Project description:Retroviral integration is catalyzed by a tetramer of integrase (IN) assembled on viral DNA ends in a stable complex, known as the intasome. How the intasome interfaces with chromosomal DNA, which exists in the form of nucleosomal arrays, is currently unknown. Here we show that the prototype foamy virus (PFV) intasome is proficient at stable capture of nucleosomes as targets for integration. Single-particle cryo-electron microscopy (EM) reveals a multivalent intasome-nucleosome interface involving both gyres of nucleosomal DNA and one H2A-H2B heterodimer. While the histone octamer remains intact, the DNA is lifted from the surface of the H2A-H2B heterodimer to allow integration at strongly preferred superhelix location (SHL) ±3.5 positions. Amino acid substitutions disrupting these contacts impinge on the ability of the intasome to engage nucleosomes in vitro and redistribute viral integration sites on the genomic scale. Our findings elucidate the molecular basis for nucleosome capture by the viral DNA recombination machinery and the underlying nucleosome plasticity that allows integration. Genomic positions of integration sites of WT and mutant PFV vectors in HT1080 cells were determined using ligation-mediated PCR and next generation sequencing. Integration sites of purified recombinant PFV intasome into deproteinized human genomic DNA were used as a reference dataset.
Project description:The N6-methyladenosine (m6A) is the most abundant internal modification in almost all eukaryotic messenger RNAs, and is dynamically regulated. Therefore, identification of m6A readers is especially important in determining the cellular function of m6A. YTHDF2 has recently been characterized as the first m6A reader that regulates the cytoplasmic stability of methylated RNA. Here we show that YTHDC1 is a nuclear m6A reader and report the crystal structure of the YTH domain of YTHDC1 bound to m6A-containing RNA. We further determined the structure of another YTH domain, YTHDF1, and found that the YTH domain utilizes a conserved aromatic cage to specifically recognize the methyl group of m6A. Our structural characterizations of the YTHDC1-m6A RNA complex also shed light on the molecular basis for the preferential binding of the GG(m6A)C sequence by YTHDC1 and confirm the YTH domain as a specific m6A RNA reader. PAR-CLIP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) was applied to human YTHDC1 protein to identify its binding sites.
Project description:Retroviral integration is catalyzed by a tetramer of integrase (IN) assembled on viral DNA ends in a stable complex, known as the intasome. How the intasome interfaces with chromosomal DNA, which exists in the form of nucleosomal arrays, is currently unknown. Here we show that the prototype foamy virus (PFV) intasome is proficient at stable capture of nucleosomes as targets for integration. Single-particle cryo-electron microscopy (EM) reveals a multivalent intasome-nucleosome interface involving both gyres of nucleosomal DNA and one H2A-H2B heterodimer. While the histone octamer remains intact, the DNA is lifted from the surface of the H2A-H2B heterodimer to allow integration at strongly preferred superhelix location (SHL) ±3.5 positions. Amino acid substitutions disrupting these contacts impinge on the ability of the intasome to engage nucleosomes in vitro and redistribute viral integration sites on the genomic scale. Our findings elucidate the molecular basis for nucleosome capture by the viral DNA recombination machinery and the underlying nucleosome plasticity that allows integration.
Project description:We used HSUR1 – a small non-coding RNA from Herpesvirus saimiri that induces degradation of host miR-27 – to validate structural insights into target-directed miRNA degradation (TDMD). While performing systematic mutagenesis of HSUR1 we noticed that HSUR1 mutants exhibiting complementarity to the extreme 3' end of miR-27, lead to generation of extended miR-27 isoforms (isomiRs). These isomiRs likely represent failed products of TDMD and could mean that features of the pairing between the TDMD target and miRNA dictate which enzymes are recruited to modify the miRNA 3′ end. Small RNA sequencing revealed that a mixture of adenylates and uridylates is added to the 3′ end of miR-27 during TDMD.
Project description:MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate protein gene expression post-transcriptionally. By base pairing with target mRNAs, miRNAs triggers mRNA degradation or translational suppression[1-4]. Abnormal miRNAs levels are associated with cancers, diabetes, neurological and other diseases[5-8]. RNA polymerase II transcribes primary miRNA (pri-miR) in the nucleus and pri-miRs are subsequently processed by Microprocessor, which is composed of Drosha and DiGeorge syndrome critical region 8 (DGCR8) proteins, to generate precursor miRNAs (pre-miRs). Pre-miRs are exported from the nucleus to the cytoplasm in a GTP-dependent manner by Exportin-5. In the cytoplasm, a pre-miRs are further processed by Dicer to generate 21-22 nt mature miRNA[4, 9]. Argonaute (Ago) protein loads one of mature miRNAs into protein and forms miRNA-induced silencing complex, which is responsible for mRNA degradation or translational suppression[2, 4].
