The characterization of cancer genomes has provided insight into somatically altered genes across tumors, transformed our understanding of cancer biology, and enabled tailoring of therapeutic strategies. However, the function of most cancer alleles remains mysterious, and many cancer features transcend their genomes. Consequently, tumor genomic characterization does not influence therapy for most patients. Approaches to understand the function and circuitry of cancer genes provide complementary approaches to elucidate both oncogene and non-oncogene dependencies. Emerging work indicates that the diversity of therapeutic targets engendered by non-oncogene dependencies is much larger than the list of recurrently mutated genes. Here we describe a framework for this expanded list of cancer targets, providing novel opportunities for clinical translation.
Retroelements, the prevalent class of plant transposons, have major impacts on host genome integrity and evolution. They produce multiple proteins from highly compact genomes and, similar to viruses, must have evolved original strategies to optimize gene expression, although this aspect has been seldom investigated thus far. Here, we have established a high-resolution transcriptome/translatome map for the near-entirety of transposons, using two distinct DNA methylation mutants in which transposon expression is broadly de-repressed. The value of this map to study potentially intact and transcriptionally active transposons in is illustrated by our comprehensive analysis of the cotranscriptional and translational features of elements, a family of young and active retroelements in plant genomes, and how such features impact their biology. Genome-wide transcript profiling revealed a unique and widely conserved alternative splicing event coupled to premature termination that allows for the synthesis of a short subgenomic RNA solely dedicated to production of the GAG structural protein and that preferentially associates with polysomes for efficient translation. Mutations engineered in a transgenic version of the element further show how alternative splicing is crucial for the appropriate coordination of full-length and subgenomic RNA transcription. We propose that this hitherto undescribed genome expression strategy, conserved among plant elements, enables an excess of structural versus catalytic components, mandatory for mobilization.
In plants, tasiRNAs form a class of endogenous secondary siRNAs produced through the action of RNA-DEPENDENT-RNA-POLYMERASE-6 (RDR6) upon microRNA-mediated cleavage of non-coding TAS RNAs. In Arabidopsis thaliana, TAS1, TAS2 and TAS4 tasiRNA production proceeds via a single cleavage event mediated by 22nt-long or/and asymmetric miRNAs in an ARGONAUTE-1 (AGO1)-dependent manner. By contrast, tasiRNA production from TAS3 seems to follow the so-called ‘two-hit’ process, where dual targeting of TAS3, specifically mediated by the 21nt-long, symmetric miR390, initiates AGO7-dependent tasiRNA production. Interestingly, features for TAS3 tasiRNA production differ in other plant species and we show here that such features also enable TAS3 tasiRNA biogenesis in Arabidopsis, and that a single miR390 targeting event is, in fact, sufficient for this process, suggesting that the ‘one-hit’ model underpins all the necessary rudiments of secondary siRNA biogenesis from plant TAS transcripts. Further results suggest that the two-hit configuration likely enhances the fidelity of tasiRNA production and, hence, the accuracy of downstream gene regulation. Finally, we show that a ‘non-cleavable one-hit’ process allows tasiRNA production from both TAS1 and TAS3 transcripts, indicating that RDR6 recruitment does not require miRNA cleavage, nor does the recruitment, as we further show, of SUPRRESSOR-OF-GENE-SILENCING-3, indispensable for tasiRNA generation.
Plant RNA silencing operates via RNA-directed DNA-methylation (RdDM) to repress transcription or by targeting mRNAs via posttranscriptional gene silencing (PTGS). These pathways rely on distinct Dicer-like (DCL) proteins that process doublestranded RNA (dsRNA) into small-interfering RNAs (siRNAs). Here, we explored the expression and subcellular localization of Arabidopsis thaliana DCL4. DCL4 expression predominates as a transcription start site isoform encoding a cytoplasmic protein, which also represents the ancestral form in plants. A longer DCL4 transcript isoform encoding a nuclear localization signal, DCL4 NLS , is present in Arabidopsis, but DNA methylation normally suppresses its expression. Hypomethylation caused by mutation, developmental reprogramming, and biotic stress correlates with enhanced DCL4 NLS expression, while hypermethylation of a DCL4 transgene causes a reduction in DCL4 NLS expression. DCL4 NLS functions in a noncanonical siRNA pathway, producing a unique set of 21-nucleotide-long "disiRNAs," for DCL4 NLS isoform-dependent siRNAs, through the nuclear RdDM dsRNA synthesis pathway. disiRNAs originate mostly from transposable elements (TEs) and TEoverlapping/proximal genes, load into the PTGS effector ARGONAUTE1 (AGO1), and display a subtle effect on transcript accumulation together with overlapping 24-nucleotide siRNAs. We propose that, via PTGS, disiRNAs could help to tighten the expression of epigenetically activated TEs and genes using the methylation-state-responsive DCL4 NLS .
Diverse classes of silencing small (s)RNAs operate via ARGONAUTE-family proteins within RNA-induced-silencing-complexes (RISCs). Here, we have streamlined various embodiments of a Q-sepharose-based RISC-purification method that relies on conserved biochemical properties of all ARGONAUTEs. We show, in multiple benchmarking assays, that the resulting 15-min benchtop extraction procedure allows simultaneous purification of all known classes of RISC-associated sRNAs without prior knowledge of the samples-intrinsic ARGONAUTE repertoires. Optimized under a user-friendly format, the method – coined ‘TraPR’ for Trans-kingdom, rapid, affordable Purification of RISCs – operates irrespectively of the organism, tissue, cell type or bio-fluid of interest, and scales to minute amounts of input material. The method is highly suited for direct profiling of silencing sRNAs, with TraPR-generated sequencing libraries outperforming those obtained via gold-standard procedures that require immunoprecipitations and/or lengthy polyacrylamide gel-selection. TraPR considerably improves the quality and consistency of silencing sRNA sample preparation including from notoriously difficult-to-handle tissues/bio-fluids such as starchy storage roots or mammalian plasma, and regardless of RNA contaminants or RNA degradation status of samples.
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