A Pro-metastatic tRNA Pathway.

tRNAs are best known as basic modules for global regulation of protein synthesis. Goodarzi et al. now show that two tRNAs upregulated in metastatic breast cancer cells enhance stability and translation of transcripts enriched with these codons, leading to specific increase in production of pro-metastatic proteins.

Noncanonical Roles of tRNAs: tRNA Fragments and Beyond.

As one of the most abundant and conserved RNA species, transfer RNAs (tRNAs) are well known for their role in reading the codons on messenger RNAs and translating them into proteins. In this review, we discuss the noncanonical functions of tRNAs. These include tRNAs as precursors to novel small RNA molecules derived from tRNAs, also called tRNA-derived fragments, that are abundant across species and have diverse functions in different biological processes, including regulating protein translation, Argonaute-dependent gene silencing, and more. Furthermore, the role of tRNAs in biosynthesis and other regulatory pathways, including nutrient sensing, splicing, transcription, retroelement regulation, immune response, and apoptosis, is reviewed. Genome organization and sequence variation of tRNA genes are also discussed in light of their noncanonical functions. Lastly, we discuss the recent applications of tRNAs in genome editing and microbiome sequencing.

The Deubiquitinase USP46 Is Essential for Proliferation and Tumor Growth of HPV-Transformed Cancers.

High-risk human papilloma viruses (HPVs) cause cervical, anal, and oropharyngeal cancers, unlike the low-risk HPVs, which cause benign lesions. E6 oncoproteins from the high-risk strains are essential for cell proliferation and transformation in HPV-induced cancers. We report that a cellular deubiquitinase, USP46, is selectively recruited by the E6 of high-risk, but not low-risk, HPV to deubiqutinate and stabilize Cdt2/DTL. Stabilization of Cdt2, a component of the CRL4Cdt2 E3 ubiquitin ligase, limits the level of Set8, an epigenetic writer, and promotes cell proliferation. USP46 is essential for the proliferation of HPV-transformed cells, but not of cells without HPV. Cdt2 is elevated in human cervical cancers and knockdown of USP46 inhibits HPV-transformed tumor growth in xenografts. Recruitment of a cellular deubiquitinase to stabilize key cellular proteins is an important activity of oncogenic E6, and the importance of E6-USP46-Cdt2-Set8 pathway in HPV-induced cancers makes USP46 a target for the therapy of such cancers.

XRN2 suppresses aberrant entry of tRNA trailers into argonaute in humans and Arabidopsis.

MicroRNAs (miRNAs) are a well-characterized class of small RNAs (sRNAs) that regulate gene expression post-transcriptionally. miRNAs function within a complex milieu of other sRNAs of similar size and abundance, with the best characterized being tRNA fragments or tRFs. The mechanism by which the RNA-induced silencing complex (RISC) selects for specific sRNAs over others is not entirely understood in human cells. Several highly expressed tRNA trailers (tRF-1s) are strikingly similar to microRNAs in length but are generally excluded from the microRNA effector pathway. This exclusion provides a paradigm for identifying mechanisms of RISC selectivity. Here, we show that 5' to 3' exoribonuclease XRN2 contributes to human RISC selectivity. Although highly abundant, tRF-1s are highly unstable and degraded by XRN2 which blocks tRF-1 accumulation in RISC. We also find that XRN mediated degradation of tRF-1s and subsequent exclusion from RISC is conserved in plants. Our findings reveal a conserved mechanism that prevents aberrant entry of a class of highly produced sRNAs into Ago2.

ASF1a Promotes Non-homologous End Joining Repair by Facilitating Phosphorylation of MDC1 by ATM at Double-Strand Breaks.

Double-strand breaks (DSBs) of DNA in eukaryotic cells are predominantly repaired by non-homologous end joining (NHEJ). The histone chaperone anti-silencing factor 1a (ASF1a) interacts with MDC1 and is recruited to sites of DSBs to facilitate the interaction of phospho-ATM with MDC1 and phosphorylation of MDC1, which are required for the recruitment of RNF8/RNF168 histone ubiquitin ligases. Thus, ASF1a deficiency reduces histone ubiquitination at DSBs, decreasing the recruitment of 53BP1, and decreases NHEJ, rendering cells more sensitive to DSBs. This role of ASF1a in DSB repair cannot be provided by the closely related ASF1b and does not require its histone chaperone activity. Homozygous deletion of ASF1A is seen in 10%-15% of certain cancers, suggesting that loss of NHEJ may be selected in some malignancies and that the deletion can be used as a molecular biomarker for cancers susceptible to radiotherapy or to DSB-inducing chemotherapy.

