Alteration of ribosome function upon 5-fluorouracil treatment favors cancer cell drug-tolerance

Mechanisms of drug-tolerance remain poorly understood and have been linked to genomic but also to non-genomic processes. 5-fluorouracil (5-FU), the most widely used chemotherapy in oncology is associated with resistance. While prescribed as an inhibitor of DNA replication, 5-FU alters all RNA pathways. Here, we show that 5-FU treatment leads to the production of fluorinated ribosomes exhibiting altered translational activities. 5-FU is incorporated into ribosomal RNAs of mature ribosomes in cancer cell lines, colorectal xenografts, and human tumors. Fluorinated ribosomes appear to be functional, yet, they display a selective translational activity towards mRNAs depending on the nature of their 5′-untranslated region. As a result, we find that sustained translation of IGF-1R mRNA, which encodes one of the most potent cell survival effectors, promotes the survival of 5-FU-treated colorectal cancer cells. Altogether, our results demonstrate that “man-made” fluorinated ribosomes favor the drug-tolerant cellular phenotype by promoting translation of survival genes.

Genome-Scale Computational Identification and Characterization of UTR Introns in Atalantia buxifolia

Accumulated evidence has shown that CDS introns (CIs) play important roles in regulating gene expression. However, research on UTR introns (UIs) is limited. In this study, UIs (including 5′UTR and 3′UTR introns (5UIs and 3UIs)) were identified from the Atalantia buxifolia genome. The length and nucleotide distribution characteristics of both 5UIs and 3UIs and the distributions of cis-acting elements and transcription factor binding sites (TFBSs) in 5UIs were investigated. Moreover, PageMan enrichment analysis was applied to show the possible roles of transcripts containing UIs (UI-Ts). In total, 1077 5UIs and 866 3UIs were identified from 897 5UI-Ts and 670 3UI-Ts, respectively. Among them, 765 (85.28%) 5UI-Ts and 527 (78.66%) 3UI-Ts contained only one UI, and 94 (6.38%) UI-Ts contained both 5UI and 3UI. The UI density was lower than that of CDS introns, but their mean and median intron sizes were ~2 times those of the CDS introns. The A. buxifolia 5UIs were rich in gene-expression-enhancement-related elements and contained many TFBSs for BBR-BPC, MIKC_MADS, AP2 and Dof TFs, indicating that 5UIs play a role in regulating or enhancing the expression of downstream genes. Enrichment analysis revealed that UI-Ts involved in ‘not assigned’ and ‘RNA’ pathways were significantly enriched. Noteworthily, 119 (85.61%) of the 3UI-Ts were genes encoding pentatricopeptide (PPR) repeat-containing proteins. These results will be helpful for the future study of the regulatory roles of UIs in A. buxifolia.

Diversification of small RNA pathways underlies germline RNAi incompetence in wild C. elegans strains

ABSTRACTThe discovery that experimental delivery of dsRNA can induce gene silencing at target genes revolutionized genetics research, by both uncovering essential biological processes and creating new tools for developmental geneticists. However, wild-type C. elegans strains vary dramatically in their response to exogenous RNAi, challenging our characterization of RNAi in the lab relative to its activity and significance in nature. Here, we investigate why some strains fail to mount a robust RNAi response to germline targets. We observe diversity in mechanism: in some strains, the response is stochastic, either on or off among individuals, while in others the response is consistent but delayed. Increased activity of the Argonaute PPW-1, which is required for germline RNAi in the laboratory strain N2, rescues the response in some strains, but dampens it further in others. Across strains, we observe variability in expression of known RNAi genes and strain-specific instances of pseudogenization and allelic divergence. Our results support the conclusions that Argonautes share overlapping functions, that germline RNAi incompetence is strain-specific but likely caused by genetic variants at common genes, and that RNAi pathways are evolving rapidly and dynamically. This work expands our understanding of RNAi by identifying conserved and variable pathway components, and it offers new access into characterizing gene function, identifying pathway interactions, and elucidating the biological significance of RNAi.

