Katharina Kramer

Photo-cross-linking and high-resolution mass spectrometry for assignment of RNA-binding sites in RNA-binding proteins

RNA-protein complexes play pivotal roles in many central biological processes. Although methods based on high-throughput sequencing have advanced our ability to identify the specific RNAs bound by a particular protein, there is a need for precise and systematic ways to identify RNA interaction sites on proteins. We have developed an experimental and computational workflow combining photo-induced cross-linking, high-resolution mass spectrometry and automated analysis of the resulting mass spectra for the identification of cross-linked peptides, cross-linking sites and the cross-linked RNA oligonucleotide moieties of such RNA-binding proteins. The workflow can be applied to any RNA-protein complex of interest or to whole proteomes. We applied the approach to human and yeast mRNA-protein complexes in vitro and in vivo, demonstrating its powerful utility by identifying 257 cross-linking sites on 124 distinct RNA-binding proteins. The open-source software pipeline developed for this purpose, RNPxl, is available as part of the OpenMS project.

A cytoplasmic complex mediates specific mRNA recognition and localization in yeast.

The localization of ash mRNA in yeast requires the binding of She2p and the myosin adaptor protein She3p to its localization element, which is highly specific and leads to the assembly of stable transport complexes.

The NHL domain of BRAT is an RNA-binding domain that directly contacts the hunchback mRNA for regulation.

The Drosophila protein brain tumor (Brat) forms a complex with Pumilio (Pum) and Nanos (Nos) to repress hunchback (hb) mRNA translation at the posterior pole during early embryonic development. It is currently thought that complex formation is initiated by Pum, which directly binds the hb mRNA and subsequently recruits Nos and Brat. Here we report that, in addition to Pum, Brat also directly interacts with the hb mRNA. We identify Brat-binding sites distinct from the Pum consensus motif and show that RNA binding and translational repression by Brat do not require Pum, suggesting so far unrecognized Pum-independent Brat functions. Using various biochemical and biophysical methods, we also demonstrate that the NHL (NCL-1, HT2A, and LIN-41) domain of Brat, a domain previously believed to mediate protein-protein interactions, is a novel, sequence-specific ssRNA-binding domain. The Brat-NHL domain folds into a six-bladed β propeller, and we identify its positively charged top surface as the RNA-binding site. Brat belongs to the functional diverse TRIM (tripartite motif)-NHL protein family. Using structural homology modeling, we predict that the NHL domains of all TRIM-NHL proteins have the potential to bind RNA, indicating that Brat is part of a conserved family of RNA-binding proteins.

Investigation of protein–RNA interactions by mass spectrometry—Techniques and applications ☆

Protein-RNA complexes play many important roles in diverse cellular functions. They are involved in a wide variety of different processes in growth and differentiation at the various stages of the cell cycle. As their function and catalytic activity are directly coupled to the structural arrangement of their components--proteins and ribonucleic acids--the investigation of protein-RNA interactions is of great functional and structural importance. Here we discuss the most prominent examples of protein-RNA complexes and describe some frequently used purification strategies. We present various techniques and applications of mass spectrometry to study protein-RNA complexes. We discuss the analysis of intact complexes as well as proteomics-based and crosslinking-based approaches in which proteins are cleaved into smaller peptides. This article is part of a Special Section entitled: Understanding genome regulation and genetic diversity by mass spectrometry.

Crystal structure of Cwc2 reveals a novel architecture of a multipartite RNA‐binding protein

The yeast splicing factor Cwc2 contacts several catalytically important RNA elements in the active spliceosome, suggesting that Cwc2 is involved in determining their spatial arrangement at the spliceosomes catalytic centre. We have determined the crystal structure of the Cwc2 functional core, revealing how a previously uncharacterized Torus domain, an RNA recognition motif (RRM) and a zinc finger (ZnF) are tightly integrated in a compact folding unit. The ZnF plays a pivotal role in the architecture of the whole assembly. UV‐induced crosslinking of Cwc2–U6 snRNA allowed the identification by mass spectrometry of six RNA‐contacting sites: four in or close to the RRM domain, one in the ZnF and one on a protruding element connecting the Torus and RRM domains. The three distinct regions contacting RNA are connected by a contiguous and conserved positively charged surface, suggesting an expanded interface for RNA accommodation. Cwc2 mutations confirmed that the connector element plays a crucial role in splicing. We conclude that Cwc2 acts as a multipartite RNA‐binding platform to bring RNA elements of the spliceosomes catalytic centre into an active conformation.

Lysine acetylome profiling uncovers novel histone deacetylase substrate proteins in Arabidopsis

Histone deacetylases have central functions in regulating stress defenses and development in plants. However, the knowledge about the deacetylase functions is largely limited to histones, although these enzymes were found in diverse subcellular compartments. In this study, we determined the proteome‐wide signatures of the RPD3/HDA1 class of histone deacetylases in Arabidopsis. Relative quantification of the changes in the lysine acetylation levels was determined on a proteome‐wide scale after treatment of Arabidopsis leaves with deacetylase inhibitors apicidin and trichostatin A. We identified 91 new acetylated candidate proteins other than histones, which are potential substrates of the RPD3/HDA1‐like histone deacetylases in Arabidopsis, of which at least 30 of these proteins function in nucleic acid binding. Furthermore, our analysis revealed that histone deacetylase 14 (HDA14) is the first organellar‐localized RPD3/HDA1 class protein found to reside in the chloroplasts and that the majority of its protein targets have functions in photosynthesis. Finally, the analysis of HDA14 loss‐of‐function mutants revealed that the activation state of RuBisCO is controlled by lysine acetylation of RuBisCO activase under low‐light conditions.

