DCL1 is the ribonuclease that carries out miRNA biogenesis in plants. The enzyme has two tandem double stranded RNA binding domains (dsRBDs) in its C-terminus. Here we show that the first of these domains binds precursor RNA fragments when isolated and cooperates with the second domain in the recognition of substrate RNA. Remarkably, despite showing RNA binding activity, this domain is intrinsically disordered. We found that it acquires a folded conformation when bound to its substrate, being the first report of a complete dsRBD folding upon binding. The free unfolded form shows tendency to adopt folded conformations, and goes through an unfolded bound state prior to the folding event. The significance of these results is discussed by comparison with the behavior of other dsRBDs.
Versatile genetic assembly system (VEGAS) to assemble pathways for expression in S. cerevisiae
We have developed a method for assembling genetic pathways for expression in Saccharomyces cerevisiae. Our pathway assembly method, called VEGAS (Versatile genetic assembly system), exploits the native capacity of S. cerevisiae to perform homologous recombination and efficiently join sequences with terminal homology. In the VEGAS workflow, terminal homology between adjacent pathway genes and the assembly vector is encoded by ‘VEGAS adapter’ (VA) sequences, which are orthogonal in sequence with respect to the yeast genome. Prior to pathway assembly by VEGAS in S. cerevisiae, each gene is assigned an appropriate pair of VAs and assembled using a previously described technique called yeast Golden Gate (yGG). Here we describe the application of yGG specifically to building transcription units for VEGAS assembly as well as the VEGAS methodology. We demonstrate the assembly of four-, five- and six-gene pathways by VEGAS to generate S. cerevisiae cells synthesizing β-carotene and violacein. Moreover, we demonstrate the capacity of yGG coupled to VEGAS for combinatorial assembly.
Post-translational environmental switch of RadA activity by extein-intein interactions in protein splicing
Post-translational control based on an environmentally sensitive intervening intein sequence is described. Inteins are invasive genetic elements that self-splice at the protein level from the flanking host protein, the exteins. Here we show in Escherichia coli and in vitro that splicing of the RadA intein located in the ATPase domain of the hyperthermophilic archaeon Pyrococcus horikoshii is strongly regulated by the native exteins, which lock the intein in an inactive state. High temperature or solution conditions can unlock the intein for full activity, as can remote extein point mutations. Notably, this splicing trap occurs through interactions between distant residues in the native exteins and the intein, in three-dimensional space. The exteins might thereby serve as an environmental sensor, releasing the intein for full activity only at optimal growth conditions for the native organism, while sparing ATP consumption under conditions of cold-shock. This partnership between the intein and its exteins, which implies coevolution of the parasitic intein and its host protein may provide a novel means of post-translational control.
DNA hairpins destabilize duplexes primarily by promoting melting rather than by inhibiting hybridization
The effect of secondary structure on DNA duplex formation is poorly understood. Using oxDNA, a nucleotide level coarse-grained model of DNA, we study how hairpins influence the rate and reaction pathways of DNA hybridzation. We compare to experimental systems studied by Gao et al. (1) and find that 3-base pair hairpins reduce the hybridization rate by a factor of 2, and 4-base pair hairpins by a factor of 10, compared to DNA with limited secondary structure, which is in good agreement with experiments. By contrast, melting rates are accelerated by factors of ~100 and ~2000. This surprisingly large speed-up occurs because hairpins form during the melting process, and significantly lower the free energy barrier for dissociation. These results should assist experimentalists in designing sequences to be used in DNA nanotechnology, by putting limits on the suppression of hybridization reaction rates through the use of hairpins and offering the possibility of deliberately increasing dissociation rates by incorporating hairpins into single strands.
Sequence-independent characterization of viruses based on the pattern of viral small RNAs produced by the host
Virus surveillance in vector insects is potentially of great benefit to public health. Large-scale sequencing of small and long RNAs has previously been used to detect viruses, but without any formal comparison of different strategies. Furthermore, the identification of viral sequences largely depends on similarity searches against reference databases. Here, we developed a sequence-independent strategy based on virus-derived small RNAs produced by the host response, such as the RNA interference pathway. In insects, we compared sequences of small and long RNAs, demonstrating that viral sequences are enriched in the small RNA fraction. We also noted that the small RNA size profile is a unique signature for each virus and can be used to identify novel viral sequences without known relatives in reference databases. Using this strategy, we characterized six novel viruses in the viromes of laboratory fruit flies and wild populations of two insect vectors: mosquitoes and sandflies. We also show that the small RNA profile could be used to infer viral tropism for ovaries among other aspects of virus biology. Additionally, our results suggest that virus detection utilizing small RNAs can also be applied to vertebrates, although not as efficiently as to plants and insects.
