The RNase III enzymes Drosha and Dicer are essential for the production of small non-coding RNAs (ncRNAs). In canonical RNAi, microRNAs (miRNAs) regulate gene expression by post-transcriptional gene ...silencing. In non-canonical RNAi, nuclear RNAi factors generate small ncRNAs that are essential for transcriptional gene silencing. Recent evidence points to the existence of additional non-canonical nuclear RNAi functions in various organisms, including in genome maintenance and editing, as well as in DNA repair. Drosha and Dicer directly regulate gene expression and RNA metabolism at different stages, such as transcriptional initiation and termination, and the processing of various RNA species, including pre-mRNAs. Furthermore, Dicer isoforms were recently discovered and attributed with roles in apoptosis, development and disease.
Dicer is a key component of RNA interference (RNAi) and well-known for its role in biogenesis of micro (mi)RNA in the cytoplasm. Increasing evidence suggests that mammalian Dicer is also present and ...active in the nucleus. We have previously shown that phosphorylated human Dicer associates with chromatin in response to DNA damage and processes double-stranded (ds)RNA in the nucleus. However, a recent study by Much et al. investigated endogenously tagged HA-Dicer both in primary mouse embryonic fibroblasts (PMEFs) as well as adult homozygous viable and fertile HA-Dicer mice under physiological conditions and concluded that murine Dicer is exclusively cytoplasmic. The authors challenged several findings, reporting functions of Dicer in mammalian nuclei. We have re-investigated this issue by applying subcellular fractionation, super-resolution microscopy followed by 3D reconstitution, and phospho-Dicer-specific antibodies using the same HA-Dicer PMEF cell line. Our data show that a small fraction of the murine HA-Dicer pool, approximately 5%, localises in the nucleus and is phosphorylated upon DNA damage. We propose that Dicer localisation is dynamic and not exclusively cytoplasmic, particularly in cells exposed to DNA damage.
The endoribonuclease Dicer is a key component of the human RNA interference pathway and is known for its role in cytoplasmic microRNA production. Recent findings suggest that noncanonical Dicer ...generates small noncoding RNA to mediate the DNA damage response (DDR). Here, we show that human Dicer is phosphorylated in the platform-Piwi/Argonaute/Zwille-connector helix cassette (S1016) upon induction of DNA damage. Phosphorylated Dicer (p-Dicer) accumulates in the nucleus and is recruited to DNA double-strand breaks. We further demonstrate that turnover of damage-induced nuclear, double-stranded (ds) RNA requires additional phosphorylation of carboxy-terminal Dicer residues (S1728 and S1852). DNA damage-induced nuclear Dicer accumulation is conserved in mammals. Dicer depletion causes endogenous DNA damage and delays the DDR by impaired recruitment of repair factors MDC1 and 53BP1. Collectively, we place Dicer within the context of the DDR by demonstrating a DNA damage-inducible phosphoswitch that causes localized processing of nuclear dsRNA by p-Dicer to promote DNA repair.
The nuclear paraspeckle assembly transcript 1 (NEAT1) locus encodes two long non-coding (lnc)RNA isoforms that are upregulated in many tumours and dynamically expressed in response to stress. NEAT1 ...transcripts form ribonucleoprotein complexes with numerous RNA-binding proteins (RBPs) to assemble paraspeckles and modulate the localisation and activity of gene regulatory enzymes as well as a subset of messenger (m)RNA transcripts. The investigation of the dynamic composition of NEAT1-associated proteins and mRNAs is critical to understand the function of NEAT1. Interestingly, a growing number of biochemical and genetic tools to assess NEAT1 interactomes has been reported. Here, we discuss the Hybridisation Proximity (HyPro) labeling technique in the context of NEAT1. HyPro labeling is a recently developed method to detect spatially ordered interactions of RNA-containing nuclear compartments in cultured human cells. After introducing NEAT1 and paraspeckles, we describe the advantages of the HyPro technology in the context of other methods to study RNA interactomes, and review the key findings in mapping NEAT1-associated RNA transcripts and protein binding partners. We further discuss the limitations and potential improvements of HyPro labeling, and conclude by delineating its applicability in paraspeckles-related cancer research.