G-quadruplex structural dynamics at MAPK12 promoter dictates transcriptional switch to determine stemness in breast cancer.

P38γ (MAPK12) is predominantly expressed in triple negative breast cancer cells (TNBC) and induces stem cell (CSC) expansion resulting in decreased survival of the patients due to metastasis. Abundance of G-rich sequences at MAPK12 promoter implied the functional probability to reverse tumorigenesis, though the formation of G-Quadruplex (G4) structures at MAPK12 promoter is elusive. Here, we identified two evolutionary consensus adjacent G4 motifs upstream of the MAPK12 promoter, forming parallel G4 structures. They exist in an equilibria between G4 and duplex, regulated by the binding turnover of Sp1 and Nucleolin that bind to these G4 motifs and regulate MAPK12 transcriptional homeostasis. To underscore the gene-regulatory functions of G4 motifs, we employed CRISPR-Cas9 system to eliminate G4s from TNBC cells and synthesized a naphthalene diimide (NDI) derivative (TGS24) which shows high-affinity binding to MAPK12-G4 and inhibits MAPK12 transcription. Deletion of G4 motifs and NDI compound interfere with the recruitment of the transcription factors, inhibiting MAPK12 expression in cancer cells. The molecular basis of NDI-induced G4 transcriptional regulation was analysed by RNA-seq analyses, which revealed that MAPK12-G4 inhibits oncogenic RAS transformation and trans-activation of NANOG. MAPK12-G4 also reduces CD44High/CD24Low population in TNBC cells and downregulates internal stem cell markers, arresting the stemness properties of cancer cells.

Germline Variants That Affect Tumor Progression.

Germline variants have a rich history of being studied in the context of cancer risk. Emerging studies now suggest that germline variants contribute not only to cancer risk but to tumor progression as well. In this opinion article, we discuss the initial discoveries associating germline variants with patient outcome and the mechanisms by which germline variants affect molecular pathways. Germline variants affect molecular pathways through amino acid changes, alteration of splicing patterns or expression of genes, influencing the selection for somatic mutations, and causing genome-wide mutational enrichment. These molecular alterations can lead to tumor phenotypes that become clinically apparent such as metastasis, alterations to the immune microenvironment, and modulation of therapeutic response. Overall, the growing body of evidence suggests that germline variants play a larger role in tumor progression than has been previously appreciated and that germline variation holds substantial potential for improving personalized medicine and patient outcomes.

The tumor-suppressive long noncoding RNA DRAIC inhibits protein translation and induces autophagy by activating AMPK.

Long noncoding RNAs (lncRNAs) are long RNA transcripts that do not code for proteins and have been shown to play a major role in cellular processes through diverse mechanisms. DRAIC, a lncRNA that is downregulated in castration-resistant advanced prostate cancer, inhibits the NF-κB pathway by inhibiting the IκBα kinase. Decreased DRAIC expression predicted poor patient outcome in gliomas and seven other cancers. We now report that DRAIC suppresses invasion, migration, colony formation and xenograft growth of glioblastoma-derived cell lines. DRAIC activates AMP-activated protein kinase (AMPK) by downregulating the NF-κB target gene GLUT1, and thus represses mTOR, leading to downstream effects, such as a decrease in protein translation and increase in autophagy. DRAIC, therefore, has an effect on multiple signal transduction pathways that are important for oncogenesis, namely, the NF-κB pathway and AMPK-mTOR-S6K/ULK1 pathway. The regulation of NF-κB, protein translation and autophagy by the same lncRNA explains the tumor-suppressive role of DRAIC in different cancers and reinforces the importance of lncRNAs as emerging regulators of signal transduction pathways. This article has an associated First Person interview with the first author of the paper.