We report a high-resolution time series study of transcriptome dynamics following antimiR-mediated inhibition of miR-9 in a Hodgkin lymphoma cell-line—the first such dynamic study of the microRNA inhibition response—revealing both general and specific aspects of the physiological response. We show miR-9 inhibition inducing a multiphasic transcriptome response, with a direct target perturbation before 4 h, earlier than previously reported, amplified by a downstream peak at ~32 h consistent with an indirect response due to secondary coherent regulation. Predictive modelling indicates a major role for miR-9 in post-transcriptional control of RNA processing and RNA binding protein regulation. Cluster analysis identifies multiple co-regulated gene regulatory modules. Functionally, we observe a shift over time from mRNA processing at early time points to translation at later time points. We validate the key observations with independent time series qPCR and we experimentally validate key predicted miR-9 targets. Methodologically, we developed sensitive functional data analytic predictive methods to analyse the weak response inherent in microRNA inhibition experiments. The methods of this study will be applicable to similar high-resolution time series transcriptome analyses and provides the context for more accurate experimental design and interpretation of future microRNA inhibition studies.
The eIF3 complex of Leishmania--subunit composition and mode of recruitment to different cap-binding complexes
Eukaryotic initiation factor 3 (eIF3) is a multi-protein complex and a key participant in the assembly of the translation initiation machinery. In mammals, eIF3 comprises 13 subunits, most of which are characterized by conserved structural domains. The trypanosomatid eIF3 subunits are poorly conserved. Here, we identify 12 subunits that comprise the Leishmania eIF3 complex (LeishIF3a-l) by combining bioinformatics with affinity purification and mass spectrometry analyses. These results highlight the strong association of LeishIF3 with LeishIF1, LeishIF2 and LeishIF5, suggesting the existence of a multi-factor complex. In trypanosomatids, the translation machinery is tightly regulated in the different life stages of these organisms as part of their adaptation and survival in changing environments. We, therefore, addressed the mechanism by which LeishIF3 is recruited to different mRNA cap-binding complexes. A direct interaction was observed in vitro between the fully assembled LeishIF3 complex and recombinant LeishIF4G3, the canonical scaffolding protein of the cap-binding complex in Leishmania promastigotes. We further highlight a novel interaction between the C-terminus of LeishIF3a and LeishIF4E1, the only cap-binding protein that efficiently binds the cap structure under heat shock conditions, anchoring a complex that is deficient of any MIF4G-based scaffolding subunit.
A fine balance: epigenetic control of cellular quiescence by the tumor suppressor PRDM2/RIZ at a bivalent domain in the cyclin a gene
Adult stem cell quiescence is critical to ensure regeneration while minimizing tumorigenesis. Epigenetic regulation contributes to cell cycle control and differentiation, but few regulators of the chromatin state in quiescent cells are known. Here we report that the tumor suppressor PRDM2/RIZ, an H3K9 methyltransferase, is enriched in quiescent muscle stem cells in vivo and controls reversible quiescence in cultured myoblasts. We find that PRDM2 associates with >4400 promoters in G0 myoblasts, 55% of which are also marked with H3K9me2 and enriched for myogenic, cell cycle and developmental regulators. Knockdown of PRDM2 alters histone methylation at key promoters such as Myogenin and CyclinA2 (CCNA2), and subverts the quiescence program via global de-repression of myogenesis, and hyper-repression of the cell cycle. Further, PRDM2 acts upstream of the repressive PRC2 complex in G0. We identify a novel G0-specific bivalent chromatin domain in the CCNA2 locus. PRDM2 protein interacts with the PRC2 protein EZH2 and regulates its association with the bivalent domain in the CCNA2 gene. Our results suggest that induction of PRDM2 in G0 ensures that two antagonistic programs—myogenesis and the cell cycle—while stalled, are poised for reactivation. Together, these results indicate that epigenetic regulation by PRDM2 preserves key functions of the quiescent state, with implications for stem cell self-renewal.
