The Rice ILI2 Locus Is a Bidirectional Target of the African Xanthomonas oryzae pv. oryzae Major Transcription Activator-like Effector TalC but Does Not Contribute to Disease Susceptibility.

Xanthomonas oryzae pv. oryzae (Xoo) strains that cause bacterial leaf blight (BLB) limit rice (Oryza sativa) production and require breeding more resistant varieties. Transcription activator-like effectors (TALEs) activate transcription to promote leaf colonization by binding to specific plant host DNA sequences termed effector binding elements (EBEs). Xoo major TALEs universally target susceptibility genes of the SWEET transporter family. TALE-unresponsive alleles of clade III OsSWEET susceptibility gene promoter created with genome editing confer broad resistance on Asian Xoo strains. African Xoo strains rely primarily on the major TALE TalC, which targets OsSWEET14. Although the virulence of a talC mutant strain is severely impaired, abrogating OsSWEET14 induction with genome editing does not confer equivalent resistance on African Xoo. To address this contradiction, we postulated the existence of a TalC target susceptibility gene redundant with OsSWEET14. Bioinformatics analysis identified a rice locus named ATAC composed of the INCREASED LEAF INCLINATION 2 (ILI2) gene and a putative lncRNA that are shown to be bidirectionally upregulated in a TalC-dependent fashion. Gain-of-function approaches with designer TALEs inducing ATAC sequences did not complement the virulence of a Xoo strain defective for SWEET gene activation. While editing the TalC EBE at the ATAC loci compromised TalC-mediated induction, multiplex edited lines with mutations at the OsSWEET14 and ATAC loci remained essentially susceptible to African Xoo strains. Overall, this work indicates that ATAC is a probable TalC off-target locus but nonetheless documents the first example of divergent transcription activation by a native TALE during infection.

Urinary extracellular vesicles contain mature transcriptome enriched in circular and long noncoding RNAs with functional significance in prostate cancer.

Long noncoding (lnc)RNAs modulate gene expression alongside presenting unexpected source of neoantigens. Despite their immense interest, their ability to be transferred and control adjacent cells is unknown. Extracellular Vesicles (EVs) offer a protective environment for nucleic acids, with pro and antitumourigenic functions by controlling the immune response. In contrast to extracellular nonvesicular RNA, few studies have addressed the full RNA content within human fluids' EVs and have compared them with their tissue of origin. Here, we performed Total RNA-Sequencing on six Formalin-Fixed-Paraffin-Embedded (FFPE) prostate cancer (PCa) tumour tissues and their paired urinary (u)EVs to provide the first whole transcriptome comparison from the same patients. UEVs contain simplified transcriptome with intron-free cytoplasmic transcripts and enriched lnc/circular (circ)RNAs, strikingly common to an independent 20 patients' urinary cohort. Our full cellular and EVs transcriptome comparison within three PCa cell lines identified a set of overlapping 14 uEV-circRNAs characterized as essential for prostate cell proliferation in vitro and 28 uEV-lncRNAs belonging to the cancer-related lncRNA census (CLC2). In addition, we found 15 uEV-lncRNAs, predicted to encode 768 high-affinity neoantigens, and for which three of the encoded-ORF produced detectable unmodified peptides by mass spectrometry. Our dual analysis of EVs-lnc/circRNAs both in urines' and in vitro's EVs provides a fundamental resource for future uEV-lnc/circRNAs phenotypic characterization involved in PCa.

HOTAIR lncRNA promotes epithelial-mesenchymal transition by redistributing LSD1 at regulatory chromatin regions.

Epithelial-to-mesenchymal transition (EMT) describes the loss of epithelial traits and gain of mesenchymal traits by normal cells during development and by neoplastic cells during cancer metastasis. The long noncoding RNA HOTAIR triggers EMT, in part by serving as a scaffold for PRC2 and thus promoting repressive histone H3K27 methylation. In addition to PRC2, HOTAIR interacts with the LSD1 lysine demethylase, an epigenetic regulator of cell fate during development and differentiation, but little is known about the role of LSD1 in HOTAIR function during EMT. Here, we show that HOTAIR requires its LSD1-interacting domain, but not its PRC2-interacting domain, to promote the migration of epithelial cells. This activity is suppressed by LSD1 overexpression. LSD1-HOTAIR interactions induce partial reprogramming of the epithelial transcriptome altering LSD1 distribution at promoter and enhancer regions. Thus, we uncover an unexpected role of HOTAIR in EMT as an LSD1 decommissioning factor, counteracting its activity in the control of epithelial identity.

Badnaviruses and banana genomes: a long association sheds light on Musa phylogeny and origin.

Badnaviruses are double-stranded DNA pararetroviruses of the family Caulimoviridae. Badnaviral sequences found in banana are distributed over three main clades of the genus Badnavirus and exhibit wide genetic diversity. Interestingly, the nuclear genome of many plants, including banana, is invaded by numerous badnaviral sequences although badnaviruses do not require an integration step to replicate, unlike animal retroviruses. Here, we confirm that banana streak viruses (BSVs) are restricted to clades 1 and 3. We also show that only BSVs from clade 3 encompassing East African viral species are not integrated into Musa genomes, unlike BSVs from clade 1. Finally, we demonstrate that sequences from clade 2 are definitively integrated into Musa genomes with no evidence of episomal counterparts; all are phylogenetically distant from BSVs known to date. Using different molecular approaches, we dissected the coevolution between badnaviral sequences of clade 2 and banana by comparing badnavirus integration patterns across a banana sampling representing major Musa speciation events. Our data suggest that primary viral integrations occurred millions of years ago in banana genomes under different possible scenarios. Endogenous badnaviral sequences can be used as powerful markers to better characterize the Musa phylogeny, narrowing down the likely geographical origin of the Musa ancestor.

