Mapping targets for small nucleolar RNAs in yeast

Background: Recent analyses implicate changes in the expression of the box C/D class of small nucleolar RNAs (snoRNAs) in several human diseases. Methods: Here we report the identification of potential novel RNA targets for box C/D snoRNAs in budding yeast, using the approach of UV crosslinking and sequencing of hybrids (CLASH) with the snoRNP proteins Nop1, Nop56 and Nop58. We also developed a bioinformatics approach to filter snoRNA-target interactions for bona fide methylation guide interactions. Results: We recovered 241,420 hybrids, out of which 190,597 were classed as reproducible, high energy hybrids. As expected, the majority of snoRNA interactions were with the ribosomal RNAs (rRNAs). Following filtering, 117,047 reproducible hybrids included 51 of the 55 reported rRNA methylation sites. The majority of interactions at methylation sites were predicted to guide methylation. However, competing, potentially regulatory, binding was also identified. In marked contrast, following CLASH performed with the RNA helicase Mtr4 only 7% of snoRNA-rRNA interactions recovered were predicted to guide methylation. We propose that Mtr4 functions in dissociating inappropriate snoRNA-target interactions. Numerous snoRNA-snoRNA interactions were recovered, indicating potential cross regulation. The snoRNAs snR4 and snR45 were recently implicated in site-directed rRNA acetylation, and hybrids were identified adjacent to the acetylation sites. We also identified 1,368 reproducible snoRNA-mRNA interactions, representing 448 sites of interaction involving 39 snoRNAs and 382 mRNAs. Depletion of the snoRNAs U3, U14 or snR4 each altered the levels of numerous mRNAs. Targets identified by CLASH were over-represented among these species, but causality has yet to be established. Conclusions: Systematic mapping of snoRNA-target binding provides a catalogue of high-confidence binding sites and indicates numerous potential regulatory interactions

A new function of GAPDH from Chlamydomonas reinhardtii: a thiol-disulfide exchange reaction with CP12,

International audienc

Mechanisms of partner recognition by intrinsically disordered proteins

10th European-Biophysical-Societies-Association (EBSA) European Biophysics Congress, Dresden, GERMANY, JUL 18-22, 2015International audienceno abstrac

Mechanisms of partner recognition by intrinsically disordered proteins

10th European-Biophysical-Societies-Association (EBSA) European Biophysics Congress, Dresden, GERMANY, JUL 18-22, 2015International audienceno abstrac

Comparative sequence analysis of CP12, a small protein involved in the formation of a Calvin cycle complex in photosynthetic organisms

CP12, a small intrinsically unstructured protein, plays an important role in the regulation of the Calvin cycle by forming a complex with phosphoribulokinase (PRK) and glyceraldehyde-3- phosphate dehydrogenase (GAPDH). An extensive search in databases revealed 129 protein sequences from: higher plants, mosses and liverworts, different groups of eukaryotic algae and cyanobacteria. CP12 was identified throughout the Plantae, apart from in the Prasinophyceae. Within the Chromalveolata, two putative CP12 proteins have been found in the genomes of the diatom Thalassiosira pseudonana and the haptophyte Emiliania huxleyi, but specific searches in further chromalveolate genomes or EST datasets did not reveal any CP12 sequences in other Prymnesiophyceae, Dinophyceae or Pelagophyceae. A species from the Euglenophyceae within the Excavata also appeared to lack CP12. Phylogenetic analysis showed a clear separation into a number of higher taxonomic clades and among different forms of CP12 in higher plants. Cyanobacteria, Chlorophyceae, Rhodophyta & Glaucophyceae, Bryophyta, and the CP12-3 forms in higher plants all form separate clades. The degree of disorder of CP12 was higher in higher plants than in eukaryotic algae and cyanobacteria apart from the green algal class Mesostigmatophyceae that is ancestral to the streptophytes. This suggests that CP12 has evolved to become more flexible and possibly take on more general roles. Different features of the CP12 sequences in the different taxonomic groups and their potential functions and interactions in the Calvin cycle are discussed