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

Hrle, Ajla; Su, Andreas A. H.; Ebert, J.; Benda, Christian; Randau, Lennart; Conti, Elena

doi:10.4161/rna.26500

Cited by 34 publications

(47 citation statements)

References 45 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The cross-linking sites within T. pendens Cas7 encircle a positively charged groove and biochemical analysis demonstrated that conserved residues in this groove contribute significantly to RNA binding [24]. Moreover, the location of the cross-linked residues within the predicted insertion domain 1 of the proteins T. tenax Cas7 and T. thermophilus Csm3 are in full agreement with previous studies on the respective Type I-A and III-A homologs, Sulfolobus solfataricus Cas7 and Methanopyrus kandleri Csm3 [23,52].…”

Section: Cross-link Sites On the Structural Model Of Cas7 Proteinssupporting

confidence: 88%

“…In vivo, several copies of Cas7 proteins are wrapped around crRNA in a sequence-unspecific helical fashion [5,30,50,51]. Crystal structures from single and complex-bound Cas7 proteins show two composite RNA-binding surfaces: a central cleft and a structurally variable insertion domain [5,23,24,52]. In all Cas7 proteins characterized to date both these domains are defined by insertions within the secondary structure elements of the central RRM domain (insertion domain 1 is b1-a1, b2-b3, a2-b4, and insertion domain 2 is a1-b2).…”

Section: Mapping the Rna Binding Interface In Cas7 Proteinsmentioning

confidence: 99%

“…These do not show primary RNA-binding sequence motifs that resemble those of eukaryotic proteins. Nonetheless, three-dimensional structures of bacterial RBPs are similar to structures of eukaryotic RBDs, for example, the bacterial HfQ protein with the characteristic Sm fold [21,22] and the prokaryotic Cas7 protein family with their RRM motifs [23,24].…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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

Sharma

Hrle

Kramer

et al. 2015

Methods

Self Cite

View full text Add to dashboard Cite

a b s t r a c tRibonucleoprotein (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 singleand 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.

show abstract

Section: Cross-link Sites On the Structural Model Of Cas7 Proteinssupporting

confidence: 88%

Section: Mapping the Rna Binding Interface In Cas7 Proteinsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

Sharma

Hrle

Kramer

et al. 2015

Methods

Self Cite

View full text Add to dashboard Cite

show abstract

“…As a result of these flanking elements, Cmr4 has a rather elongated structure (about 60 3 25 Å ), with an extended b sheet effectively sandwiched between two layers of a helices. Structural comparison with Mk Csm3 (Hrle et al, 2013), the putative Cmr4-like subunit in type III-A effector complexes, shows large differences in the structural elements that sandwich the central b sheet.…”

Section: Cmr4 and Cmr5 Assemble Into Backbone-like Superhelical Strucmentioning

confidence: 99%

“…Interestingly, not only is Pf D26 Cmr4 conserved in Cmr orthologs from type III-B systems ( Figures 5D and S4A), but it is also one of the few amino acid residues to be conserved in the Csm3 proteins (e.g., Mk D35 Csm3 ; Hrle et al, 2013), the putative backbone protein in type III-A systems (Figures 5D and S4B). …”

Section: The Nuclease Active Site Of the Cmr Complex Resides In Cmr4mentioning

confidence: 99%

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

et al. 2014

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Annotation and Classification of CRISPR-Cas Systems

Makarova

Koonin

2015

Methods in Molecular Biology

352

271

View full text Add to dashboard Cite

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins) is a prokaryotic adaptive immune system that is represented in most archaea and many bacteria. Among the currently known prokaryotic defense systems, the CRISPR-Cas genomic loci show unprecedented complexity and diversity. Classification of CRISPR-Cas variants that would capture their evolutionary relationships to the maximum possible extent is essential for comparative genomic and functional characterization of this theoretically and practically important system of adaptive immunity. To this end, a multipronged approach has been developed that combines phylogenetic analysis of the conserved Cas proteins with comparison of gene repertoires and arrangements in CRISPR-Cas loci. This approach led to the current classification of CRISPR-Cas systems into three distinct types and ten subtypes for each of which signature genes have been identified. Comparative genomic analysis of the CRISPR-Cas systems in new archaeal and bacterial genomes performed over the 3 years elapsed since the development of this classification makes it clear that new types and subtypes of CRISPR-Cas need to be introduced. Moreover, this classification system captures only part of the complexity of CRISPR-Cas organization and evolution, due to the intrinsic modularity and evolutionary mobility of these immunity systems, resulting in numerous recombinant variants. Moreover, most of the cas genes evolve rapidly, complicating the family assignment for many Cas proteins and the use of family profiles for the recognition of CRISPR-Cas subtype signatures. Further progress in the comparative analysis of CRISPR-Cas systems requires integration of the most sensitive sequence comparison tools, protein structure comparison, and refined approaches for comparison of gene neighborhoods.

show abstract

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

Cited by 34 publications

References 45 publications

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

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

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

Annotation and Classification of CRISPR-Cas Systems

Contact Info

Product

Resources

About