MicroDNA levels are dependent on MMEJ, repressed by c-NHEJ pathway, and stimulated by DNA damage.

Extrachromosomal circular DNA (eccDNA) are present within all eukaryotic organisms and actively contribute to gene expression changes. MicroDNA (200-1000bp) are the most abundant type of eccDNA and can amplify tRNA, microRNA, and novel si-like RNA sequences. Due to the heterogeneity of microDNA and the limited technology to directly quantify circular DNA molecules, the specific DNA repair pathways that contribute to microDNA formation have not been fully elucidated. Using a sensitive and quantitative assay that quantifies eight known abundant microDNA, we report that microDNA levels are dependent on resection after double-strand DNA break (DSB) and repair by Microhomology Mediated End Joining (MMEJ). Further, repair of DSB without resection by canonical Non-Homologous End Joining (c-NHEJ) diminishes microDNA formation. MicroDNA levels are induced locally even by a single site-directed DSB, suggesting that excision of genomic DNA by two closely spaced DSB is not necessary for microDNA formation. Consistent with all this, microDNA levels accumulate as cells undergo replication in S-phase, when DNA breaks and repair are elevated, and microDNA levels are decreased if DNA synthesis is prevented. Thus, formation of microDNA occurs during the repair of endogenous or induced DNA breaks by resection-based DNA repair pathways.

Identification and characterization of extrachromosomal circular DNA in maternal plasma.

We explored the presence of extrachromosomal circular DNA (eccDNA) in the plasma of pregnant women. Through sequencing following either restriction enzyme or Tn5 transposase treatment, we identified eccDNA molecules in the plasma of pregnant women. These eccDNA molecules showed bimodal size distributions peaking at ∼202 and ∼338 bp with distinct 10-bp periodicity observed throughout the size ranges within both peaks, suggestive of their nucleosomal origin. Also, the predominance of the 338-bp peak of eccDNA indicated that eccDNA had a larger size distribution than linear DNA in human plasma. Moreover, eccDNA of fetal origin were shorter than the maternal eccDNA. Genomic annotation of the overall population of eccDNA molecules revealed a preference of these molecules to be generated from 5'-untranslated regions (5'-UTRs), exonic regions, and CpG island regions. Two sets of trinucleotide repeat motifs flanking the junctional sites of eccDNA supported multiple possible models for eccDNA generation. This work highlights the topologic analysis of plasma DNA, which is an emerging direction for circulating nucleic acid research and applications.

Targeted CRISPR screening identifies PRMT5 as synthetic lethality combinatorial target with gemcitabine in pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most challenging cancers to treat. Due to the asymptomatic nature of the disease and lack of curative treatment modalities, the 5-y survival rate of PDAC patients is one of the lowest of any cancer type. The recurrent genetic alterations in PDAC are yet to be targeted. Therefore, identification of effective drug combinations is desperately needed. Here, we performed an in vivo CRISPR screen in an orthotopic patient-derived xenograft (PDX) model to identify gene targets whose inhibition creates synergistic tumor growth inhibition with gemcitabine (Gem), a first- or second-line chemotherapeutic agent for PDAC treatment. The approach revealed protein arginine methyltransferase gene 5 (PRMT5) as an effective druggable candidate whose inhibition creates synergistic vulnerability of PDAC cells to Gem. Genetic depletion and pharmacological inhibition indicate that loss of PRMT5 activity synergistically enhances Gem cytotoxicity due to the accumulation of excessive DNA damage. At the molecular level, we show that inhibition of PRMT5 results in RPA depletion and impaired homology-directed DNA repair (HDR) activity. The combination (Gem + PRMT5 inhibition) creates conditional lethality and synergistic reduction of PDAC tumors in vivo. The findings demonstrate that unbiased genetic screenings combined with a clinically relevant model system is a practical approach in identifying synthetic lethal drug combinations for cancer treatment.

Sequential replication-coupled destruction at G1/S ensures genome stability.

