Christian Benda

Sequestration by IFIT1 Impairs Translation of 2 ' O-unmethylated Capped RNA

Viruses that generate capped RNA lacking 2′O methylation on the first ribose are severely affected by the antiviral activity of Type I interferons. We used proteome-wide affinity purification coupled to mass spectrometry to identify human and mouse proteins specifically binding to capped RNA with different methylation states. This analysis, complemented with functional validation experiments, revealed that IFIT1 is the sole interferon-induced protein displaying higher affinity for unmethylated than for methylated capped RNA. IFIT1 tethers a species-specific protein complex consisting of other IFITs to RNA. Pulsed stable isotope labelling with amino acids in cell culture coupled to mass spectrometry as well as in vitro competition assays indicate that IFIT1 sequesters 2′O-unmethylated capped RNA and thereby impairs binding of eukaryotic translation initiation factors to 2′O-unmethylated RNA template, which results in inhibition of translation. The specificity of IFIT1 for 2′O-unmethylated RNA serves as potent antiviral mechanism against viruses lacking 2′O-methyltransferase activity and at the same time allows unperturbed progression of the antiviral program in infected cells.

Interaction of Circadian Clock Proteins CRY1 and PER2 Is Modulated by Zinc Binding and Disulfide Bond Formation

Period (PER) proteins are essential components of the mammalian circadian clock. They form complexes with cryptochromes (CRY), which negatively regulate CLOCK/BMAL1-dependent transactivation of clock and clock-controlled genes. To define the roles of mammalian CRY/PER complexes in the circadian clock, we have determined the crystal structure of a complex comprising the photolyase homology region of mouse CRY1 (mCRY1) and a C-terminal mouse PER2 (mPER2) fragment. mPER2 winds around the helical mCRY1 domain covering the binding sites of FBXL3 and CLOCK/BMAL1, but not the FAD binding pocket. Our structure revealed an unexpected zinc ion in one interface, which stabilizes mCRY1-mPER2 interactions in vivo. We provide evidence that mCRY1/mPER2 complex formation is modulated by an interplay of zinc binding and mCRY1 disulfide bond formation, which may be influenced by the redox state of the cell. Our studies may allow for the development of circadian and metabolic modulators.

Structural and Functional Organization of the Ska Complex, a Key Component of the Kinetochore-Microtubule Interface.

The Ska complex is an essential mitotic component required for accurate cell division in human cells. It is composed of three subunits that function together to establish stable kinetochore-microtubule interactions in concert with the Ndc80 network. We show that the structure of the Ska core complex is a W-shaped dimer of coiled coils, formed by intertwined interactions between Ska1, Ska2, and Ska3. The C-terminal domains of Ska1 and Ska3 protrude at each end of the homodimer, bind microtubules in vitro when connected to the central core, and are essential in vivo. Mutations disrupting the central coiled coil or the dimerization interface result in chromosome congression failure followed by cell death. The Ska complex is thus endowed with bipartite and cooperative tubulin-binding properties at the ends of a 350 Å-long molecule. We discuss how this symmetric architecture might complement and stabilize the Ndc80-microtubule attachments with analogies to the yeast Dam1/DASH complex.

Structural Model of a CRISPR RNA-Silencing Complex Reveals the RNA-Target Cleavage Activity in Cmr4

The Cmr complex is an RNA-guided endonuclease that cleaves foreign RNA targets as part of the CRISPR prokaryotic defense system. We investigated the molecular architecture of the P. furiosus Cmr complex using an integrative structural biology approach. We determined crystal structures of P. furiosus Cmr1, Cmr2, Cmr4, and Cmr6 and combined them with known structural information to interpret the cryo-EM map of the complex. To support structure determination, we obtained residue-specific interaction data using protein crosslinking and mass spectrometry. The resulting pseudoatomic model reveals how the superhelical backbone of the complex is defined by the polymerizing principles of Cmr4 and Cmr5 and how it is capped at the extremities by proteins of similar folds. The inner surface of the superhelix exposes conserved residues of Cmr4 that we show are required for target-cleavage activity. The structural and biochemical data thus identify Cmr4 as the conserved endoribonuclease of the Cmr complex.

HOT1 is a mammalian direct telomere repeat‐binding protein contributing to telomerase recruitment

Telomeres are repetitive DNA structures that, together with the shelterin and the CST complex, protect the ends of chromosomes. Telomere shortening is mitigated in stem and cancer cells through the de novo addition of telomeric repeats by telomerase. Telomere elongation requires the delivery of the telomerase complex to telomeres through a not yet fully understood mechanism. Factors promoting telomerase–telomere interaction are expected to directly bind telomeres and physically interact with the telomerase complex. In search for such a factor we carried out a SILAC‐based DNA–protein interaction screen and identified HMBOX1, hereafter referred to as homeobox telomere‐binding protein 1 (HOT1). HOT1 directly and specifically binds double‐stranded telomere repeats, with the in vivo association correlating with binding to actively processed telomeres. Depletion and overexpression experiments classify HOT1 as a positive regulator of telomere length. Furthermore, immunoprecipitation and cell fractionation analyses show that HOT1 associates with the active telomerase complex and promotes chromatin association of telomerase. Collectively, these findings suggest that HOT1 supports telomerase‐dependent telomere elongation.