Context-specific role of SOX9 in NF-Y mediated gene regulation in colorectal cancer cells
Roles for SOX9 have been extensively studied in development and particular emphasis has been placed on SOX9 roles in cell lineage determination in a number of discrete tissues. Aberrant expression of SOX9 in many cancers, including colorectal cancer, suggests roles in these diseases as well and recent studies have suggested tissue- and context-specific roles of SOX9. Our genome wide approach by chromatin immunoprecipitation sequencing (ChIP-seq) in human colorectal cancer cells identified a number of physiological targets of SOX9, including ubiquitously expressed cell cycle regulatory genes, such as CCNB1 and CCNB2, CDK1, and TOP2A. These novel high affinity-SOX9 binding peaks precisely overlapped with binding sites for histone-fold NF-Y transcription factor. Furthermore, our data showed that SOX9 is recruited by NF-Y to these promoters of cell cycle regulatory genes and that SOX9 is critical for the full function of NF-Y in activation of the cell cycle genes. Mutagenesis analysis and in vitro binding assays provided additional evidence to show that SOX9 affinity is through NF-Y and that SOX9 DNA binding domain is not necessary for SOX9 affinity to those target genes. Collectively, our results reveal possibly a context-dependent, non-classical regulatory role for SOX9.
PRC2 regulates RNA polymerase III transcribed non-translated RNA gene transcription through EZH2 and SUZ12 interaction with TFIIIC complex
Polycomb repression complex 2 (PRC2) component EZH2 tri-methylates H3K27 and exerts epigenetic repression on target gene expression. EZH2-mediated epigenetic control of RNA polymerase II (Pol II) transcribed coding gene transcription has been well established. However, little is known about EZH2-mediated epigenetic regulation of RNA polymerase III (Pol III) transcription. Here we present a paradigm that EZH2 is involved in the repression of Pol III transcription via interaction with transcriptional factor complex IIIC (TFIIIC). EZH2 and H3K27me3 co-occupy the promoter of tRNATyr, 5S rRNA and 7SL RNA genes. Depletion of EZH2 or inhibition of EZH2 methyltransferase activity led to upregulation of Pol III target gene transcription. EZH2-mediated repression of Pol III transcribed gene expression requires presence of SUZ12. SUZ12 was able to interact with TFIIIC complex and knockdown of SUZ12 decreased occupancy of EZH2 and H3K27me3 at the promoter of Pol III target genes. Our findings pointed out a previously unidentified role of PRC2 complex in suppressing transcription of Pol III transcribed non-translated RNA genes, putting Pol III on a new layer of epigenetic regulation.
TBP-like protein (TLP) interferes with Taspase1-mediated processing of TFIIA and represses TATA box gene expression
TBP-TFIIA interaction is involved in the potentiation of TATA box-driven promoters. TFIIA activates transcription through stabilization of TATA box-bound TBP. The precursor of TFIIA is subjected to Taspase1-directed processing to generate α and β subunits. Although this processing has been assumed to be required for the promoter activation function of TFIIA, little is known about how the processing is regulated. In this study, we found that TBP-like protein (TLP), which has the highest affinity to TFIIA among known proteins, affects Taspase1-driven processing of TFIIA. TLP interfered with TFIIA processing in vivo and in vitro, and direct binding of TLP to TFIIA was essential for inhibition of the processing. We also showed that TATA box promoters are specifically potentiated by processed TFIIA. Processed TFIIA, but not unprocessed TFIIA, associated with the TATA box. In a TLP-knocked-down condition, not only the amounts of TATA box-bound TFIIA but also those of chromatin-bound TBP were significantly increased, resulting in the stimulation of TATA box-mediated gene expression. Consequently, we suggest that TLP works as a negative regulator of the TFIIA processing and represses TFIIA-governed and TATA-dependent gene expression through preventing TFIIA maturation.
Bacteriophage Xp10 anti-termination factor p7 induces forward translocation by host RNA polymerase
Regulation of transcription elongation is based on response of RNA polymerase (RNAP) to various pause signals and is modulated by various accessory factors. Here we report that a 7 kDa protein p7 encoded by bacteriophage Xp10 acts as an elongation processivity factor of RNAP of host bacterium Xanthomonas oryzae, a major rice pathogen. Our data suggest that p7 stabilizes the upstream DNA duplex of the elongation complex thus disfavouring backtracking and promoting forward translocated states of the elongation complex. The p7-induced ‘pushing’ of RNAP and modification of RNAP contacts with the upstream edge of the transcription bubble lead to read-through of various types of pauses and termination signals and generally increase transcription processivity and elongation rate, contributing for transcription of an extremely long late genes operon of Xp10. Forward translocation was observed earlier upon the binding of unrelated bacterial elongation factor NusG, suggesting that this may be a general pathway of regulation of transcription elongation.
