roots and shoots of several accessions that were maintained on different concentrations of Zn. Quantitative real-time polymerase chain reaction results revealed that ZIP1 transcript levels are elevated with decreasing Zn supply in all accessions. Particularly, ZIP1 transcript accumulation was lower in the roots of accession MM 5/4 while the susceptible, 19-36 accession, has elevated levels of ZIP1 transcript, revealing a Zn deficiency response for this genotype. We also identified and cloned a full-length ZIP1 transporter, named TdZIP1, and further analyzed the corresponding protein sequence for structural attributes. Under Zn deficiency, deleting the last 20 amino acids from the last transmembrane domain of TdZIP1 and tagging with GFP resulted in endoplasmic reticulum localization. Functional expression of the isolated TdZIP1 using Zn-uptake defective Saccharomyces cerevisiae strains on limiting Zn media showed that it could indeed transport Zn. However, overexpression of this transporter causes excess accumulation of Zn in the cells, thus generating a toxic environment. Overall, our results indicate the possibility of using Triticum dicoccoides for the genetic improvement of zinc deficiency tolerance in wheat

Targeted profiling of human extrachromosomal DNA by CRISPR-CATCH

Extrachromosomal DNA (ecDNA) is a common mode of oncogene amplification but is challenging to analyze. Here, we adapt CRISPR-CATCH, in vitro CRISPR-Cas9 treatment and pulsed field gel electrophoresis of agarose-entrapped genomic DNA, previously developed for bacterial chromosome segments, to isolate megabase-sized human ecDNAs. We demonstrate strong enrichment of ecDNA molecules containing EGFR, FGFR2 and MYC from human cancer cells and NRAS ecDNA from human metastatic melanoma with acquired therapeutic resistance. Targeted enrichment of ecDNA versus chromosomal DNA enabled phasing of genetic variants, identified the presence of an EGFRvIII mutation exclusively on ecDNAs and supported an excision model of ecDNA genesis in a glioblastoma model. CRISPR-CATCH followed by nanopore sequencing enabled single-molecule ecDNA methylation profiling and revealed hypomethylation of the EGFR promoter on ecDNAs. We distinguished heterogeneous ecDNA species within the same sample by size and sequence with base-pair resolution and discovered functionally specialized ecDNAs that amplify select enhancers or oncogene-coding sequences