The backbone of prokaryotic adaptive immunity

CRISPR/Cas is the prokaryotic adaptive immune response to viral invasion. Its mechanism is reminiscent of the eukaryotic RNA interference. The host actively incorporates short sequences from invading genetic elements (viruses or plasmids) into a region of its genome that is characterized by clustered regularly interspaced short palindromic repeats (CRISPRs) and a number of CRISPR-associated (cas) genes. The molecular memory of previous infections can be transcribed and processed into small RNAs (crRNAs) that guide a multiprotein–nucleic acid interference complex to recognize and cleave incoming foreign genetic material. Three pathways (I, II, III) are defined by their protein machinery and target specificity (DNA vs. RNA). In types I and III, the main protagonist of the interference complex is the Cas7 protein. Up to six copies of Cas7 constitute the complex’s main building block that assembles around the crRNA and provides a platform for protein interactions and target binding. During my PhD work, I solved the crystal structures of two Cas7 orthologs from different archaeal species, at 1.8 Å for Thermofilum pendens (Tp) Csc2 and at 2.37 Å for Meth- anopyrus kandleri (Mk) Csm3. The crystal structures of Mk Csm3 and Tp Csc2 were solved by experimental phasing and revealed a core RRM-like domain with a β1-α1-β2-β3-α2-β4 arrangement of secondary structure elements. The core is flanked by three peripheral domains that are defined by insertions within the core. Structural superposition of the RRM-like core domains of Mk Csm3 and Tp Csc2 with the representatives of other Cas families (5/6/7) revealed the highest homology beyond the RRM with a Cas7 family homolog. Thus I showed that Cas7 family proteins share equivalent insertions, forming homologous peripheral domains. Using the information obtained from structural data, I investigated the RNA binding properties Mk Csm3, Tp Csc2 and a Cas7 protein from subtype I-A, Thermoproteus tenax (Tt) Csa2. All orthologs bound RNA in a sequence-independent manner, according to their physiological function of spacer binding. Furthermore, a combined approach consisting of mutation analysis, UV-based protein–RNA crosslinking, mass spectrometry and fluorescence anisotropy mapped the RNA interacting regions to two structurally highly conserved positively charged surfaces. Taken together, this thesis describes a comprehensive structural study of the Cas7 family, defining the family’s structural features. These structural data from single proteins and the mapped RNA binding interfaces agree with protein–RNA interactions observed in the Escherichia coli interference complex

The backbone of prokaryotic adaptive immunity

CRISPR/Cas is the prokaryotic adaptive immune response to viral invasion. Its mechanism is reminiscent of the eukaryotic RNA interference. The host actively incorporates short sequences from invading genetic elements (viruses or plasmids) into a region of its genome that is characterized by clustered regularly interspaced short palindromic repeats (CRISPRs) and a number of CRISPR-associated (cas) genes. The molecular memory of previous infections can be transcribed and processed into small RNAs (crRNAs) that guide a multiprotein–nucleic acid interference complex to recognize and cleave incoming foreign genetic material. Three pathways (I, II, III) are defined by their protein machinery and target specificity (DNA vs. RNA). In types I and III, the main protagonist of the interference complex is the Cas7 protein. Up to six copies of Cas7 constitute the complex’s main building block that assembles around the crRNA and provides a platform for protein interactions and target binding. During my PhD work, I solved the crystal structures of two Cas7 orthologs from different archaeal species, at 1.8 Å for Thermofilum pendens (Tp) Csc2 and at 2.37 Å for Meth- anopyrus kandleri (Mk) Csm3. The crystal structures of Mk Csm3 and Tp Csc2 were solved by experimental phasing and revealed a core RRM-like domain with a β1-α1-β2-β3-α2-β4 arrangement of secondary structure elements. The core is flanked by three peripheral domains that are defined by insertions within the core. Structural superposition of the RRM-like core domains of Mk Csm3 and Tp Csc2 with the representatives of other Cas families (5/6/7) revealed the highest homology beyond the RRM with a Cas7 family homolog. Thus I showed that Cas7 family proteins share equivalent insertions, forming homologous peripheral domains. Using the information obtained from structural data, I investigated the RNA binding properties Mk Csm3, Tp Csc2 and a Cas7 protein from subtype I-A, Thermoproteus tenax (Tt) Csa2. All orthologs bound RNA in a sequence-independent manner, according to their physiological function of spacer binding. Furthermore, a combined approach consisting of mutation analysis, UV-based protein–RNA crosslinking, mass spectrometry and fluorescence anisotropy mapped the RNA interacting regions to two structurally highly conserved positively charged surfaces. Taken together, this thesis describes a comprehensive structural study of the Cas7 family, defining the family’s structural features. These structural data from single proteins and the mapped RNA binding interfaces agree with protein–RNA interactions observed in the Escherichia coli interference complex

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

Analysis of protein-RNA interactions in CRISPR proteins and effector complexes by UV-induced cross-linking and mass spectrometry

Ribonucleoprotein (RNP) complexes play important roles in the cell by mediating basic cellular processes, including gene expression and its regulation. Understanding the molecular details of these processes requires the identification and characterization of protein-RNA interactions. Over the years various approaches have been used to investigate these interactions, including computational analyses to look for RNA binding domains, gel-shift mobility assays on recombinant and mutant proteins as well as co-crystallization and NMR studies for structure elucidation. Here we report a more specialized and direct approach using UV-induced cross-linking coupled with mass spectrometry. This approach permits the identification of cross-linked peptides and RNA moieties and can also pin-point exact RNA contact sites within the protein. The power of this method is illustrated by the application to different single- and multi-subunit RNP complexes belonging to the prokaryotic adaptive immune system, CRISPR-Cas (CRISPR: clustered regularly interspaced short palindromic repeats; Cas: CRISPR associated). In particular, we identified the RNA-binding sites within three Cas7 protein homologs and mapped the cross-linking results to reveal structurally conserved Cas7 - RNA binding interfaces. These results demonstrate the strong potential of UV-induced cross-linking coupled with mass spectrometry analysis to identify RNA interaction sites on the RNA binding proteins.</p

Structural analyses of the CRISPR protein Csc2 reveal the RNA-binding interface of the type I-D Cas7 family

<div><p>Upon pathogen invasion, bacteria and archaea activate an RNA-interference-like mechanism termed CRISPR (clustered regularly interspaced short palindromic repeats). A large family of Cas (CRISPR-associated) proteins mediates the different stages of this sophisticated immune response. Bioinformatic studies have classified the Cas proteins into families, according to their sequences and respective functions. These range from the insertion of the foreign genetic elements into the host genome to the activation of the interference machinery as well as target degradation upon attack. Cas7 family proteins are central to the type I and type III interference machineries as they constitute the backbone of the large interference complexes. Here we report the crystal structure of <i>Thermofilum pendens</i> Csc2, a Cas7 family protein of type I-D. We found that Csc2 forms a core RRM-like domain, flanked by three peripheral insertion domains: a lid domain, a Zinc-binding domain and a helical domain. Comparison with other Cas7 family proteins reveals a set of similar structural features both in the core and in the peripheral domains, despite the absence of significant sequence similarity. <i>T. pendens</i> Csc2 binds single-stranded RNA in vitro in a sequence-independent manner. Using a crosslinking - mass-spectrometry approach, we mapped the RNA-binding surface to a positively charged surface patch on <i>T. pendens</i> Csc2. Thus our analysis of the key structural and functional features of <i>T. pendens</i> Csc2 highlights recurring themes and evolutionary relationships in type I and type III Cas proteins.</p></div