20 research outputs found
Purification of cross-linked RNA-protein complexes by phenol-toluol extraction
Recent methodological advances allowed the identification of an increasing number of RNA-binding proteins (RBPs) and their RNA-binding sites. Most of those methods rely, however, on capturing proteins associated to polyadenylated RNAs which neglects RBPs bound to non-adenylate RNA classes (tRNA, rRNA, pre-mRNA) as well as the vast majority of species that lack poly-A tails in their mRNAs (including all archea and bacteria). We have developed the Phenol Toluol extraction (PTex) protocol that does not rely on a specific RNA sequence or motif for isolation of cross-linked ribonucleoproteins (RNPs), but rather purifies them based entirely on their physicochemical properties. PTex captures RBPs that bind to RNA as short as 30 nt, RNPs directly from animal tissue and can be used to simplify complex workflows such as PAR-CLIP. Finally, we provide a global RNA-bound proteome of human HEK293 cells and the bacterium Salmonella Typhimurium
Evidence for a cytoplasmic pool of ribosome-free mRNAs encoding inner membrane proteins in Escherichia coli
The cysteineâfree single mutant C32S of APEX2 is a highly expressed and active fusion tag for proximity labeling applications
Multiple capsid protein binding sites mediate selective packaging of the alphavirus genomic RNA
Proximity-CLIP provides a snapshot of protein-occupied RNA elements in subcellular compartments
Identification of functional tetramolecular RNA G-quadruplexes derived from transfer RNAs
Novel biomarker ZCCHC13 revealed by integrating DNA methylation and mRNA expression data in non-obstructive azoospermia
N6-Methyladenosine methyltransferase ZCCHC4 mediates ribosomal RNA methylation
International audienceN6-Methyladenosine (m6A) RNA modification is present in messenger RNAs (mRNA), ribosomal RNAs (rRNA), and spliceosomal RNAs (snRNA) in humans. Although mRNA m6A modifications have been extensively studied and shown to play critical roles in many cellular processes, the identity of m6A methyltransferases for rRNAs and the function of rRNA m6A modifications are unknown. Here we report a new m6A methyltransferase, ZCCHC4, which primarily methylates human 28S rRNA and also interacts with a subset of mRNAs. ZCCHC4 knockout eliminates m6A4220 modification in 28S rRNA, reduces global translation, and inhibits cell proliferation. We also find that ZCCHC4 protein is overexpressed in hepatocellular carcinoma tumors, and ZCCHC4 knockout significantly reduces tumor size in a xenograft mouse model. Our results highlight the functional significance of an rRNA m6A modification in translation and in tumor biology