A number of authors have investigated the impact of early childhood education and care programs on the development of children. Often they have focused on the effects on children from ...socio-economically disadvantaged families. To assess the effects of various preschool programs on cognitive development, recent key studies were reviewed. In addition, the extent to which these programs could establish equal educational opportunities for children from different social backgrounds was evaluated. Program start, intensity, and duration were considered. The findings indicate that the vast majority of recent early education and care programs had considerable positive short-term effects and somewhat smaller long-term effects on cognitive development and that in relative terms children from socio-economically disadvantaged families made as much or slightly more progress than their more advantaged peers. Despite this, early childhood education and care cannot compensate completely for developmental deficits due to unfavorable learning conditions in disadvantaged milieus. Implications for research and policy are discussed.
Accumulating evidence indicates that students' sense of school belonging has a substantial positive effect on educational attainment. At the same time, life course and life span developmental ...theories suggest that the benefits of a sense of school belonging could be weakened by the channeling effects of education systems that assign students to distinct educational tracks that lead otherwise similar students to quite different educational destinations. The current study analyzed the extent to which the sense of school belonging predicted educational trajectories in a system that partially channels students into distinct tracks. It assessed educational trajectories as they relate to transitions at two critical junctures of the system-the transition from lower- to upper-secondary education, and from upper-secondary to tertiary (university) education. The study used data from a nationally representative panel survey that followed participants from age 15 to 30 (N = 4,986, 44% male, 12.9% immigrants). Findings indicated that students with a stronger sense of school belonging were more likely to continue in or transition into academic tracks. However, the benefits of students' sense of belonging were bounded by the system's channeling structure. While for students in academic tracks, the sense of school belonging strongly predicted the probability of continuing in academic tracks, it only marginally predicted the probability of moving into academic tracks for those whose educational career began in more vocationally oriented tracks. Hence the sense of school belonging may influence academic trajectories only inasmuch as institutional structures allow it to, because these structures differentially enable and constrain such trajectories.
Drugs for cancer therapy belong to different categories of chemical substances. The cellular targets for the therapeutic efficacy are often not unambiguously identified. Here, we describe the process ...of ribosome biogenesis as a target of a large variety of chemotherapeutic drugs. We determined the inhibitory concentration of 36 chemotherapeutic drugs for transcription and processing of ribosomal RNA by in vivo labeling experiments. Inhibitory drug concentrations were correlated to the loss of nucleolar integrity. The synergism of drugs inhibiting ribosomal RNA synthesis at different levels was studied. Drugs inhibited ribosomal RNA synthesis either at the level of (i) rRNA transcription (e.g. oxaliplatin, doxorubicin, mitoxantrone, methotrexate), (ii) early rRNA processing (e.g. camptothecin, flavopiridol, roscovitine), or (iii) late rRNA processing (e.g. 5-fluorouracil, MG-132, homoharringtonine). Blockage of rRNA transcription or early rRNA processing steps caused nucleolar disintegration, whereas blockage of late rRNA processing steps left the nucleolus intact. Flavopiridol and 5-fluorouracil showed a strong synergism for inhibition of rRNA processing. We conclude that inhibition of ribosome biogenesis by chemotherapeutic drugs potentially may contribute to the efficacy of therapeutic regimens.
The production and processing of ribosomal RNA is a complex and well-coordinated nucleolar process for ribosome biogenesis. Progress in understanding nucleolar structure and function has lead to the ...unexpected discovery of the nucleolus as a highly sensitive sensor of cellular stress and an important regulator of the tumor suppressor p53. Inhibition of ribosomal RNA metabolism has been shown to activate a signaling pathway for p53 induction. This review elucidates the potential of classical and recently developed chemotherapeutic drugs to stabilize p53 by inhibiting nucleolar functions.
Alternative cleavage and polyadenylation (APA) plays a crucial role in the regulation of gene expression across eukaryotes. Although APA is extensively studied, its regulation within cellular ...compartments and its physiological impact remains largely enigmatic. Here, we used a rigorous subcellular fractionation approach to compare APA profiles of cytoplasmic and nuclear RNA fractions from human cell lines. This approach allowed us to extract APA isoforms that are subjected to differential regulation and provided us with a platform to interrogate the molecular regulatory pathways that shape APA profiles in different subcellular locations. Here, we show that APA isoforms with shorter 3' UTRs tend to be overrepresented in the cytoplasm and appear to be cell-type-specific events. Nuclear retention of longer APA isoforms occurs and is partly a result of incomplete splicing contributing to the observed cytoplasmic bias of transcripts with shorter 3' UTRs. We demonstrate that the endoribonuclease III, DICER1, contributes to the establishment of subcellular APA profiles not only by expected cytoplasmic miRNA-mediated destabilization of APA mRNA isoforms, but also by affecting polyadenylation site choice.
PDF