Nematode Small RNA Pathways in the Absence of piRNAs

Small RNA pathways play diverse regulatory roles in the nematode C. elegans. However, our understanding of small RNA pathways, their conservation, and their roles in other nematodes is limited. Here, we analyzed small RNA pathways in the parasitic nematode Ascaris. Ascaris has ten Argonautes with five worm-specific Argonautes (WAGOs) that are associated with secondary 5'-triphosphate small RNAs (22-24G-RNAs). These Ascaris WAGOs and their small RNAs target repetitive sequences (WAGO-1, WAGO-2, WAGO-3, and NRDE-3) or mature mRNAs (CSR-1, NRDE-3, and WAGO-3) and are similar to the C. elegans mutator, nuclear, and CSR-1 small RNA pathways. Ascaris CSR-1 likely functions to "license" gene expression in the absence of an Ascaris piRNA pathway. Ascaris ALG-4 and its associated 26G-RNAs target and appear to repress specific mRNAs during meiosis in the testes. Notably, Ascaris WAGOs (WAGO-3 and NRDE-3) small RNAs change their targets between repetitive sequences and mRNAs during spermatogenesis or in early embryos illustrating target plasticity of these WAGOs. We provide a unique and comprehensive view of mRNA and small RNA expression throughout nematode spermatogenesis that illustrates the dynamics and flexibility of small RNA pathways. Overall, our study provides key insights into the conservation and divergence of nematode small RNA pathways.

Lost and Found: Piwi and Argonaute Pathways in Flatworms

Platyhelminthes comprise one of the major phyla of invertebrate animals, inhabiting a wide range of ecosystems, and one of the most successful in adapting to parasitic life. Small non-coding RNAs have been implicated in regulating complex developmental transitions in model parasitic species. Notably, parasitic flatworms have lost Piwi RNA pathways but gained a novel Argonaute gene. Herein, we analyzed, contrasted and compared the conservation of small RNA pathways among several free-living species (a paraphyletic group traditionally known as ‘turbellarians’) and parasitic species (organized in the monophyletic clade Neodermata) to disentangle possible adaptations during the transition to parasitism. Our findings showed that complete miRNA and RNAi pathways are present in all analyzed free-living flatworms. Remarkably, whilst all ‘turbellarians’ have Piwi proteins, these were lost in parasitic Neodermantans. Moreover, two clusters of Piwi class Argonaute genes are present in all ‘turbellarians’. Interestingly, we identified a divergent Piwi class Argonaute in free living flatworms exclusively, which we named ‘Fliwi’. In addition, other key proteins of the Piwi pathways were conserved in ‘turbellarians’, while none of them were detected in Neodermatans. Besides Piwi and the canonical Argonaute proteins, a flatworm-specific class of Argonautes (FL-Ago) was identified in the analyzed species confirming its ancestrallity to all Platyhelminthes. Remarkably, this clade was expanded in parasitic Neodermatans, but not in free-living species. These phyla-specific Argonautes showed lower sequence conservation compared to other Argonaute proteins, suggesting that they might have been subjected to high evolutionary rates. However, key residues involved in the interaction with the small RNA and mRNA cleavage in the canonical Argonautes were more conserved in the FL-Agos than in the Piwi Argonautes. Whether this is related to specialized functions and adaptations to parasitism in Neodermatans remains unclear. In conclusion, differences detected in gene conservation, sequence and structure of the Argonaute family suggest tentative biological and evolutionary diversifications that are unique to Platyhelminthes. The remarkable divergencies in the small RNA pathways between free-living and parasitic flatworms indicate that they may have been involved in the adaptation to parasitism of Neodermatans.

