A comprehensive thermodynamic model for RNA binding by the Saccharomyces cerevisiae Pumilio protein PUF4.

Genomic methods have been valuable for identifying RNA-binding proteins (RBPs) and the genes, pathways, and processes they regulate. Nevertheless, standard motif descriptions cannot be used to predict all RNA targets or test quantitative models for cellular interactions and regulation. We present a complete thermodynamic model for RNA binding to the S.

How to measure and evaluate binding affinities.

Quantitative measurements of biomolecule associations are central to biological understanding and are needed to build and test predictive and mechanistic models. Given the advances in high-throughput technologies and the projected increase in the availability of binding data, we found it especially timely to evaluate the current standards for performing and reporting binding measurements. A review of 100 studies revealed that in most cases essential controls for establishing the appropriate incubation time and concentration regime were not documented, making it impossible to determine measurement reliability.

A Quantitative and Predictive Model for RNA Binding by Human Pumilio Proteins.

High-throughput methodologies have enabled routine generation of RNA target sets and sequence motifs for RNA-binding proteins (RBPs). Nevertheless, quantitative approaches are needed to capture the landscape of RNA-RBP interactions responsible for cellular regulation. We have used the RNA-MaP platform to directly measure equilibrium binding for thousands of designed RNAs and to construct a predictive model for RNA recognition by the human Pumilio proteins PUM1 and PUM2.

Blind tests of RNA-protein binding affinity prediction.

Interactions between RNA and proteins are pervasive in biology, driving fundamental processes such as protein translation and participating in the regulation of gene expression. Modeling the energies of RNA-protein interactions is therefore critical for understanding and repurposing living systems but has been hindered by complexities unique to RNA-protein binding. Here, we bring together several advances to complete a calculation framework for RNA-protein binding affinities, including a unified free energy function for bound complexes, automated Rosetta modeling of mutations, and use of secondary structure-based energetic calculations to model unbound RNA states.

Demonstration of protein cooperativity mediated by RNA structure using the human protein PUM2.

Posttranslational gene regulation requires a complex network of RNA-protein interactions. Cooperativity, which tunes response sensitivities, originates from protein-protein interactions in many systems. For RNA-binding proteins, cooperativity can also be mediated through RNA structure.

Lso2 is a conserved ribosome-bound protein required for translational recovery in yeast.

Ribosome-binding proteins function broadly in protein synthesis, gene regulation, and cellular homeostasis, but the complete complement of functional ribosome-bound proteins remains unknown. Using quantitative mass spectrometry, we identified late-annotated short open reading frame 2 (Lso2) as a ribosome-associated protein that is broadly conserved in eukaryotes. Genome-wide crosslinking and immunoprecipitation of Lso2 and its human ortholog coiled-coil domain containing 124 (CCDC124) recovered 25S ribosomal RNA in a region near the A site that overlaps the GTPase activation center.

Pseudouridine and -methyladenosine modifications weaken PUF protein/RNA interactions.

RNA modifications are ubiquitous in biology, with over 100 distinct modifications. While the vast majority were identified and characterized on abundant noncoding RNA such as tRNA and rRNA, the advent of sensitive sequencing-based approaches has led to the discovery of extensive and regulated modification of eukaryotic messenger RNAs as well. The two most abundant mRNA modifications-pseudouridine (Ψ) and -methyladenosine (mA)-affect diverse cellular processes including mRNA splicing, localization, translation, and decay and modulate RNA structure.

Protein kinase A regulates gene-specific translational adaptation in differentiating yeast.

Cellular differentiation is driven by coordinately regulated changes in gene expression. Recent discoveries suggest that translation contributes as much as transcription to regulating protein abundance, but the role of translational regulation in cellular differentiation is largely unexplored. Here we investigate translational reprogramming in yeast during cellular adaptation to the absence of glucose, a stimulus that induces invasive filamentous differentiation.

RluD, a highly conserved pseudouridine synthase, modifies 50S subunits more specifically and efficiently than free 23S rRNA.

Pseudouridine modifications in helix 69 (H69) of 23S ribosomal RNA are highly conserved among all organisms. H69 associates with helix 44 of 16S rRNA to form bridge B2a, which plays a vital role in bridging the two ribosomal subunits and stabilizing the ribosome. The three pseudouridines in H69 were shown earlier to play an important role in 50S subunit assembly and in its association with the 30S subunit.