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Supplementary Figure 3. Protein blot from AGO proteins from nuclear and cytoplasmic portions of A2780.

Dysregulation of miRNA expression is associated with multiple diseases, including cancers, in which small RNAs can have either oncogenic or tumor suppressive functions. Here we investigated the potential tumor suppressive function of miR-450a, one of the most significantly downregulated miRNAs in ovarian cancer. RNA-seq analysis of the ovarian cancer cell line A2780 revealed that overexpression of miR-450a suppressed multiple genes involved in the epithelial-to-mesenchymal transition (EMT). Overexpression of miR-450a reduced tumor migration and invasion and increased anoikis in A2780 and SKOV-3 cell lines and reduced tumor growth in an ovarian tumor xenographic model. Combined AGO-PAR-CLIP and RNA-seq analysis identified a panel of potential miR-450a targets, of which many, including TIMMDC1, MT-ND2, ACO2, and ATP5B, regulate energetic metabolism. Following glutamine withdrawal, miR-450a overexpression decreased mitochondrial membrane potential but increased glucose uptake and viability, characteristics of less invasive ovarian cancer cell lines. In summary, we propose that miR-450a acts as a tumor suppressor in ovarian cancer cells by modulating targets associated with glutaminolysis, which leads to decreased production of lipids, amino acids, and nucleic acids, as well as inhibition of signaling pathways associated with EMT.Significance:miR-450a limits the metastatic potential of ovarian cancer cells by targeting a set of mitochondrial mRNAs to reduce glycolysis and glutaminolysis.

Supplementary Table 1. List of potential total miRNA and miR-450a only binding sites from A2780 cell line and expressing miR-450a

Supplementary Figure 2. miR-450b similar seed sequence and effects after its overexpression.

Supplementary Figure 1. miR-450a overexpression and effects in SKOV-3 ovarian cancer cell line.

Supplementary Figure 4. miR-450a effects on glutamine uptake and glutamate production.

Supplementary Table 2. List of targets from miR-450a derived from PAR-CLIP analysis.

Contains detailed Supplementary Figure and Table legends

Natural killer (NK) cells play key roles in immune surveillance against tumors and viral infection. NK cells distinguish abnormal cells from healthy cells by cell–cell interaction with cell surface proteins and then attack target cells via multiple mechanisms. In addition, extracellular vesicles (EVs) derived from NK cells (NK-EVs), including exosomes, possess cytotoxic capacity against tumor cells, but their characteristics and regulation by cytokines remain unknown. Here, we report that EVs derived from human NK-92 cells stimulated with IL-15 + IL-21 show enhanced cytotoxic capacity against tumor cells. Major cytolytic granules, granzyme B and granzyme H, are enriched by IL-15 + IL-21 stimulation in NK-EVs; however, knockout experiments reveal those cytolytic granules are independent of enhanced cytotoxic capacity. To find out the key molecules, mass spectrometry analyses were performed with different cytokine conditions, no cytokine, IL-15, IL-21, or IL-15 + IL-21. We then found that CD226 (DNAM-1) on NK-EVs is enriched by IL-15 + IL-21 stimulation and that blocking antibodies against CD226 reduced the cytolytic activity of NK-EVs. We also show NK-EVs are taken up by target cells via macropinocytosis. Collectively, our findings elucidate the novel properties of NK-EVs and the mechanism of their incorporation into target cells.

Retinoids are widely used in diseases spanning from dermatological lesions to cancer, but exhibit severe adverse effects. A novel all-trans-Retinoic Acid (atRA)-spermine conjugate (termed RASP) has shown previously optimal in vitro and in vivo anti-inflammatory and anticancer efficacy, with undetectable teratogenic and toxic side-effects. To get insights, we treated HaCaT cells which resemble human epidermis with IC50 concentration of RASP and analyzed their miRNA expression profile. Gene ontology analysis of their predicted targets indicated dynamic networks involved in cell proliferation, signal transduction and apoptosis. Furthermore, DNA microarrays analysis verified that RASP affects the expression of the same categories of genes. A protein-protein interaction map produced using the most significant common genes, revealed hub genes of nodal functions. We conclude that RASP is a synthetic retinoid derivative with improved properties, which possess the beneficial effects of retinoids without exhibiting side-effects and with potential beneficial effects against skin diseases including skin cancer.