Characterization of the RNA-interference pathway as a tool for reverse genetic analysis in the nascent phototrophic endosymbiosis, Paramecium bursaria

Endosymbiosis was fundamental for the evolution of eukaryotic complexity. Endosymbiotic interactions can be dissected through forward- and reverse-genetic experiments, such as RNA-interference (RNAi). However, distinguishing small (s)RNA pathways in a eukaryote–eukaryote endosymbiotic interaction is challenging. Here, we investigate the repertoire of RNAi pathway protein-encoding genes in the model nascent endosymbiotic system, Paramecium bursaria–Chlorella spp. Using comparative genomics and transcriptomics supported by phylogenetics, we identify essential proteome components of the small interfering (si)RNA, scan (scn)RNA and internal eliminated sequence (ies)RNA pathways. Our analyses reveal that copies of these components have been retained throughout successive whole genome duplication (WGD) events in the Paramecium clade. We validate feeding-induced siRNA-based RNAi in P. bursaria via knock-down of the splicing factor, u2af1 , which we show to be crucial to host growth. Finally, using simultaneous knock-down ‘paradox’ controls to rescue the effect of u2af1 knock-down, we demonstrate that feeding-induced RNAi in P. bursaria is dependent upon a core pathway of host-encoded Dcr1 , Piwi and Pds1 components. Our experiments confirm the presence of a functional, host-derived RNAi pathway in P. bursaria that generates 23-nt siRNA, validating the use of the P. bursaria – Chlorella spp. system to investigate the genetic basis of a nascent endosymbiosis.

Ultrastructural immunocytochemistry shows impairment of RNA pathways in skeletal muscle nuclei of old mice: A link to sarcopenia?

During aging, skeletal muscle is affected by sarcopenia, a progressive decline in muscle mass, strength and endurance that leads to loss of function and disability. Cell nucleus dysfunction is a possible factor contributing to sarcopenia because aging-associated alterations in mRNA and rRNA transcription/maturation machinery have been shown in several cell types including muscle cells. In this study, the distribution and density of key molecular factors involved in RNA pathways namely, nuclear actin (a motor protein and regulator of RNA transcription), 5-methyl cytosine (an epigenetic regulator of gene transcription), and ribonuclease A (an RNA degrading enzyme) were compared in different nuclear compartments of late adult and old mice myonuclei by means of ultrastructural immunocytochemistry. In all nuclear compartments, an age-related decrease of nuclear actin suggested altered chromatin structuring and impaired nucleus-to-cytoplasm transport of both mRNA and ribosomal subunits, while a decrease of 5-methyl cytosine and ribonuclease A in the nucleoli of old mice indicated an age-dependent loss of rRNA genes. These findings provide novel experimental evidence that, in the aging skeletal muscle, nuclear RNA pathways undergo impairment, likely hindering protein synthesis and contributing to the onset and progression of sarcopenia.

RNA-silencing induces target gene relocalization toward a specialized nuage domain

Argonaute small RNA pathways maintain genome integrity and fertility by enforcing the transgenerational silencing of transposons as well as many developmentally regulated germline genes 1. To propagate silencing, Argonaute pathways coordinate heterochromatin silencing with cycles of small RNA amplification 2. In animal germlines, mRNA surveillance is thought to occur within cytoplasmic perinuclear domains called nuage 3. In C. elegans 20-50 nuage droplets called P granules surround each pachytene germline nucleus. P granules are known to host many of the Argonaute small RNA systems that carry out transcriptome surveillance, but what if any specific roles P granules might play in Argonaute silencing have remained mysterious. Here we show that RNAi triggers the expansion of a unique P granule, which accumulates large amounts of the target RNA. As transcriptional silencing ensues, both alleles of the target gene relocate near the inner nuclear membrane (INM) directly adjacent this enlarged P granule. Similarly, during piRNA-mediated silencing, both alleles of a target gene reside adjacent to a P granule containing target RNA sequences. In an Argonaute mutant defective in piRNA silencing, the target RNA is released from nuage, and the target alleles dissociate from each other and from the INM. Our findings suggest that transcriptome-surveillance tasks are sub-divided between nuage domains that become specialized to coordinate small RNA silencing signals to their heterochromatin targets.