Timely ubiquitin-mediated protein degradation is fundamental to cell cycle control, but the precise degradation order at each cell cycle phase transition is still unclear. We investigated the degradation order among substrates of a single human E3 ubiquitin ligase, CRL4(Cdt2), which mediates the S-phase degradation of key cell cycle proteins, including Cdt1, PR-Set7, and p21. Our analysis of synchronized cells and asynchronously proliferating live single cells revealed a consistent order of replication-coupled destruction during both S-phase entry and DNA repair; Cdt1 is destroyed first, whereas p21 destruction is always substantially later than that of Cdt1. These differences are attributable to the CRL4(Cdt2) targeting motif known as the PIP degron, which binds DNA-loaded proliferating cell nuclear antigen (PCNA(DNA)) and recruits CRL4(Cdt2). Fusing Cdt1's PIP degron to p21 causes p21 to be destroyed nearly concurrently with Cdt1 rather than consecutively. This accelerated degradation conferred by the Cdt1 PIP degron is accompanied by more effective Cdt2 recruitment by Cdt1 even though p21 has higher affinity for PCNA(DNA). Importantly, cells with artificially accelerated p21 degradation display evidence of stalled replication in mid-S phase and sensitivity to replication arrest. We therefore propose that sequential degradation ensures orderly S-phase progression to avoid replication stress and genome instability.

The H19 long noncoding RNA gives rise to microRNAs miR-675-3p and miR-675-5p to promote skeletal muscle differentiation and regeneration.

Regulated expression of the H19 long noncoding RNA gene has been well characterized as a paradigm for genomic imprinting, but the H19 RNA's biological function remains largely unclear. H19 is abundantly expressed maternally in embryonic tissues but is strongly repressed after birth, and significant transcription persists only in skeletal muscle. Thus, we examined the role of the H19 RNA in skeletal muscle differentiation and regeneration. Knockdown of H19 RNA in myoblast cells and H19 knockout mouse satellite cells decreases differentiation. H19 exon1 encodes two conserved microRNAs, miR-675-3p and miR-675-5p, both of which are induced during skeletal muscle differentiation. The inhibition of myogenesis by H19 depletion during myoblast differentiation is rescued by exogenous expression of miR-675-3p and miR-675-5p. H19-deficient mice display abnormal skeletal muscle regeneration after injury, which is rectified by reintroduction of miR-675-3p and miR-675-5p. miR-675-3p and miR-675-5p function by directly targeting and down-regulating the anti-differentiation Smad transcription factors critical for the bone morphogenetic protein (BMP) pathway and the DNA replication initiation factor Cdc6. Therefore, the H19 long noncoding RNA has a critical trans-regulatory function in skeletal muscle differentiation and regeneration that is mediated by the microRNAs encoded within H19.

ATR checkpoint kinase and CRL1ßTRCP collaborate to degrade ASF1a and thus repress genes overlapping with clusters of stalled replication forks.

Many agents used for chemotherapy, such as doxorubicin, interfere with DNA replication, but the effect of this interference on transcription is largely unknown. Here we show that doxorubicin induces the firing of dense clusters of neoreplication origins that lead to clusters of stalled replication forks in gene-rich parts of the genome, particularly on expressed genes. Genes that overlap with these clusters of stalled forks are actively dechromatinized, unwound, and repressed by an ATR-dependent checkpoint pathway. The ATR checkpoint pathway causes a histone chaperone normally associated with the replication fork, ASF1a, to degrade through a CRL1(ßTRCP)-dependent ubiquitination/proteasome pathway, leading to the localized dechromatinization and gene repression. Therefore, a globally active checkpoint pathway interacts with local clusters of stalled forks to specifically repress genes in the vicinity of the stalled forks, providing a new mechanism of action of chemotherapy drugs like doxorubicin. Finally, ASF1a-depleted cancer cells are more sensitive to doxorubicin, suggesting that the 7%-10% of prostate adenocarcinomas and adenoid cystic carcinomas reported to have homozygous deletion or significant underexpression of ASF1a should be tested for high sensitivity to doxorubicin.

Alternative Sigma Factor of Staphylococcus aureus Interacts with the Cognate Antisigma Factor Primarily Using Its Domain 3.

