Identification of RNAs bound by Hfq reveals widespread RNA partners and a sporulation regulator in the human pathogen Clostridioides difficile.

Noncoding RNAs (ncRNA) have emerged as important components of regulatory networks governing bacterial physiology and virulence. Previous deep-sequencing analysis identified a large diversity of ncRNAs in the human enteropathogen Clostridioides (Clostridium) difficile. Some of them are trans-encoded RNAs that could require the RNA chaperone protein Hfq for their action. Recent analysis suggested a pleiotropic role of Hfq in C. difficile with the most pronounced effect on sporulation, a key process during the infectious cycle of this pathogen. However, a global view of RNAs interacting with C. difficile Hfq is missing. In the present study, we performed RNA immunoprecipitation high-throughput sequencing (RIP-Seq) to identify Hfq-associated RNAs in C. difficile. Our work revealed a large set of Hfq-interacting mRNAs and ncRNAs, including mRNA leaders and coding regions, known and potential new ncRNAs. In addition to trans-encoded RNAs, new categories of Hfq ligands were found including cis-antisense RNAs, riboswitches and CRISPR RNAs. ncRNA-mRNA and ncRNA-ncRNA pairings were postulated through computational predictions. Investigation of one of the Hfq-associated ncRNAs, RCd1, suggests that this RNA contributes to the control of late stages of sporulation in C. difficile. Altogether, these data provide essential molecular basis for further studies of post-transcriptional regulatory network in this enteropathogen.

cBAF generates subnucleosomes that expand OCT4 binding and function beyond DNA motifs at enhancers.

The canonical BRG/BRM-associated factor (cBAF) complex is essential for chromatin opening at enhancers in mammalian cells. However, the nature of the open chromatin remains unclear. Here, we show that, in addition to producing histone-free DNA, cBAF generates stable hemisome-like subnucleosomal particles containing the four core histones associated with 50-80 bp of DNA. Our genome-wide analysis indicates that cBAF makes these particles by targeting and splitting fragile nucleosomes. In mouse embryonic stem cells, these subnucleosomes become an in vivo binding substrate for the master transcription factor OCT4 independently of the presence of OCT4 DNA motifs. At enhancers, the OCT4-subnucleosome interaction increases OCT4 occupancy and amplifies the genomic interval bound by OCT4 by up to one order of magnitude compared to the region occupied on histone-free DNA. We propose that cBAF-dependent subnucleosomes orchestrate a molecular mechanism that projects OCT4 function in chromatin opening beyond its DNA motifs.

Homozygous iMycCα transgenic mice as a model of plasma B-cell lymphomas.

The 3' regulatory region (3'RR) located downstream from the Cα gene is the conductor of transcription, accessibility, and remodeling of the IgH locus at mature B-cell stages. Convincing demonstrations of the essential contributions of the 3'RR in B-cell lymphomagenesis have been provided by mouse models which bring the oncogene c-Myc under the 3'RR transcriptional control. In this study, we developed a mouse model of CD138+ plasma B-cell lymphomas. If the KI of c-myc directly into Cα just 5' to the 3'RR in iMycCα mice produced B-cell lymphomas with low kinetics, we enforced c-myc production in iMycCα mice by the generation of homozygous c-myc transgenic mice. Our results show that homozygous iMycCα mice lead to a mouse model of plasma CD138+ B-cell lymphomas with interesting and wide transcriptomic similarities to human multiple myeloma and appropriated emergence kinetics that can be used to test new experimental therapeutic approaches.

DE-kupl: exhaustive capture of biological variation in RNA-seq data through k-mer decomposition.

We introduce a k-mer-based computational protocol, DE-kupl, for capturing local RNA variation in a set of RNA-seq libraries, independently of a reference genome or transcriptome. DE-kupl extracts all k-mers with differential abundance directly from the raw data files. This enables the retrieval of virtually all variation present in an RNA-seq data set. This variation is subsequently assigned to biological events or entities such as differential long non-coding RNAs, splice and polyadenylation variants, introns, repeats, editing or mutation events, and exogenous RNA. Applying DE-kupl to human RNA-seq data sets identified multiple types of novel events, reproducibly across independent RNA-seq experiments.

RNA-binding proteins are a major target of silica nanoparticles in cell extracts.

Upon contact with biological fluids, nanoparticles (NPs) are readily coated by cellular compounds, particularly proteins, which are determining factors for the localization and toxicity of NPs in the organism. Here, we improved a methodological approach to identify proteins that adsorb on silica NPs with high affinity. Using large-scale proteomics and mixtures of soluble proteins prepared either from yeast cells or from alveolar human cells, we observed that proteins with large unstructured region(s) are more prone to bind on silica NPs. These disordered regions provide flexibility to proteins, a property that promotes their adsorption. The statistical analyses also pointed to a marked overrepresentation of RNA-binding proteins (RBPs) and of translation initiation factors among the adsorbed proteins. We propose that silica surfaces, which are mainly composed of Si-O- and Si-OH groups, mimic ribose-phosphate molecules (rich in -O- and -OH) and trap the proteins able to interact with ribose-phosphate containing molecules. Finally, using an in vitro assay, we showed that the sequestration of translation initiation factors by silica NPs results in an inhibition of the in vitro translational activity. This result demonstrates that characterizing the protein corona of various NPs would be a relevant approach to predict their potential toxicological effects.