Matrin3 is an RNA-binding protein that affects diverse RNA-related processes, including mRNA splicing. While Matrin3 has been intensively studied in neurodegenerative diseases, its function in cancer remains unclear. Here, we discovered Matrin3-mediated regulation of mitotic spindle dynamics in colorectal cancer (CRC) cells. We identified bound and regulated Matrin3-target RNAs transcriptome-wide in CRC cells and found that Matrin3 broadly modulates mRNA splicing patterns. Among the top Matrin3 targets, we focused on CDC14B and found that Matrin3 loss increased inclusion of an exon containing a premature termination codon into the CDC14B transcript and simultaneous down-regulation of the standard CDC14B transcript. Selective knockdown of the CDC14B standard transcript phenocopied Matrin3 knockdown and exhibited reduced proliferation and defects in mitotic spindle formation, suggesting that CDC14B is a key downstream effector of Matrin3. Collectively, these data reveal an important role for the Matrin3/CDC14B axis in control of CRC cell proliferation and mitotic spindle formation.

p53 is an intensely studied tumor-suppressive transcription factor. Recent studies suggest that the RNA-binding protein (RBP) ZMAT3 is important in mediating the tumor-suppressive effects of p53. Here, we globally identify ZMAT3-regulated RNAs and their binding sites at nucleotide resolution in intact colorectal cancer (CRC) cells. ZMAT3 binds to thousands of mRNA precursors, mainly at intronic uridine-rich sequences and affects their splicing. The strongest alternatively spliced ZMAT3 target was CD44, a cell adhesion gene and stem cell marker that controls tumorigenesis. Silencing ZMAT3 increased inclusion of CD44 variant exons, resulting in significant up-regulation of oncogenic CD44 isoforms (CD44v) and increased CRC cell growth that was rescued by concurrent knockdown of CD44v. Silencing p53 phenocopied the loss of ZMAT3 with respect to CD44 alternative splicing, suggesting that ZMAT3-mediated regulation of CD44 splicing is vital for p53 function. Collectively, our findings uncover a p53–ZMAT3–CD44 axis in growth suppression in CRC cells.

Heterogeneous nuclear ribonucleoprotein K (HNRNPK) regulates pre-mRNA processing and long non-coding RNA localization in the nucleus. It was previously shown that shuttling of HNRNPK to the cytoplasm promotes cell proliferation and cancer metastasis. However, the mechanism of HNRNPK cytoplasmic localization, its cytoplasmic RNA ligands, and impact on posttranscriptional gene regulation remain uncharacterized. Here we show that the intermediate filament protein Keratin 19 (K19) directly interacts with HNRNPK and sequesters it in the cytoplasm. Correspondingly, in K19 knockout breast cancer cells, HNRNPK does not localize in the cytoplasm, resulting in reduced cell proliferation. We mapped cytoplasmic HNRNPK target mRNAs using PAR-CLIP where transcriptome data to show that, in the cytoplasm, HNRNPK stabilizes target mRNAs bound to the 3’ untranslated region at the expected C-rich sequence elements. Furthermore, these mRNAs are typically involved in cancer progression and include the p53 signaling pathway that is dysregulated upon HNRNPK knockdown or K19 knockout. This study identifies how a cytoskeletal protein can directly regulate gene expression by controlling subcellular localization of RNA binding proteins to support pathways involved in cancer progression.