The SAGA complex in the rice pathogen Fusarium fujikuroi: structure and functional characterization

Post‐translational modification of histones is a crucial mode of transcriptional regulation in eukaryotes. A well‐described acetylation modifier of certain lysine residues is the Spt‐Ada‐Gcn5 acetyltransferase (SAGA) complex assembled around the histone acetyltransferase Gcn5 in Saccharomyces cerevisiae. We identified and characterized the SAGA complex in the rice pathogen Fusarium fujikuroi, well‐known for producing a large variety of secondary metabolites (SMs). By using a co‐immunoprecipitation approach, almost all of the S. cerevisiae SAGA complex components have been identified, except for the ubiquitinating DUBm module and the chromodomain containing Chd1. Deletion of GCN5 led to impaired growth, loss of conidiation and alteration of SM biosynthesis. Furthermore, we show that Gcn5 is essential for the acetylation of several histone 3 lysines in F. fujikuroi, that is, H3K4, H3K9, H3K18 and H3K27. A genome‐wide microarray analysis revealed differential expression of about 30% of the genome with an enrichment of genes involved in primary and secondary metabolism, transport and histone modification. HPLC‐based analysis of known SMs revealed significant alterations in the Δgcn5 mutant. While most SM genes were activated by Gcn5 activity, the biosynthesis of the pigment bikaverin was strongly increased upon GCN5 deletion underlining the diverse roles of the SAGA complex in F. fujikuroi.

A practice-based clinical evaluation of the survival and success of metal-ceramic and zirconia molar crowns: 5-year results

This practice-based study evaluates the survival and success of conventionally luted metal-ceramic and zirconia molar crowns fabricated by using a prolonged cooling period for the veneering porcelain. Fifty-three patients were treated from 07/2008 to 07/2009 with either metal-ceramic crowns (MCC) or zirconia crowns (ZC). Forty-five patients (26 female) with 91 restorations (obser-vational period: 64.0 ± 4.8 months) participated in a clinical follow-up examination and were included in the study. Estimated cumulative survival (ECSv), success (ECSc) and veneering ceramic success (ECVCSc) were calculated (Kaplan-Meier) and analysed by the crown fabrication technique and the position of the restoration (Cox regression model) (P < 0.05). Five complete failures (MCC: 2, ZC: 3) were recorded (5-year ECSv: MCC: 97.6%, (95% confidence interval (95%-CI): [93%; 100%]/ZC: 94.0%, (95%-CI): [87%; 100%]). Of the MCCs (n = 41), 85.0%, [95%-CI: (77%; 96%)] remained event-free, whereas the ECSc for the ZCs (n = 50) was 74.3% (95%-CI): [61%; 87%]. No significant differences in ECSv (P = 0.51), ECSc (P = 0.43) and ECVCSc (P = 0.36) were detected between the two fabrication techniques. Restorations placed on terminal abutments (n = 44) demonstrated a significantly lower ECVCSc (P = 0.035), (5-year VCF-rate: 14.8%) than crowns placed on tooth-neighboured abutments (n = 47), (5-year VCF-rate: 4.3%). In the present study, zirconia molar crowns demonstrated a 5-year ECSv, ECSc and ECVCSc comparable to MCCs. Irrespective of the fabrication technique, crowns on terminal abutments bear a significantly increased risk for VCFs. Clinical investigations with an increased number of restorations are needed.

Sequence Polymorphisms at the REDUCED DORMANCY5 Pseudophosphatase Underlie Natural Variation in Arabidopsis Dormancy

DELAY OF GERMINATION18 QTL encodes a pseudophosphatase, involved in seed dormancy regulation. Seed dormancy controls the timing of germination, which regulates the adaptation of plants to their environment and influences agricultural production. The time of germination is under strong natural selection and shows variation within species due to local adaptation. The identification of genes underlying dormancy quantitative trait loci is a major scientific challenge, which is relevant for agricultural and ecological goals. In this study, we describe the identification of the DELAY OF GERMINATION18 (DOG18) quantitative trait locus, which was identified as a factor in natural variation for seed dormancy in Arabidopsis (Arabidopsis thaliana). DOG18 encodes a member of the clade A of the type 2C protein phosphatases family, which we previously identified as the REDUCED DORMANCY5 (RDO5) gene. DOG18/RDO5 shows a relatively high frequency of loss-of-function alleles in natural accessions restricted to northwestern Europe. The loss of dormancy in these loss-of-function alleles can be compensated for by genetic factors like DOG1 and DOG6, and by environmental factors such as low temperature. RDO5 does not have detectable phosphatase activity. Analysis of the phosphoproteome in dry and imbibed seeds revealed a general decrease in protein phosphorylation during seed imbibition that is enhanced in the rdo5 mutant. We conclude that RDO5 acts as a pseudophosphatase that inhibits dephosphorylation during seed imbibition.

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.