Phosphorylation of the yeast γ-tubulin Tub4 regulates microtubule function.

The yeast γ-tubulin Tub4 is assembled with Spc97 and Spc98 into the small Tub4 complex. The Tub4 complex binds via the receptor proteins Spc72 and Spc110 to the spindle pole body (SPB), the functional equivalent of the mammalian centrosome, where the Tub4 complex organizes cytoplasmic and nuclear microtubules. Little is known about the regulation of the Tub4 complex. Here, we isolated the Tub4 complex with the bound receptors from yeast cells. Analysis of the purified Tub4 complex by mass spectrometry identified more than 50 phosphorylation sites in Spc72, Spc97, Spc98, Spc110 and Tub4. To examine the functional relevance of the phosphorylation sites, phospho-mimicking and non-phosphorylatable mutations in Tub4, Spc97 and Spc98 were analyzed. Three phosphorylation sites in Tub4 were found to be critical for Tub4 stability and microtubule organization. One of the sites is highly conserved in γ-tubulins from yeast to human.

Molecular basis of PRC1 targeting to Polycomb response elements by PhoRC.

Polycomb group (PcG) protein complexes repress transcription by modifying target gene chromatin. In Drosophila, this repression requires association of PcG protein complexes with cis-regulatory Polycomb response elements (PREs), but the interactions permitting formation of these assemblies are poorly understood. We show that the Sfmbt subunit of the DNA-binding Pho-repressive complex (PhoRC) and the Scm subunit of the canonical Polycomb-repressive complex 1 (PRC1) directly bind each other through their SAM domains. The 1.9 Å crystal structure of the Scm-SAM:Sfmbt-SAM complex reveals the recognition mechanism and shows that Sfmbt-SAM lacks the polymerization capacity of the SAM domains of Scm and its PRC1 partner subunit, Ph. Functional analyses in Drosophila demonstrate that Sfmbt-SAM and Scm-SAM are essential for repression and that PhoRC DNA binding is critical to initiate PRC1 association with PREs. Together, this suggests that PRE-tethered Sfmbt-SAM nucleates PRC1 recruitment and that Scm-SAM/Ph-SAM-mediated polymerization then results in the formation of PRC1-compacted chromatin.

Structure and RNA-binding properties of the Type III-A CRISPR-associated protein Csm3

The prokaryotic adaptive immune system is based on the incorporation of genome fragments of invading viral genetic elements into clusters of regulatory interspaced short palindromic repeats (CRISPRs). The CRISPR loci are transcribed and processed into crRNAs, which are then used to target the invading nucleic acid for degradation. The large family of CRISPR-associated (Cas) proteins mediates this interference response. We have characterized Methanopyrus kandleri Csm3, a protein of the type III-A CRISPR-Cas complex. The 2.4 Å resolution crystal structure shows an elaborate four-domain fold organized around a core RRM-like domain. The overall architecture highlights the structural homology to Cas7, the Cas protein that forms the backbone of type I interference complexes. Csm3 binds unstructured RNAs in a sequence non-specific manner, suggesting that it interacts with the variable spacer sequence of the crRNA. The structural and biochemical data provide insights into the similarities and differences in this group of Cas proteins.

mRNA export through an additional cap-binding complex consisting of NCBP1 and NCBP3.

The flow of genetic information from DNA to protein requires polymerase-II-transcribed RNA characterized by the presence of a 5′-cap. The cap-binding complex (CBC), consisting of the nuclear cap-binding protein (NCBP) 2 and its adaptor NCBP1, is believed to bind all capped RNA and to be necessary for its processing and intracellular localization. Here we show that NCBP1, but not NCBP2, is required for cell viability and poly(A) RNA export. We identify C17orf85 (here named NCBP3) as a cap-binding protein that together with NCBP1 forms an alternative CBC in higher eukaryotes. NCBP3 binds mRNA, associates with components of the mRNA processing machinery and contributes to poly(A) RNA export. Loss of NCBP3 can be compensated by NCBP2 under steady-state conditions. However, NCBP3 becomes pivotal under stress conditions, such as virus infection. We propose the existence of an alternative CBC involving NCBP1 and NCBP3 that plays a key role in mRNA biogenesis.

DNA binding by PHF1 prolongs PRC2 residence time on chromatin and thereby promotes H3K27 methylation

Polycomb repressive complex 2 (PRC2) trimethylates histone H3 at lysine 27 to mark genes for repression. We measured the dynamics of PRC2 binding on recombinant chromatin and free DNA at the single-molecule level using total internal reflection fluorescence (TIRF) microscopy. PRC2 preferentially binds free DNA with multisecond residence time and midnanomolar affinity. PHF1, a PRC2 accessory protein of the Polycomblike family, extends PRC2 residence time on DNA and chromatin. Crystallographic and functional studies reveal that Polycomblike proteins contain a winged-helix domain that binds DNA in a sequence-nonspecific fashion. DNA binding by this winged-helix domain accounts for the prolonged residence time of PHF1–PRC2 on chromatin and makes it a more efficient H3K27 methyltranferase than PRC2 alone. Together, these studies establish that interactions with DNA provide the predominant binding affinity of PRC2 for chromatin. Moreover, they reveal the molecular basis for how Polycomblike proteins stabilize PRC2 on chromatin and stimulate its activity.