Mitogen-activated protein kinase phosphatase (Mkp)-1 exerts its anti-inflammatory activities during Gram-negative sepsis by deactivating p38 and c-Jun N-terminal kinase (JNK). We have previously shown that Mkp-1+/+ mice, but not Mkp-1&minus, /&minus, mice, exhibit hypertriglyceridemia during severe sepsis. However, the regulation of hepatic lipid stores and the underlying mechanism of lipid dysregulation during sepsis remains an enigma. To understand the molecular mechanism underlying the sepsis-associated metabolic changes and the role of Mkp-1 in the process, we infected Mkp-1+/+ and Mkp-1&minus, mice with Escherichia coli i.v., and assessed the effects of Mkp-1 deficiency on tissue lipid contents. We also examined the global gene expression profile in the livers via RNA-seq. We found that in the absence of E. coli infection, Mkp-1 deficiency decreased liver triglyceride levels. Upon E. coli infection, Mkp-1+/+ mice, but not Mkp-1&minus, mice, developed hepatocyte ballooning and increased lipid deposition in the livers. E. coli infection caused profound changes in the gene expression profile of a large number of proteins that regulate lipid metabolism in wildtype mice, while these changes were substantially disrupted in Mkp-1&minus, mice. Interestingly, in Mkp-1+/+ mice E. coli infection resulted in downregulation of genes that facilitate fatty acid synthesis but upregulation of Cd36 and Dgat2, whose protein products mediate fatty acid uptake and triglyceride synthesis, respectively. Taken together, our studies indicate that sepsis leads to a substantial change in triglyceride metabolic gene expression programs and Mkp-1 plays an important role in this process.

The activities of RNA polymerase and the spliceosome are responsible for the heterogeneity in the abundance and isoform composition of mRNA in human cells. However, the dynamics of these megadalton enzymatic complexes working in concert on endogenous genes have not been described. Here, we establish a quasi-genome-scale platform for observing synthesis and processing kinetics of single nascent RNA molecules in real time. We find that all observed genes show transcriptional bursting. We also observe large kinetic variation in intron removal for single introns in single cells, which is inconsistent with deterministic splice site selection. Transcriptome-wide footprinting of the U2AF complex, nascent RNA profiling, long-read sequencing, and lariat sequencing further reveal widespread stochastic recursive splicing within introns. We propose and validate a unified theoretical model to explain the general features of transcription and pervasive stochastic splice site selection.

Crosslinking and immunoprecipitation (CLIP) methods are powerful techniques to interrogate direct protein-RNA interactions and dissect posttranscriptional gene regulatory networks. One widely used CLIP variant is photoactivatable ribonucleoside enhanced CLIP (PAR-CLIP) that involves in vivo labeling of nascent RNAs with the photoreactive nucleosides 4-thiouridine (4SU) or 6-thioguanosine (6SG), which can efficiently crosslink to interacting proteins using UVA and UVB light. Crosslinking of 4SU or 6SG to interacting amino acids changes their base-pairing properties and results in characteristic mutations in cDNA libraries prepared for high-throughput sequencing, which can be computationally exploited to remove abundant background from non-crosslinked sequences and help pinpoint RNA binding protein binding sites at nucleotide resolution on a transcriptome-wide scale. Here we present a streamlined protocol for fluorescence-based PAR-CLIP (fPAR-CLIP) that eliminates the need to use radioactivity. It is based on direct ligation of a fluorescently labeled adapter to the 3′end of crosslinked RNA on immobilized ribonucleoproteins, followed by isolation of the adapter-ligated RNA and efficient conversion into cDNA without the previously needed size fractionation on denaturing polyacrylamide gels. These improvements cut the experimentation by half to 2 days and increases sensitivity by 10–100-fold.

Nuclear localization of the pyruvate kinase M2 isoform (PKM2) is widely observed in cancer and correlates with its aggressiveness, but its contribution to gene regulation and pathogenesis remains unclear. We find that PKM2 is a non-canonical RNA binding protein that specifically recognizes folded RNA G-quadruplex (rG4) structures and binds thousands of precursor mRNAs (pre-mRNA) in the nucleus of human cells. Translocation of PKM2 to the nucleus increases expression of pre-mRNAs with rG4s elements within 200 nt of splice sites, likely by promoting mRNA processing through competition with repressive rG4 binding proteins, such as HNRNPF. The same rG4 mRNAs were upregulated in a cancer model undergoing epithelial-to-mesenchymal transition (EMT), highlighting the PKM2-G4 interaction as a potential drug target for EMT-associated tumorigenicity. Our results show a gene-regulatory role for rG4 formation on pre-mRNA and propose that rG4s allow for collective co-transcriptional regulation of related mRNAs by competing RBPs.