σB, an alternative sigma factor, is usually employed to tackle the general stress response in Staphylococcus aureus and other Gram-positive bacteria. This protein, involved in S. aureus-mediated pathogenesis, is typically blocked by RsbW, an antisigma factor having serine kinase activity. σB, a σ70-like sigma factor, harbors three conserved domains designated σB2, σB3, and σB4. To better understand the interaction between RsbW and σB or its domains, we have studied their recombinant forms, rRsbW, rσB, rσB2, rσB3, and rσB4, using different probes. The results show that none of the rσB domains, unlike rσB, showed binding to a cognate DNA in the presence of a core RNA polymerase. However, both rσB2 and rσB3, like rσB, interacted with rRsbW, and the order of their rRsbW binding affinity looks like rσB > rσB3 > rσB2. Furthermore, the reaction between rRsbW and rσB or rσB3 was exothermic and occurred spontaneously. rRsbW and rσB3 also associate with each other at a stoichiometry of 2:1, and different types of noncovalent bonds might be responsible for their interaction. A structural model of the RsbW-σB3 complex that has supported our experimental results indicated the binding of rσB3 at the putative dimeric interface of RsbW. A genetic study shows that the tentative dimer-forming region of RsbW is crucial for preserving its rσB binding ability, serine kinase activity, and dimerization ability. Additionally, a urea-induced equilibrium unfolding study indicated a notable thermodynamic stabilization of σB3 in the presence of RsbW. Possible implications of the stabilization data in drug discovery were discussed at length.

A human cancer cell line initiates DNA replication normally in the absence of ORC5 and ORC2 proteins.

The origin recognition complex (ORC), composed of six subunits, ORC1-6, binds to origins of replication as a ring-shaped heterohexameric ATPase that is believed to be essential to recruit and load MCM2-7, the minichromosome maintenance protein complex, around DNA and initiate DNA replication. We previously reported the creation of viable cancer cell lines that lacked detectable ORC1 or ORC2 protein without a reduction in the number of origins firing. Here, using CRISPR-Cas9-mediated mutations, we report that human HCT116 colon cancer cells also survive when ORC5 protein expression is abolished via a mutation in the initiator ATG of the ORC5 gene. Even if an internal methionine is used to produce an undetectable, N terminally deleted ORC5, the protein would lack 80% of the AAA+ ATPase domain, including the Walker A motif. The ORC5-depleted cells show normal chromatin binding of MCM2-7 and initiate replication from a similar number of origins as WT cells. In addition, we introduced a second mutation in ORC2 in the ORC5 mutant cells, rendering both ORC5 and ORC2 proteins undetectable in the same cells and destabilizing the ORC1, ORC3, and ORC4 proteins. Yet the double mutant cells grow, recruit MCM2-7 normally to chromatin, and initiate DNA replication with normal number of origins. Thus, in these selected cancer cells, either a crippled ORC lacking ORC2 and ORC5 and present at minimal levels on the chromatin can recruit and load enough MCM2-7 to initiate DNA replication, or human cell lines can sometimes recruit MCM2-7 to origins independent of ORC.

Discoveries of Extrachromosomal Circles of DNA in Normal and Tumor Cells.

While the vast majority of cellular DNA in eukaryotes is contained in long linear strands in chromosomes, we have long recognized some exceptions like mitochondrial DNA, plasmids in yeasts, and double minutes (DMs) in cancer cells where the DNA is present in extrachromosomal circles. In addition, specialized extrachromosomal circles of DNA (eccDNA) have been noted to arise from repetitive genomic sequences like telomeric DNA or rDNA. Recently eccDNA arising from unique (nonrepetitive) DNA have been discovered in normal and malignant cells, raising interesting questions about their biogenesis, function and clinical utility. Here, we review recent results and future directions of inquiry on these new forms of eccDNA.

miR-206 family is important for mitochondrial and muscle function, but not essential for myogenesis in vitro.

miR-206, miR-1a-1, and miR-1a-2 are induced during differentiation of skeletal myoblasts and promote myogenesis in vitro. miR-206 is required for skeletal muscle regeneration in vivo. Although this miRNA family is hypothesized to play an essential role in differentiation, a triple knock-out (tKO) of the three genes has not been done to test this hypothesis. We report that tKO C2C12 myoblasts generated using CRISPR/Cas9 method differentiate despite the expected derepression of the miRNA targets. Surprisingly, their mitochondrial function is diminished. tKO mice demonstrate partial embryonic lethality, most likely due to the role of miR-1a in cardiac muscle differentiation. Two tKO mice survive and grow normally to adulthood with smaller myofiber diameter, diminished physical performance, and an increase in PAX7 positive satellite cells. Thus, unlike other miRNAs important in other differentiation pathways, the miR-206 family is not absolutely essential for myogenesis and is instead a modulator of optimal differentiation of skeletal myoblasts.

