Processing of a Dicistronic tRNA-snoRNA Precursor: Combined Analysis in Vitro and in Vivo Reveals Alternate Pathways and Coupling to Assembly of snoRNP1

The C/D box small nucleolar RNAs (snoRNAs) represent an essential class of small nucleolar RNAs that guide 2′-O-Rib methylation of ribosomal RNAs and other RNAs in eukaryotes. In Arabidopsis (Arabidopsis thaliana), >100 C/D snoRNAs have been identified, most of them encoded by polycistronic gene clusters, but little is known on the factors controlling their biogenesis. Here, we focus on the identification of factors controlling the processing of tRNA-snoRNA dicistronic precursors (pre-tsnoRNA) synthesized by RNA polymerase III and producing tRNAGly and C/D snoR43. We produced radiolabeled RNA probes corresponding to different pre-tsnoRNA mutants to test their impact on processing in vitro by a recombinant tRNAse Z, the Arabidopsis endonuclease that processes the 3′end of tRNAs, and by nuclear extracts from cauliflower (Brassica oleracea) inflorescences that accurately process the pre-tsnoRNA. This was coupled to an in vivo analysis of the processing of tagged pre-tsnoRNA mutants expressed in Arabidopsis. Our results strongly implicate tRNase Z in endonucleolytic cleavage of the pre-tsnoRNA. In addition, they reveal an alternate pathway that could depend on a tRNA decay surveillance mechanism. Finally, we provide arguments showing that processing of pre-tsnoRNA, both in planta and by nuclear extracts, is coupled to the assembly of snoRNA with core proteins forming the functional snoRNP (for small nucleolar ribonucleoprotein complex)

AtNUFIP, an essential protein for plant development, reveals the impact of snoRNA gene organisation on the assembly of snoRNPs and rRNA methylation in Arabidopsis thaliana.

International audienceIn all eukaryotes, C/D small nucleolar ribonucleoproteins (C/D snoRNPs) are essential for methylation and processing of ribosomal RNAs. They consist of a box C/D small nucleolar RNA (C/D snoRNA) associated with four highly conserved nucleolar proteins. Recent data in HeLa cells and yeast have revealed that assembly of these snoRNPs is directed by NUFIP protein and other auxiliary factors. Nevertheless, the precise function and biological importance of NUFIP and the other assembly factors remains unknown. In plants, few studies have focused on RNA methylation and snoRNP biogenesis. Here, we identify and characterise the AtNUFIP gene that directs assembly of C/D snoRNP. To elucidate the function of AtNUFIP in planta, we characterized atnufip mutants. These mutants are viable but have severe developmental phenotypes. Northern blot analysis of snoRNA accumulation in atnufip mutants revealed a specific degradation of C/D snoRNAs and this situation is correlated with a reduction in rRNA methylation. Remarkably, the impact of AtNUFIP depends on the structure of snoRNA genes: it is essential for the accumulation of those C/D snoRNAs encoded by polycistronic genes, but not by monocistronic or tsnoRNA genes. We propose that AtNUFIP controls the kinetics of C/D snoRNP assembly on nascent precursors to overcome snoRNA degradation of aberrant RNPs. Finally, we show that AtNUFIP has broader RNP targets, controlling the accumulation of scaRNAs that direct methylation of spliceosomal snRNA in Cajal bodies

Inventory, evolution and expression profiling diversity of the LEA (late embryogenesis abundant) protein gene family in Arabidopsis thaliana

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Mapping rRNA 2’-O-methylations and identification of C/D snoRNAs in Arabidopsis thaliana plants

International audienceIn all eukaryotic cells, the most abundant modification of ribosomal RNA (rRNA) is methylation at the ribose moiety (2ʹ-O-methylation). Ribose methylation at specific rRNA sites is guided by small nucleolar RNAs (snoRNAs) of C/D-box type (C/D snoRNA) and achieved by the methyltransferase Fibrillarin (FIB). Here we used the Illumina-based RiboMethSeq approach for mapping rRNA 2ʹ-O-methylation sites in A. thaliana Col-0 (WT) plants. This analysis detected novel C/D snoRNA-guided rRNA 2ʹ-O-methylation positions and also some orphan sites without a matching C/D snoRNA. Furthermore, immunoprecipitation of Arabidopsis FIB2 identified and demonstrated expression of C/D snoRNAs corresponding to majority of mapped rRNA sites. On the other hand, we show that disruption of Arabidopsis Nucleolin 1 gene (NUC1), encoding a major nucleolar protein, decreases 2ʹ-O-methylation at specific rRNA sites suggesting functional/structural interconnections of 2ʹ-O-methylation with nucleolus organization and plant development. Finally, based on our findings and existent database sets, we introduce a new nomenclature system for C/D snoRNA in Arabidopsis plant

Sequencing the extrachromosomal circular mobilome reveals retrotransposon activity in plants