During the course of their life cycle, most RNAs move between several cellular environments where they associate with different RNA binding proteins (RBPs). Reciprocally, a significant portion of RBPs reside in more than a single cellular compartment, where they can interact with discrete RNAs and even exert distinct biological roles. Proximity-CLIP combines proximity biotinylation of proteins with photoactivatable ribonucleoside-enhanced protein-RNA crosslinking to simultaneously profile the proteome, including RBPs and the RBP-bound transcriptome, in any given subcellular compartment. Here we provide a detailed experimental protocol for Proximity-CLIP along with a simplified non-radioactive, small-RNA cDNA library preparation protocol. Published 2020 U.S. Government. Basic Protocol 1: Cell culture, 4SU labeling, proximity biotinylation, and crosslinking Basic Protocol 2: Cell extraction, streptavidin affinity purification, and on-beads trypsinization Basic Protocol 3: RNA footprints cDNA library preparation Support Protocol: Preparation of RNA-seq libraries from intact RNA.

Methods for the systematic study of subcellular RNA localization are limited, and their development has lagged behind that of proteomic tools. We combined APEX2-mediated proximity biotinylation of proteins with photoactivatable ribonucleoside-enhanced crosslinking to simultaneously profile the proteome and the transcriptome bound by RNA-binding proteins in any given subcellular compartment. Our approach is fractionation independent and allows study of the localization of RNA processing intermediates, as well as the identification of regulatory RNA cis-acting elements occupied by proteins, in a cellular-compartment-specific manner. We used our method, Proximity-CLIP, to profile RNA and protein in the nucleus, in the cytoplasm, and at cell–cell interfaces. Among other insights, we observed frequent transcriptional readthrough continuing for several kilobases downstream of the canonical cleavage and polyadenylation site and a differential RBP occupancy pattern for mRNAs in the nucleus and cytoplasm. We observed that mRNAs localized to cell–cell interfaces often encoded regulatory proteins and contained protein-occupied CUG sequence elements in their 3′ untranslated region. Proximity-CLIP, a method that combines proximity-based protein biotinylation and UV crosslinking, profiles RNAs and ribonucleoproteins in any cellular compartment.

The alphavirus capsid protein (Cp) selectively packages genomic RNA (gRNA) into the viral nucleocapsid to produce infectious virus. Using photoactivatable ribonucleoside crosslinking and an innovative biotinylated Cp retrieval method, here we comprehensively define binding sites for Semliki Forest virus (SFV) Cp on the gRNA. While data in infected cells demonstrate Cp binding to the proposed genome packaging signal (PS), mutagenesis experiments show that PS is not required for production of infectious SFV or Chikungunya virus. Instead, we identify multiple Cp binding sites that are enriched on gRNA-specific regions and promote infectious SFV production and gRNA packaging. Comparisons of binding sites in cytoplasmic vs. viral nucleocapsids demonstrate that budding causes discrete changes in Cp-gRNA interactions. Notably, Cp’s top binding site is maintained throughout virus assembly, and specifically binds and assembles with Cp into core-like particles in vitro. Together our data suggest a model for selective alphavirus genome recognition and assembly., Alphaviruses need to selectively package genomic viral RNA for transmission, but the packaging mechanism remains unclear. Here, Brown et al. combine PAR-CLIP with biotinylated capsid protein (Cp) retrieval and identify multiple Cp binding sites on genomic viral RNA that promote virion formation.

