The pentatricopeptide repeat (PPR) is a helical repeat motif found in an exceptionally large family of RNA–binding proteins that functions in mitochondrial and chloroplast gene expression. PPR proteins harbor between 2 and 30 repeats and typically bind single-stranded RNA in a sequence-specific fashion. However, the basis for sequence-specific RNA recognition by PPR tracts has been unknown. We used computational methods to infer a code for nucleotide recognition involving two amino acids in each repeat, and we validated this model by recoding a PPR protein to bind novel RNA sequences in vitro. Our results show that PPR tracts bind RNA via a modular recognition mechanism that differs from previously described RNA–protein recognition modes and that underpins a natural library of specific protein/RNA partners of unprecedented size and diversity. These findings provide a significant step toward the prediction of native binding sites of the enormous number of PPR proteins found in nature. Furthermore, the extraordinary evolutionary plasticity of the PPR family suggests that the PPR scaffold will be particularly amenable to redesign for new sequence specificities and functions.

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A Combinatorial Amino Acid Code for RNA Recognition by Pentatricopeptide Repeat Proteins

Long non-coding RNAs (lncRNAs) are associated to a plethora of cellular functions, most of which require the interaction with one or more RNA-binding proteins (RBPs); similarly, RBPs are often able to bind a large number of different RNAs. The currently available knowledge is already drawing an intricate network of interactions, whose deregulation is frequently associated to pathological states. Several different techniques were developed in the past years to obtain protein–RNA binding data in a high-throughput fashion. In parallel, in silico inference methods were developed for the accurate computational prediction of the interaction of RBP–lncRNA pairs. The field is growing rapidly, and it is foreseeable that in the near future, the protein–lncRNA interaction network will rise, offering essential clues for a better understanding of lncRNA cellular mechanisms and their disease-associated perturbations.

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Revealing protein–lncRNA interaction

3′-untranslated regions (3′-UTRs) are the noncoding parts of mRNAs. Compared to yeast, in humans, median 3′-UTR length has expanded approximately tenfold alongside an increased generation of alternative 3′-UTR isoforms. In contrast, the number of coding genes, as well as coding region length, has remained similar. This suggests an important role for 3′-UTRs in the biology of higher organisms. 3′-UTRs are best known to regulate diverse fates of mRNAs, including degradation, translation, and localization, but they can also function like long noncoding or small RNAs, as has been shown for whole 3′-UTRs as well as for cleaved fragments. Furthermore, 3′-UTRs determine the fate of proteins through the regulation of protein–protein interactions. They facilitate cotranslational protein complex formation, which establishes a role for 3′-UTRs as evolved eukaryotic operons. Whereas bacterial operons promote the interaction of two subunits, 3′-UTRs enable the formation of protein complexes with diverse compositions. ...

Regulation by 3'-Untranslated Regions.

RNA Bind-n-Seq: Quantitative Assessment of the Sequence and Structural Binding Specificity of RNA Binding Proteins

3' untranslated regions (3' UTRs) of messenger RNAs (mRNAs) are best known to regulate mRNA-based processes, such as mRNA localization, mRNA stability, and translation In addition, 3' UTRs can establish 3' UTR-mediated protein-protein interactions (PPIs), and thus can transmit genetic information encoded in 3' UTRs to proteins This function has been shown to regulate diverse protein features, including protein complex formation or posttranslational modifications, but is also expected to alter protein conformations Therefore, 3' UTR-mediated information transfer can regulate protein features that are not encoded in the amino acid sequence This review summarizes both 3' UTR functions-the regulation of mRNA and protein-based processes-and highlights how each 3' UTR function was discovered with a focus on experimental approaches used and the concepts that were learned This review also discusses novel approaches to study 3' UTR functions in the future by taking advantage of recent advances in technology

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What Are 3′ UTRs Doing?

https://biochem.wisc.edu/sites/default/files/labs/wickens/pdfs/91.pdf

Cooperativity in RNA-Protein Interactions: Global Analysis of RNA Binding Specificity

Programmable protein scaffolds that target DNA are invaluable tools for genome engineering and designer control of transcription. RNA manipulation provides broad new opportunities for control, including changes in translation. PUF proteins are an attractive platform for that purpose because they bind specific single-stranded RNA sequences by using short repeated modules, each contributing three amino acids that contact an RNA base. Here, we identified the specificities of natural and designed combinations of those three amino acids, using a large randomized RNA library. The resulting specificity code reveals the RNA binding preferences of natural proteins and enables the design of new specificities. Using the code and a translational activation domain, we designed a protein that targets endogenous cyclin B1 mRNA in human cells, increasing sensitivity to chemotherapeutic drugs. Our study provides a guide for rational design of engineered mRNA control, including translational stimulation.

/pdf/a-protein-rna-specificity-code-enables-targeted-activation-1pj3u5bikq.pdf

A protein-RNA specificity code enables targeted activation of an endogenous human transcript

mRNA control hinges on the specificity and affinity of proteins for their RNA binding sites. Regulatory proteins must bind their own sites and reject even closely related noncognate sites. In the PUF [Pumilio and fem-3 binding factor (FBF)] family of RNA binding proteins, individual proteins discriminate differences in the length and sequence of binding sites, allowing each PUF to bind a distinct battery of mRNAs. Here, we show that despite these differences, the pattern of RNA interactions is conserved among PUF proteins: the two ends of the PUF protein make critical contacts with the two ends of the RNA sites. Despite this conserved “two-handed” pattern of recognition, the RNA sequence is flexible. Among the binding sites of yeast Puf4p, RNA sequence dictates the pattern in which RNA bases are flipped away from the binding surface of the protein. Small differences in RNA sequence allow new modes of control, recruiting Puf5p in addition to Puf4p to a single site. This embedded information adds a new layer of biological meaning to the connections between RNA targets and PUF proteins.

/pdf/patterns-and-plasticity-in-rna-protein-interactions-enable-43ju5vz6z0.pdf

Cary T. Valley

Papers

Cooperativity in RNA-Protein Interactions: Global Analysis of RNA Binding Specificity

A protein-RNA specificity code enables targeted activation of an endogenous human transcript

Patterns and plasticity in RNA-protein interactions enable recruitment of multiple proteins through a single site