International audienceRetrotransposons are mobile genetic elements abundant in plant and animal genomes. While efficiently silenced by the epigenetic machinery, they can be reactivated upon stress or during development. Their level of transcription not reflecting their transposition ability, it is thus difficult to evaluate their contribution to the active mobilome. Here we applied a simple methodology based on the high throughput sequencing of extrachromosomal circular DNA (eccDNA) forms of active retrotransposons to characterize the repertoire of mobile retrotransposons in plants. This method successfully identified known active retrotransposons in both Arabidopsis and rice material where the epigenome is destabilized. When applying mobilome-seq to developmental stages in wild type rice, we identified PopRice as a highly active retrotransposon producing eccDNA forms in the wild type endosperm. The mobilome-seq strategy opens new routes for the characterization of a yet unexplored fraction of plant genomes

Large tandem duplications affect gene expression, 3D organization, and plant–pathogen response

Rapid plant genome evolution is crucial to adapt to environmental changes. Chromosomal rearrangements and gene copy number variation (CNV) are two important tools for genome evolution and sources for the creation of new genes. However, their emergence takes many generations. In this study, we show that in Arabidopsis thaliana, a significant loss of ribosomal RNA (rRNA) genes with a past history of a mutation for the chromatin assembly factor 1 (CAF1) complex causes rapid changes in the genome structure. Using long-read sequencing and microscopic approaches, we have identified up to 15 independent large tandem duplications in direct orientation (TDDOs) ranging from 60 kb to 1.44 Mb. Our data suggest that these TDDOs appeared within a few generations, leading to the duplication of hundreds of genes. By subsequently focusing on a line only containing 20% of rRNA gene copies (20rDNA line), we investigated the impact of TDDOs on 3D genome organization, gene expression, and cytosine methylation. We found that duplicated genes often accumulate more transcripts. Among them, several are involved in plant–pathogen response, which could explain why the 20rDNA line is hyper-resistant to both bacterial and nematode infections. Finally, we show that the TDDOs create gene fusions and/or truncations and discuss their potential implications for the evolution of plant genomes

Evidence for ARGONAUTE4–DNA interactions in RNA-directed DNA methylation in plants

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The mobilome-seq approach in plants.

<p>A schematic view of the main steps involved in the selection and amplification of the extrachromosomal circular molecules in plants. After DNA extraction, linear DNA molecules are digested and circular molecules are randomly amplified using rolling circle amplification. This DNA material is used for high-throughput sequencing. Mobilome-seq data analysis consists in characterizing the depth of coverage (DOC) of mapped reads and the presence of split reads (SRs) at TE loci and the detection of <i>de novo</i> assembled scaffolds corresponding to these TEs.</p

Mobilome-seq detection of <i>Tos17</i>, a known active retrotransposon in rice callus.

<p>(<b>A</b>) Abundance of reads mapping at TE-annotated loci in the <i>O</i>. <i>sativa</i> WT callus mobilome-seq library. Each dot represents the normalized coverage per million mapped reads per all TE-containing 100bp windows obtained after aligning the sequenced reads on the <i>O</i>. <i>sativa</i> reference genome. Green dots indicate the windows corresponding to annotated <i>Tos17</i> genomic loci. (<b>B</b>) Detail of the depth of coverage of total mapped reads (DOC) and split reads (SRs) abundance of the <i>O</i>. <i>sativa</i> WT callus and leaf mobilome-seq library at the <i>Tos17</i> locus on chromosome 7 (<i>green bar</i>). Legend as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006630#pgen.1006630.g002" target="_blank">Fig 2C</a>. (<b>C</b>) Detection of circular forms of <i>Tos17</i> using inverse PCR with primers localization depicted on the right (<i>black bar</i>: <i>Tos17</i> element, <i>arrows</i>: PCR primers, <i>grey boxes</i>: LTRs). Upper gel: PCR amplification of <i>Tos17</i> circles, middle gel: control PCR for <i>Tos17</i> detection, lower gel: PCR using <i>eEF1α</i> primers as loading control. (<b>D</b>) Dotter alignment of the scaffold #29 obtained after <i>de novo</i> assembly of callus mobilome-seq library and <i>Tos17</i>.</p

Mobilome-seq detection of a novel active retrotransposon in rice seeds.

<p>(<b>A</b>) Genome-wide analysis of mobilome-seq data identifies the <i>PopRice</i> retrotransposon family as the most represented active family in WT rice seeds. Legend as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006630#pgen.1006630.g003" target="_blank">Fig 3A</a>. Pink dots indicate the windows corresponding to <i>Osr4</i> and <i>PopRice</i> loci. (<b>B</b>) Detail of the depth of coverage of total mapped reads and split reads abundance of the <i>O</i>. <i>sativa</i> WT seeds mobilome-seq library at the <i>PopRice</i> locus on chromosome 2 (<i>pink bar</i>) for callus and leaf mobilome-seq data. Legend as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006630#pgen.1006630.g002" target="_blank">Fig 2C</a>. (<b>C</b>) Phylogenic tree showing that <i>PopRice</i> is a distinct subfamily of <i>Osr4</i> LTR-RT. The relative DOC calculated from two biological replicates in WT seed mobilome-seq data is indicated as a heatmap. (<b>D</b>) Dotter alignment of the scaffold #17 obtained after <i>de novo</i> assembly of WT seed mobilome-seq library and a <i>PopRice</i> element.</p