Dysregulation of miRNA expression is associated with multiple diseases, including cancers, in which small RNAs can have either oncogenic or tumor suppressive functions. Here we investigated the potential tumor suppressive function of miR-450a, one of the most significantly downregulated miRNAs in ovarian cancer. RNA-seq analysis of the ovarian cancer cell line A2780 revealed that overexpression of miR-450a suppressed multiple genes involved in the epithelial-to-mesenchymal transition (EMT). Overexpression of miR-450a reduced tumor migration and invasion and increased anoikis in A2780 and SKOV-3 cell lines and reduced tumor growth in an ovarian tumor xenographic model. Combined AGO-PAR-CLIP and RNA-seq analysis identified a panel of potential miR-450a targets, of which many, including TIMMDC1, MT-ND2, ACO2, and ATP5B, regulate energetic metabolism. Following glutamine withdrawal, miR-450a overexpression decreased mitochondrial membrane potential but increased glucose uptake and viability, characteristics of less invasive ovarian cancer cell lines. In summary, we propose that miR-450a acts as a tumor suppressor in ovarian cancer cells by modulating targets associated with glutaminolysis, which leads to decreased production of lipids, amino acids, and nucleic acids, as well as inhibition of signaling pathways associated with EMT. Significance: miR-450a limits the metastatic potential of ovarian cancer cells by targeting a set of mitochondrial mRNAs to reduce glycolysis and glutaminolysis.

Keratin 19 (K19) belongs to the keratin family of proteins, which maintains structural integrity of epithelia. In cancer, K19 is highly expressed in several types where it serves as a diagnostic marker. Despite the positive correlation between higher expression of K19 in tumor and worse patient survival, the role of K19 in breast cancer remains unclear. Therefore, we ablated K19 expression in MCF7 breast cancer cells and found that K19 was required for cell proliferation. Transcriptome analyses of KRT19 knockout cells identified defects in cell cycle progression and levels of target genes of E2F1, a key transcriptional factor for the transition into S phase. Furthermore, proper levels of cyclin dependent kinases (CDKs) and cyclins, including D-type cyclins critical for E2F1 activation, were dependent on K19 expression, and K19-cyclin D co-expression was observed in human breast cancer tissues. Importantly, K19 interacts with cyclin D3, and a loss of K19 resulted in decreased protein stability of cyclin D3 and sensitivity of cells towards CDK inhibitor-induced cell death. Overall, these findings reveal a novel function of K19 in the regulation of cell cycle program and suggest that K19 may be used to predict the efficacy of CDK inhibitors for treatments of breast cancer.

Simple Summary The profiles of miRNAs, tRNA-derived fragments and tRNAs from lung cancer biopsy specimens indicate involvement of gene networks that modulate signaling and translation initiation. The current study highlights the important role of several regulatory small non-coding RNAs in aberrant signaling and translation deregulation in lung cancer. Abstract Transcriptomics profiles of miRNAs, tRNAs or tRFs are used as biomarkers, after separate examination of several cancer cell lines, blood samples or biopsies. However, the possible contribution of all three profiles on oncogenic signaling and translation as a net regulatory effect, is under investigation. The present analysis of miRNAs and tRFs from lung cancer biopsies indicated putative targets, which belong to gene networks involved in cell proliferation, transcription and translation regulation. In addition, we observed differential expression of specific tRNAs along with several tRNA-related genes with possible involvement in carcinogenesis. Transfection of lung adenocarcinoma cells with two identified tRFs and subsequent NGS analysis indicated gene targets that mediate signaling and translation regulation. Broader analysis of all major signaling and translation factors in several biopsy specimens revealed a crosstalk between the PI3K/AKT and MAPK pathways and downstream activation of eIF4E and eEF2. Subsequent polysome profile analysis and 48S pre-initiation reconstitution experiments showed increased global translation rates and indicated that aberrant expression patterns of translation initiation factors could contribute to elevated protein synthesis. Overall, our results outline the modulatory effects that possibly correlate the expression of important regulatory non-coding RNAs with aberrant signaling and translation deregulation in lung cancer.

The activities of RNA polymerase and the spliceosome are responsible for the heterogeneity in the abundance and isoform composition of mRNA in human cells. However, the dynamics of these megadalton enzymatic complexes working in concert on endogenous genes have not been described. Here, we establish a quasi-genome-scale platform for observing synthesis and processing kinetics of single nascent RNA molecules in real time. We find that all observed genes show transcriptional bursting. We also observe large kinetic variation in intron removal for single introns in single cells which is inconsistent with deterministic splice site selection. Transcriptome-wide footprinting of the U2AF complex and nascent RNA sequencing further reveals widespread splice site selection within introns. We propose and validate a unified theoretical model to explain the general feature of transcription and pervasive stochastic splice site selection.

