Translation fidelity and efficiency require multiple ribosomal (r)RNA modifications that are mostly mediated by small nucleolar (sno)RNPs during ribosome production. Overlapping basepairing of snoRNAs with pre-rRNAs often necessitates sequential and efficient association and dissociation of the snoRNPs, however, how such hierarchy is established has remained unknown so far. Here, we identify several late-acting snoRNAs that bind pre-40S particles in human cells and show that their association and function in pre-40S complexes is regulated by the RNA helicase DDX21. We map DDX21 crosslinking sites on pre-rRNAs and show their overlap with the basepairing sites of the affected snoRNAs. While DDX21 activity is required for recruitment of the late-acting snoRNAs SNORD56 and SNORD68, earlier snoRNAs are not affected by DDX21 depletion. Together, these observations provide an understanding of the timing and ordered hierarchy of snoRNP action in pre-40S maturation and reveal a novel mode of regulation of snoRNP function by an RNA helicase in human cells.

Cysteine modifications emerge as important players in cellular signaling and homeostasis. Here, we present a chemical proteomics strategy for quantitative analysis of reversibly modified Cysteines using bioorthogonal cleavable-linker and switch technique (Cys-BOOST). Compared to iodoTMT for total Cysteine analysis, Cys-BOOST shows a threefold higher sensitivity and considerably higher specificity and precision. Analyzing S-nitrosylation (SNO) in S-nitrosoglutathione (GSNO)-treated and non-treated HeLa extracts Cys-BOOST identifies 8,304 SNO sites on 3,632 proteins covering a wide dynamic range of the proteome. Consensus motifs of SNO sites with differential GSNO reactivity confirm the relevance of both acid-base catalysis and local hydrophobicity for NO targeting to particular Cysteines. Applying Cys-BOOST to SH-SY5Y cells, we identify 2,151 SNO sites under basal conditions and reveal significantly changed SNO levels as response to early nitrosative stress, involving neuro(axono)genesis, glutamatergic synaptic transmission, protein folding/translation, and DNA replication. Our work suggests SNO as a global regulator of protein function akin to phosphorylation and ubiquitination.

No abstract

Study of the bacterial membrane proteome is a field of growing interest in the research of nutrient transport and processing. Pseudomonas sp. strain phDV1, a Gram-negative bacterium selected for its ability to degrade aromatic compounds, was monitored under different growth substrate conditions, using lysogeny broth medium (LB), glucose, and phenol as sole carbon source. The aim of this study was to characterize the membrane subproteomes of the Pseudomonas strain by proteomic means to assess the protein composition of this subcellular compartments, which appears fundamental for the biodegradation of aromatic compounds. A total number of 129 different proteins have been identified by MALDI-TOF/TOF, 19 of which are membrane proteins that belong to the inner membrane and 10 that belong to the outer membrane. Two membrane proteins were only expressed in the presence of the aromatic substrate. We identified a membrane protein involved in aromatic hydrocarbon degradation as well as a probable porin which may, in fact, function as an aromatic compound-specific porin. Although the presence of different transporters have been reported for different aromatic compounds such as toluene and benzoic acid, to our knowledge, these are the first phenol-inducible membrane transporters identified.