Mechanism of heat stress-induced cellular senescence elucidates the exclusive vulnerability of early S-phase cells to mild genotoxic stress
Heat stress is one of the best-studied cellular stress factors; however, little is known about its delayed effects. Here, we demonstrate that heat stress induces p21-dependent cellular senescence-like cell cycle arrest. Notably, only early S-phase cells undergo such an arrest in response to heat stress. The encounter of DNA replication forks with topoisomerase I-generated single-stranded DNA breaks resulted in the generation of persistent double-stranded DNA breaks was found to be a primary cause of heat stress-induced cellular senescence in these cells. This investigation of heat stress-induced cellular senescence elucidates the mechanisms underlying the exclusive sensitivity of early S-phase cells to ultra-low doses of agents that induce single-stranded DNA breaks.
SERBP1 affects homologous recombination-mediated DNA repair by regulation of CtIP translation during S phase
DNA double-strand breaks (DSBs) are the most severe type of DNA damage and are primarily repaired by non-homologous end joining (NHEJ) and homologous recombination (HR) in the G1 and S/G2 phase, respectively. Although CtBP-interacting protein (CtIP) is crucial in DNA end resection during HR following DSBs, little is known about how CtIP levels increase in an S phase-specific manner. Here, we show that Serpine mRNA binding protein 1 (SERBP1) regulates CtIP expression at the translational level in S phase. In response to camptothecin-mediated DNA DSBs, CHK1 and RPA2 phosphorylation, which are hallmarks of HR activation, was abrogated in SERBP1-depleted cells. We identified CtIP mRNA as a binding target of SERBP1 using RNA immunoprecipitation-coupled RNA sequencing, and confirmed SERBP1 binding to CtIP mRNA in S phase. SERBP1 depletion resulted in reduction of polysome-associated CtIP mRNA and concomitant loss of CtIP expression in S phase. These effects were reversed by reconstituting cells with wild-type SERBP1, but not by SERBP1 RGG, an RNA binding defective mutant, suggesting regulation of CtIP translation by SERBP1 association with CtIP mRNA. These results indicate that SERBP1 affects HR-mediated DNA repair in response to DNA DSBs by regulation of CtIP translation in S phase.
Targeted DNA damage at individual telomeres disrupts their integrity and triggers cell death
Cellular DNA is organized into chromosomes and capped by a unique nucleoprotein structure, the telomere. Both oxidative stress and telomere shortening/dysfunction cause aging-related degenerative pathologies and increase cancer risk. However, a direct connection between oxidative damage to telomeric DNA, comprising <1% of the genome, and telomere dysfunction has not been established. By fusing the KillerRed chromophore with the telomere repeat binding factor 1, TRF1, we developed a novel approach to generate localized damage to telomere DNA and to monitor the real time damage response at the single telomere level. We found that DNA damage at long telomeres in U2OS cells is not repaired efficiently compared to DNA damage in non-telomeric regions of the same length in heterochromatin. Telomeric DNA damage shortens the average length of telomeres and leads to cell senescence in HeLa cells and cell death in HeLa, U2OS and IMR90 cells, when DNA damage at non-telomeric regions is undetectable. Telomere-specific damage induces chromosomal aberrations, including chromatid telomere loss and telomere associations, distinct from the damage induced by ionizing irradiation. Taken together, our results demonstrate that oxidative damage induces telomere dysfunction and underline the importance of maintaining telomere integrity upon oxidative damage.
We have recently found that DNA packaged in phage undergoes a disordering transition triggered by temperature, which results in increased genome mobility. This solid-to-fluid like DNA transition markedly increases the number of infectious particles facilitating infection. However, the structural transition strongly depends on temperature and ionic conditions in the surrounding medium. Using titration microcalorimetry combined with solution X-ray scattering, we mapped both energetic and structural changes associated with transition of the encapsidated -DNA. Packaged DNA needs to reach a critical stress level in order for transition to occur. We varied the stress on DNA in the capsid by changing the temperature, packaged DNA length and ionic conditions. We found striking evidence that the intracapsid DNA transition is ‘switched on’ at the ionic conditions mimicking those in vivo and also at the physiologic temperature of infection at 37°C. This ion regulated on-off switch of packaged DNA mobility in turn affects viral replication. These results suggest a remarkable adaptation of phage to the environment of its host bacteria in the human gut. The metastable DNA state in the capsid provides a new paradigm for the physical evolution of viruses.