INTRODUCTION: Laboratory mice are a mainstay of biomedical research and have been instrumental for many important discoveries in the field of immunology. However, there are also major limitations, including conflicting results rooted in divergent microbiota among research facilities and the limited ability to predict the complex immune responses of humans. Recent studies have shown that conventional laboratory mice are too far removed from natural environmental conditions to faithfully mirror the physiology of free-living mammals like humans. Mammals and their immune systems evolved to survive and thrive in a microbial world and behave differently in a sanitized environment. RATIONALE: To generate a mouse model that more closely resembles the natural mammalian meta-organism with co-evolved microbes and pathogens, we transferred C57BL/6 embryos into wild mice. This resulted in a colony of C57BL/6 mice, which we call “wildlings”. RESULTS: Wildlings resembled wild mice and differed significantly from conventional laboratory mice with regards to their bacterial microbiome at important epithelial barrier sites (gut, skin, and vagina), their gut mycobiome and virome, and their level of pathogen exposure. The natural microbiota of wildlings were stable over multiple generations and resilient against antibiotic, dietary, and microbial challenges. Next, we delineated the immune landscape of wildlings, wild mice, and laboratory mice at immunologically important barrier sites (gut, skin, and vagina), a central non-lymphoid organ (liver), and a central lymphoid organ (spleen) by mass cytometry. Additionally, we characterized the blood immune cell profile by RNA sequencing. The differential contribution of microbial and host genomes in shaping the immune phenotype varied among tissues. Wildlings closely mirrored the wild mouse immune phenotype in the spleen and blood. Finally, we tested the translational research value of wildlings in a retrospective bench-to-bedside approach. This required well-documented, rodent-based studies that had failed upon transitioning to clinical trials in humans. We chose the CD28-superagonist (CD28SA) trial as representative for treatments targeting adaptive immune responses. Although CD28SA expanded anti-inflammatory regulatory T cells (T(regs)) in laboratory mice and showed therapeutic effects in multiple models of autoimmune/inflammatory diseases, the first phase I clinical trial resulted in life-threatening activation of inflammatory T cells and cytokine storms. Similarly, the CD28-superagonist treatment of wildlings, but not laboratory mice, resulted in an inflammatory cytokine response and lack of T(reg) expansion. As representative for trials targeting innate immune responses, we chose anti-TNF-α treatment (anti-TNF-α or TNF-receptor:Fc fusion protein) during septic shock, which was successful in animal models, but failed in humans. Anti-TNF-α treatment during lethal endotoxemia rescued laboratory mice, but not wildlings. Thus, wildlings better phenocopied human immune responses than conventional laboratory mice in the two models studied. CONCLUSION: The wildling model combines resilient natural microbiota and pathogens at all body sites and the tractable genetics of C57BL/6. Given the wide-ranging effects of microbiota on host physiology, natural microbiota-based models may benefit different research fields (e.g. metabolism and neurodegenerative diseases) and may also be applicable to other animals. Such models may enhance the validity and reproducibility of biomedical studies among research institutes, facilitate the discovery of disease mechanisms and treatments that cannot be studied in regular laboratory mice, and increase the translatability of immunological results to humans.

Protein synthesis is a central and dynamic process, frequently deregulated in cancer through aberrant activation or expression of translation initiation factors and tRNAs. The discovery of tRNA-derived fragments, a new class of abundant and, in some cases stress-induced, small Noncoding RNAs has perplexed the epigenomics landscape and highlights the emerging regulatory role of tRNAs in translation and beyond. Skin is the biggest organ in human body, which maintains homeostasis of its multilayers through regulatory networks that induce translational reprogramming, and modulate tRNA transcription, modification and fragmentation, in response to various stress signals, like UV irradiation. In this review, we summarize recent knowledge on the role of translation regulation and tRNA biology in the alarming prevalence of skin cancer.