Ruthenium-Benzothiadiazole Building Block Derived Dynamic Heterometallic Ru-Ag Coordination Polymer and Its Enhanced Water-Splitting Feature.

This article deals with the development of the unprecedented redox-mediated heterometallic coordination polymer {[RuIII(acac)2(µ-bis-η1-N,η1-N-BTD)2AgI(ClO4)]ClO4}n (3) via the oxidation of the monomeric building block cis-[RuII(acac)2(η1-N-BTD)2] (1) by AgClO4 (BTD = exodentate 2,1,3-benzothiadiazole, acac = acetylacetonate). Monomeric cis-[RuII(acac)2(η1-N-BTD)2] (1) and [RuII(acac)2(η1-N-BTD)(CH3CN)] (2) were simultaneously obtained from the electron-deficient BTD heterocycle and the electron-rich metal precursor RuII(acac)2(CH3CN)2 in refluxing CH3CN. Molecular identities of 1-3 were authenticated by their single-crystal X-ray structures as well as by solution spectral features. These results also reflected the elusive trigonal-planar geometry of the Ag ion in Ru-Ag-derived polymeric 3. Ru(III) (S = 1/2)-derived 3 displayed metal-based anisotropic EPR with ⟨g⟩/Δg = 2.12/0.56 and paramagnetically shifted 1H NMR. Spectroelectrochemistry in combination with DFT/TD-DFT calculations of 1n and 2n (n = 1+, 0, 1-) determined a metal-based (RuII/RuIII) oxidation and BTD-based reduction (BTD/BTD•-). The drastic decrease in the emission intensity and quantum yield but insignificant change in the lifetime of 3 with respect to 1 could be addressed in terms of static quenching and/or a paramagnetism-induced phenomenon. A homogeneously dispersed dumbbell-shaped morphology and the particle diameter of 3 were established by microscopic (TEM-EDX/SEM) and DLS analysis, respectively. Moreover, the dynamic nature of polymeric 3 was highlighted by its degradation to the η1-N-BTD coordinated monomeric fragment 1, which could also be followed spectrophotometrically in polar protic EtOH. Interestingly, both monomeric 1 and polymeric 3 exhibited efficient electrocatalytic activity toward water oxidation processes (OER, HER) on immobilization on an FTO support, which also divulged the better intrinsic water oxidation activity of 3 in comparison to 1.

CRL1-FBXO11 promotes Cdt2 ubiquitylation and degradation and regulates Pr-Set7/Set8-mediated cellular migration.

The Cul4-Cdt2 (CRL4(Cdt2)) E3 ubiquitin ligase is a master regulator of cell-cycle progression and genome stability. Despite its central role in the degradation of many cell-cycle regulators, e.g., Cdt1, p21, and Pr-Set7/Set8, little is known about the regulation of its activity. We report that Cdt2 is autoubiquitylated by the CRL4A E3 ubiquitin ligase. Cdt2 is additionally polyubiquitylated and degraded by Cul1-FBXO11 (CRL1(FBXO11)). CRL1(FBXO11)-mediated degradation of Cdt2 stabilizes p21 and Set8, and this is important during the response to TGF-ß, with the Set8 induction being important for turning off the activation of Smad2. The migration of epithelial cells is also stimulated by CRL1(FBXO11)-mediated downregulation of Cdt2 and the consequent stabilization of Set8. This is an interesting example of cross-regulation between specific Cullin 4 and Cullin 1 E3 ubiquitin ligases and highlights the role of ubiquitylation in regulating cellular responses to TGF-ß and the migration of epithelial cells.