Landscape of the Noncoding Transcriptome Response of Two Arabidopsis Ecotypes to Phosphate Starvation.

Root architecture varies widely between species; it even varies between ecotypes of the same species, despite strong conservation of the coding portion of their genomes. By contrast, noncoding RNAs evolve rapidly between ecotypes and may control their differential responses to the environment, since several long noncoding RNAs (lncRNAs) are known to quantitatively regulate gene expression. Roots from ecotypes Columbia and Landsberg erecta of Arabidopsis (Arabidopsis thaliana) respond differently to phosphate starvation. Here, we compared transcriptomes (mRNAs, lncRNAs, and small RNAs) of root tips from these two ecotypes during early phosphate starvation. We identified thousands of lncRNAs that were largely conserved at the DNA level in these ecotypes. In contrast to coding genes, many lncRNAs were specifically transcribed in one ecotype and/or differentially expressed between ecotypes independent of phosphate availability. We further characterized these ecotype-related lncRNAs and studied their link with small interfering RNAs. Our analysis identified 675 lncRNAs differentially expressed between the two ecotypes, including antisense RNAs targeting key regulators of root-growth responses. Misregulation of several lincRNAs showed that at least two ecotype-related lncRNAs regulate primary root growth in ecotype Columbia. RNA-sequencing analysis following deregulation of lncRNA NPC48 revealed a potential link with root growth and transport functions. This exploration of the noncoding transcriptome identified ecotype-specific lncRNA-mediated regulation in root apexes. The noncoding genome may harbor further mechanisms involved in ecotype adaptation of roots to different soil environments.

Reference-free transcriptome exploration reveals novel RNAs for prostate cancer diagnosis.

The use of RNA-sequencing technologies held a promise of improved diagnostic tools based on comprehensive transcript sets. However, mining human transcriptome data for disease biomarkers in clinical specimens are restricted by the limited power of conventional reference-based protocols relying on unique and annotated transcripts. Here, we implemented a blind reference-free computational protocol, DE-kupl, to infer yet unreferenced RNA variations from total stranded RNA-sequencing datasets of tissue origin. As a bench test, this protocol was powered for detection of RNA subsequences embedded into putative long noncoding (lnc)RNAs expressed in prostate cancer. Through filtering of 1,179 candidates, we defined 21 lncRNAs that were further validated by NanoString for robust tumor-specific expression in 144 tissue specimens. Predictive modeling yielded a restricted probe panel enabling more than 90% of true-positive detections of cancer in an independent The Cancer Genome Atlas cohort. Remarkably, this clinical signature made of only nine unannotated lncRNAs largely outperformed PCA3, the only used prostate cancer lncRNA biomarker, in detection of high-risk tumors. This modular workflow is highly sensitive and can be applied to any pathology or clinical application.

DE-kupl: exhaustive capture of biological variation in RNA-seq data through k-mer decomposition.

We introduce a k-mer-based computational protocol, DE-kupl, for capturing local RNA variation in a set of RNA-seq libraries, independently of a reference genome or transcriptome. DE-kupl extracts all k-mers with differential abundance directly from the raw data files. This enables the retrieval of virtually all variation present in an RNA-seq data set. This variation is subsequently assigned to biological events or entities such as differential long non-coding RNAs, splice and polyadenylation variants, introns, repeats, editing or mutation events, and exogenous RNA. Applying DE-kupl to human RNA-seq data sets identified multiple types of novel events, reproducibly across independent RNA-seq experiments.

In plants, decapping prevents RDR6-dependent production of small interfering RNAs from endogenous mRNAs.

Cytoplasmic degradation of endogenous RNAs is an integral part of RNA quality control (RQC) and often relies on the removal of the 5' cap structure and their subsequent 5' to 3' degradation in cytoplasmic processing (P-)bodies. In parallel, many eukaryotes degrade exogenous and selected endogenous RNAs through post-transcriptional gene silencing (PTGS). In plants, PTGS depends on small interfering (si)RNAs produced after the conversion of single-stranded RNAs to double-stranded RNAs by the cellular RNA-dependent RNA polymerase 6 (RDR6) in cytoplasmic siRNA-bodies. PTGS and RQC compete for transgene-derived RNAs, but it is unknown whether this competition also occurs for endogenous transcripts. We show that the lethality of decapping mutants is suppressed by impairing RDR6 activity. We establish that upon decapping impairment hundreds of endogenous mRNAs give rise to a new class of rqc-siRNAs, that over-accumulate when RQC processes are impaired, a subset of which depending on RDR6 for their production. We observe that P- and siRNA-bodies often are dynamically juxtaposed, potentially allowing for cross-talk of the two machineries. Our results suggest that the decapping of endogenous RNA limits their entry into the PTGS pathway. We anticipate that the rqc-siRNAs identified in decapping mutants represent a subset of a larger ensemble of endogenous siRNAs.