In mammals, gene silencing by the RNA-induced silencing complex (RISC) is a well-understood cytoplasmic posttranscriptional gene regulatory mechanism. Here, we show that embryonic stem cells (ESCs) contain high levels of nuclear AGO proteins and that in ESCs nuclear AGO protein activity allows for the onset of differentiation. In the nucleus, AGO proteins interact with core RISC components, including the TNRC6 proteins and the CCR4-NOT deadenylase complex. In contrast to cytoplasmic miRNA-mediated gene silencing that mainly operates on cis-acting elements in mRNA 3' untranslated (UTR) sequences, in the nucleus AGO binding in the coding sequence and potentially introns also contributed to post-transcriptional gene silencing. Thus, nuclear localization of AGO proteins in specific cell types leads to a previously unappreciated expansion of the miRNA-regulated transcriptome.

Background RNase P-mediated cleavage of target RNAs has been proposed as a promising tool for gene silencing. Ets-2 proto-oncogene controls the expression of a wide variety of genes involved in cancer and immunity. Objective Construction of a functional RNase P-based ribozyme (M1GS303) that targets Ets-2 mRNA. Methods The accessible sites for targeting of Ets-2 mRNA were identified by footprinting analysis. M1GS303 ribozyme was constructed by cloning. The activity of the ribozyme in the presence or absence of spiramysin in E. coli cells and human cell lines was quantified by RT-PCR. The efficiency of the ribozyme in silencing the endogenous expression of Ets-2 in human cell lines was examined by RT-PCR, western blot and immunofluorescence analysis. Results In E. coli cells co-transformed with plasmids bearing M1GS303 and the ets-2 target gene, Ets-2 mRNA was decreased by 93% 12h after IPTG induction in the absence, and after 4h in the presence of spiramycin. Ets-2 was rapidly downregulated in the human embryonic kidney cell line HEK293 and the T-cell line Jurkat transfected with an M1GS303 plasmid; the silencing effect of M1GS303 was considerably faster when the cells were cultured with spiramycin. In Jurkat cells, Ets-2-downregulation resulted in upregulation of the expression of IL-2, IL-4 and IFN-α cytokine genes that have Ets-2 binding sites on their promoters, whereas it had no effect on the expression of the IL-10 gene that lacks Ets-2 binding sites on its promoter. Conclusions M1GS303 ribozyme cleaves effectively Ets-2 mRNA in bacteria and mammalian cells, and its activity is enhanced by spiramycin. Downregulation of ets-2 gene in the T-cell line Jurkat upregulates IL-2, IL-4 and IFN-α cytokine genes. M1GS technology may be a better alternative to conventional gene-interference therapies and the delineation of the effects of gene silencing in various pathologies.

PNLDC1 is a homologue of poly(A) specific ribonuclease (PARN), a known deadenylase with additional role in processing of non-coding RNAs. Both enzymes were reported recently to participate in piRNA biogenesis in silkworm and C. elegans, respectively. To get insights on the role of mammalian PNLDC1, we characterized the human and mouse enzymes. PNLDC1 shows limited conservation compared to PARN and represents an evolutionary related but distinct group of enzymes. It is expressed specifically in mouse embryonic stem cells, human and mouse testes and during early mouse embryo development, while it fades during differentiation. Its expression in differentiated cells, is suppressed through methylation of its promoter by the de novo methyltransferase DNMT3B. Both enzymes are localized mainly in the ER and exhibit in vitro specificity restricted solely to 3' RNA or DNA polyadenylates. Knockdown of Pnldc1 in mESCs and subsequent NGS analysis showed that although the expression of the remaining deadenylases remains unaffected, it affects genes involved mainly in reprogramming, cell cycle and translational regulation. Mammalian PNLDC1 is a novel deadenylase expressed specifically in cell types which share regulatory mechanisms required for multipotency maintenance. Moreover, it could be involved both in posttranscriptional regulation through deadenylation and genome surveillance during early development.

