Specific recognition and ubiquitination of translating ribosomes by mammalian CCR4-NOT.

Translation affects messenger RNA stability and, in yeast, this is mediated by the Ccr4-Not deadenylation complex. The details of this process in mammals remain unclear. Here, we use cryogenic electron microscopy (cryo-EM) and crosslinking mass spectrometry to show that mammalian CCR4-NOT specifically recognizes ribosomes that are stalled during translation elongation in an in vitro reconstituted system with rabbit and human components.

Gel-Based Analysis of Protein-Nucleic Acid Interactions.

Electrophoretic mobility shift assays (EMSAs) are among the most frequently used and straightforward experiments for studying protein-nucleic acid interactions. EMSAs rely on the principle that protein-nucleic acid complexes have reduced electrophoretic mobility in a native gel matrix compared to free nucleic acid due to their larger size and reduced negative charge. Therefore, bands for the protein-nucleic acid complexes are shifted in a gel and can be distinguished from free nucleic acids.

Author Correction: The intrinsic structure of poly(A) RNA determines the specificity of Pan2 and Caf1 deadenylases.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The intrinsic structure of poly(A) RNA determines the specificity of Pan2 and Caf1 deadenylases.

The 3' poly(A) tail of messenger RNA is fundamental to regulating eukaryotic gene expression. Shortening of the poly(A) tail, termed deadenylation, reduces transcript stability and inhibits translation. Nonetheless, the mechanism for poly(A) recognition by the conserved deadenylase complexes Pan2-Pan3 and Ccr4-Not is poorly understood.

RNA-binding proteins distinguish between similar sequence motifs to promote targeted deadenylation by Ccr4-Not.

The Ccr4-Not complex removes mRNA poly(A) tails to regulate eukaryotic mRNA stability and translation. RNA-binding proteins contribute to specificity by interacting with both Ccr4-Not and target mRNAs, but this is not fully understood. Here, we reconstitute accelerated and selective deadenylation of RNAs containing AU-rich elements (AREs) and Pumilio-response elements (PREs).

mRNA Deadenylation Is Coupled to Translation Rates by the Differential Activities of Ccr4-Not Nucleases.

Translation and decay of eukaryotic mRNAs is controlled by shortening of the poly(A) tail and release of the poly(A)-binding protein Pab1/PABP. The Ccr4-Not complex contains two exonucleases-Ccr4 and Caf1/Pop2-that mediate mRNA deadenylation. Here, using a fully reconstituted biochemical system with proteins from the fission yeast Schizosaccharomyces pombe, we show that Pab1 interacts with Ccr4-Not, stimulates deadenylation, and differentiates the roles of the nuclease enzymes.

A low-complexity region in the YTH domain protein Mmi1 enhances RNA binding.

Mmi1 is an essential RNA-binding protein in the fission yeast that eliminates meiotic transcripts during normal vegetative growth. Mmi1 contains a YTH domain that binds specific RNA sequences, targeting mRNAs for degradation. The YTH domain of Mmi1 uses a noncanonical RNA-binding surface that includes contacts outside the conserved fold.

Analysis of mRNA deadenylation by multi-protein complexes.

Poly(A) tails are found at the 3' end of almost every eukaryotic mRNA and are important for the stability of mRNAs and their translation into proteins. Thus, removal of the poly(A) tail, a process called deadenylation, is critical for regulation of gene expression. Most deadenylation enzymes are components of large multi-protein complexes.

Reconstitution of Targeted Deadenylation by the Ccr4-Not Complex and the YTH Domain Protein Mmi1.

Ccr4-Not is a conserved protein complex that shortens the 3' poly(A) tails of eukaryotic mRNAs to regulate transcript stability and translation into proteins. RNA-binding proteins are thought to facilitate recruitment of Ccr4-Not to certain mRNAs, but lack of an in-vitro-reconstituted system has slowed progress in understanding the mechanistic details of this specificity. Here, we generate a fully recombinant Ccr4-Not complex that removes poly(A) tails from RNA substrates.

Major reorientation of tRNA substrates defines specificity of dihydrouridine synthases.

The reduction of specific uridines to dihydrouridine is one of the most common modifications in tRNA. Increased levels of the dihydrouridine modification are associated with cancer. Dihydrouridine synthases (Dus) from different subfamilies selectively reduce distinct uridines, located at spatially unique positions of folded tRNA, into dihydrouridine.