Project description:The N6-methyladenosine (m6A) is the most abundant internal modification in almost all eukaryotic messenger RNAs, and is dynamically regulated. Therefore, identification of m6A readers is especially important in determining the cellular function of m6A. YTHDF2 has recently been characterized as the first m6A reader that regulates the cytoplasmic stability of methylated RNA. Here we show that YTHDC1 is a nuclear m6A reader and report the crystal structure of the YTH domain of YTHDC1 bound to m6A-containing RNA. We further determined the structure of another YTH domain, YTHDF1, and found that the YTH domain utilizes a conserved aromatic cage to specifically recognize the methyl group of m6A. Our structural characterizations of the YTHDC1-m6A RNA complex also shed light on the molecular basis for the preferential binding of the GG(m6A)C sequence by YTHDC1 and confirm the YTH domain as a specific m6A RNA reader.
Project description:The bacterium, Francisella tularensis (Ft), is one of the most infectious agents known and classified as a category A bioweapon. Ft virulence is controlled by a unique set of transcription regulators, the MglA-SspA heterodimer, PigR, and the stress signal, ppGpp. These factors activate Francisella pathogenicity island (FPI) gene expression, which is required for virulence. MglA-SspA is expressed during infection and constitutively associates with the σ70 associated RNAP holoenzyme (RNAPσ70), indicating that RNAPσ70-(MglA-SspA) is a virulence specific polymerase. How virulence activation is mediated by these components, however, is unknown. Here we report cryo-EM structures of FtRNAPσ70, FtRNAPσ70-(MglA-SspA) and RNAPσ70-(MglA-SspA)-ppGpp-PigR complexes with promoter DNA. FtRNAPσ70-DNA and FtRNAPσ70-(MglA-SspA)-DNA structures and RT-PCR analyses show MglA-SspA stabilizes σ70 binding to DNA to regulate FPI-independent, virulence-enhancing genes. Strikingly, an Escherichia coli RNAPσ70 complex with EcSspA suggests this is a general mechanism for SspA-like regulation of bacterial RNAPσ70. Finally, our FtRNAP-σ70-(MglA-SspA)-ppGpp-PigR-DNA structure reveals that ppGpp binds to MglA-SspA to tether the DNA-binding activator, PigR, to FPI promoters. PigR in turn recruits FtRNAP CTDs to two DNA upstream (UP) elements, generating stable FPI transcription complexes. Thus, these studies unveil a novel paradigm for pathogenesis in Ft involving a virulence-specific RNAP that employs two (MglA-SspA)-based strategies to activate virulence genes.
Project description:The interactions between a retrovirus and host cell chromatin that underlie integration and provirus expression are poorly understood. The prototype foamy virus (PFV) structural protein GAG associates with chromosomes via a chromatin-binding sequence (CBS) located within its C-terminal region. Here, we show that the PFV CBS is essential and sufficient for a direct interaction with nucleosomes, and present a crystal structure of the CBS bound to a mono-nucleosome. The CBS interacts with the histone octamer, engaging the H2A-H2B acidic patch, in a manner similar to other acidic patch binding proteins such as herpesvirus latency-associated nuclear antigen (LANA). Substitutions of the invariant arginine anchor residue in GAG result in global re-distribution of PFV and macaque simian foamy virus (SFVmac) integration sites towards centromeres, dampening the resulting proviral expression without affecting the overall efficiency of integration. Our findings underscore the importance of retroviral structural proteins for integration site selection and the avoidance of genomic junkyards.
Project description:MALAT1 lncRNA plays key roles in regulating transcription, splicing, and tumorigenesis. Its maturation and stabilization require precise processing by RNase P, which simultaneously initiates the biogenesis of a 3′ cytoplasmic mascRNA. mascRNA was proposed to fold into a tRNA-like secondary structure, but lacks eight conserved linking residues required by the canonical tRNA fold. Here, we report crystal structures of human mascRNA before and after processing, which reveal an ultracompact, quasi-tRNA-like structure. Despite lacking all linker residues, mascRNA faithfully recreates the characteristic “elbow” feature of tRNAs to recruit RNase P and ELAC2 for processing, which exhibit distinct substrate specificities. Rotation and repositioning of the D-stem and anticodon regions preclude mascRNA from aminoacylation, avoiding interference with translation. Therefore, a class of metazoan lncRNAs employ a previously unrecognized, unusually streamlined quasi-tRNA architecture to recruit select tRNA-processing enzymes while excluding others, to drive bespoke RNA biogenesis, processing, and maturation.