Deadenylation is the exoribonucleolytic shortening of eukaryotic poly(A) tails. It is often the first and rate-limiting step for mRNA decay and translational silencing. The process is catalysed by a diversity of deadenylases, which provide robust and flexible means to control mRNA levels and gene expression. Poly(A)-specific ribonuclease (PARN) is a major mammalian deadenylase and the only known to concurrently bind the 5' cap-structure and the 3' poly(A), thus enhancing the degradation rate and amplifying its processivity. PARN is important during oocyte maturation, embryogenesis, early development, DNA damage, and in cell-cycle progression, but also in processes beyond mRNA metabolism, such as the maturation of snoRNAs. The enzyme also participates in nonsense-mediated mRNA decay and in the regulation of cytoplasmic polyadenylation. Importantly, PARN is involved in the degradation of several cancer-related genes, while its expression is altered in cancer. Apart from the direct interaction with the cap structure, several strategies regulate PARN activity, such as phosphorylation, interaction with RNA-binding proteins (RBPs), and natural nucleotides. Recent studies have focused on the regulation of its activity by synthetic nucleoside analogues with therapeutic potential. In this context, the wide repertoire of RBPs and molecules that regulate PARN activity, together with the established role of deadenylases in miRNA-mediated regulation of mRNA expression, suggest that mRNA turnover is more complex than it was previously thought and PARN holds a key role in this process. In this review, we highlight the importance of PARN during RNA's lifecycle and discuss clinical perspectives of modulating its activity.

The enhancer regions of the myogenic master regulator MyoD give rise to at least two enhancer RNAs. Core enhancer eRNA (CEeRNA) regulates transcription of the adjacent MyoD gene, whereas DRReRNA affects expression of Myogenin in trans. We found that DRReRNA is recruited at the Myogenin locus, where it colocalizes with Myogenin nascent transcripts. DRReRNA associates with the cohesin complex, and this association correlates with its transactivating properties. Despite being expressed in undifferentiated cells, cohesin is not loaded on Myogenin until the cells start expressing DRReRNA, which is then required for cohesin chromatin recruitment and maintenance. Functionally, depletion of either cohesin or DRReRNA reduces chromatin accessibility, prevents Myogenin activation, and hinders muscle cell differentiation. Thus, DRReRNA ensures spatially appropriate cohesin loading in trans to regulate gene expression.

Poly(A)-specific ribonuclease (PARN) is a cap-interacting deadenylase that mediates, together with other exonucleases, the eukaryotic mRNA turnover and thus is actively involved in the regulation of gene expression. Aminoglycosides and natural nucleotides are the only reported modulators of human PARN activity, so far. In the present study, we show that synthetic nucleoside analogues bearing a fluoro-glucopyranosyl sugar moiety and benzoyl-modified cytosine or adenine as a base can effectively inhibit human PARN. Such nucleoside analogues exhibited substantial inhibitory effects, when tested against various cancer cell lines, as has been previously reported. Kinetic analysis showed that the inhibition of PARN is competitive and could not be released by altering Mg(II) concentration. Moreover, substitution of the 2', 4', or 6'-OH of the sugar moiety with acetyl and/or trityl groups was crucial for inhibitory efficacy. To understand how the nucleosides fit into the active site of PARN, we performed molecular docking experiments followed by molecular dynamics simulations. The in silico analysis showed that these compounds can efficiently dock into the active site of PARN. Our results support the idea that the sugar moiety mediates the stabilization of the nucleoside into the active site through interactions with catalytic amino acid residues. Taken together, our in vitro and in silico data suggest that human PARN is among the molecular targets of these compounds and could act therapeutically by lowering the mRNA turnover rate, thus explaining their known in vivo inhibitory effect at the molecular level.

Poly(A)-specific ribonuclease (PARN) is a cap-interacting and poly(A)-specific 3'-exoribonuclease that efficiently degrades mRNA poly(A) tails. Based on the enzyme's preference for its natural substrates, we examined the role of purine nucleotides as potent effectors of human PARN activity. We found that all purine nucleotides tested can reduce poly(A) degradation by PARN. Detailed kinetic analysis revealed that RTP nucleotides behave as non-competitive inhibitors while RDP and RMP exhibit competitive inhibition. Mg(2 + ) which is a catalytically important mediator of PARN activity can release inhibition of RTP and RDP but not RMP. Although many strategies have been proposed for the regulation of PARN activity, very little is known about the modulation of PARN activity by small molecule effectors, such as nucleotides. Our data imply that PARN activity can be modulated by purine nucleotides in vitro, providing an additional simple regulatory mechanism.