Christopher P. Lapointe

RNA regulatory networks diversified through curvature of the PUF protein scaffold.

Proteins bind and control mRNAs, directing their localization, translation and stability. Members of the PUF family of RNA-binding proteins control multiple mRNAs in a single cell, and play key roles in development, stem cell maintenance and memory formation. Here we identified the mRNA targets of a S. cerevisiae PUF protein, Puf5p, by ultraviolet-crosslinking-affinity purification and high-throughput sequencing (HITS-CLIP). The binding sites recognized by Puf5p are diverse, with variable spacer lengths between two specific sequences. Each length of site correlates with a distinct biological function. Crystal structures of Puf5p–RNA complexes reveal that the protein scaffold presents an exceptionally flat and extended interaction surface relative to other PUF proteins. In complexes with RNAs of different lengths, the protein is unchanged. A single PUF protein repeat is sufficient to induce broadening of specificity. Changes in protein architecture, such as alterations in curvature, may lead to evolution of mRNA regulatory networks.

The nucleic acid-binding domain and translational repression activity of a Xenopus terminal uridylyl transferase.

Background: TUT7 adds uridines to and regulates RNAs. Results: Xenopus TUT7 (XTUT7) uridylates RNAs, possesses a basic region that binds nucleic acids, and represses translation of a polyadenylated RNA. Conclusion: XTUT7 contains a nucleic acid-binding domain important for activity and can repress translation. Significance: The basic region of XTUT7 may bind RNA in vivo. XTUT7 may control mRNAs by occluding poly(A). Terminal uridylyl transferases (TUTs) catalyze the addition of uridines to the 3′ ends of RNAs and are implicated in the regulation of both messenger RNAs and microRNAs. To better understand how TUTs add uridines to RNAs, we focused on a putative TUT from Xenopus laevis, XTUT7. We determined that XTUT7 catalyzed the addition of uridines to RNAs. Mutational analysis revealed that a truncated XTUT7 enzyme, which contained solely the nucleotidyl transferase and poly(A) polymerase-associated domains, was sufficient for catalytic activity. XTUT7 activity decreased upon removal of the CCHC zinc finger domains and a short segment of basic amino acids (the basic region). This basic region bound nucleic acids in vitro. We also demonstrated that XTUT7 repressed translation of a polyadenylated RNA, to which it added a distinct number of uridines. We generated a predicted structure of the XTUT7 catalytic core that indicated histidine 1269 was likely important for uridine specificity. Indeed, mutation of histidine 1269 broadened the nucleotide specificity of XTUT7 and abolished XTUT7-dependent translational repression. Our data reveal key aspects of how XTUT7 adds uridines to RNAs, highlight the role of the basic region, illustrate that XTUT7 can repress translation, and identify an amino acid important for uridine specificity.

Recurrent rewiring and emergence of RNA regulatory networks.

Significance Regulatory networks change during evolution. A protein that controls many genes in one species may control a different set of genes in another. We examined how mRNA networks evolve, focusing on the PUF (Pumilio and FBF) family of RNA-binding proteins. These govern stability and translation of hundreds of mRNAs and enable coordinate regulation of discrete biological outcomes. To understand how RNA networks evolve, we used knowledge of the RNA specificity of each PUF protein to predict its mRNA targets and directly identified mRNAs bound to each protein in divergent fungi via biochemical methods. We find networks controlled by one protein switch during evolution to be controlled by another and that proteins with different specificities can share, gain, or lose batteries of mRNAs. Alterations in regulatory networks contribute to evolutionary change. Transcriptional networks are reconfigured by changes in the binding specificity of transcription factors and their cognate sites. The evolution of RNA–protein regulatory networks is far less understood. The PUF (Pumilio and FBF) family of RNA regulatory proteins controls the translation, stability, and movements of hundreds of mRNAs in a single species. We probe the evolution of PUF–RNA networks by direct identification of the mRNAs bound to PUF proteins in budding and filamentous fungi and by computational analyses of orthologous RNAs from 62 fungal species. Our findings reveal that PUF proteins gain and lose mRNAs with related and emergent biological functions during evolution. We demonstrate at least two independent rewiring events for PUF3 orthologs, independent but convergent evolution of PUF4/5 binding specificity and the rewiring of the PUF4/5 regulons in different fungal lineages. These findings demonstrate plasticity in RNA regulatory networks and suggest ways in which their rewiring occurs.

Unbiased screen of RNA tailing enzymes at single-nucleotide resolution reveals a poly(UG) polymerase required for genome integrity and RNA silencing

Ribonucleotidyl transferases (rNTases) add non-templated ribonucleotides to diverse RNAs. We developed a screening strategy in S. cerevisiae to identify sequences added by candidate enzymes from different organisms at single-nucleotide resolution. The rNTase activities of 19 previously unexplored enzymes were determined. In addition to poly(A)- and poly(U)-adding enzymes, we identified a C-adding enzyme that is likely part of a two-enzyme system that adds CCA to tRNAs in a eukaryote; a nucleotidyl transferase that adds nucleotides to RNA without apparent nucleotide preference; and a poly(UG) polymerase, C. elegans MUT-2, which adds alternating U and G nucleotides to form poly(UG) tails. MUT-2 is known to be required for certain forms of RNA silencing, and mutations in the enzyme that are defective in silencing also fail to add poly(UG) tails in our assay. We propose that MUT-2 poly(UG) polymerase activity is required to promote genome integrity and RNA silencing.

How Messenger RNA and Nascent Chain Sequences Regulate Translation Elongation

Translation elongation is a highly coordinated, multistep, multifactor process that ensures accurate and efficient addition of amino acids to a growing nascent-peptide chain encoded in the sequence of translated messenger RNA (mRNA). Although translation elongation is heavily regulated by external factors, there is clear evidence that mRNA and nascent-peptide sequences control elongation dynamics, determining both the sequence and structure of synthesized proteins. Advances in methods have driven experiments that revealed the basic mechanisms of elongation as well as the mechanisms of regulation by mRNA and nascent-peptide sequences. In this review, we highlight how mRNA and nascent-peptide elements manipulate the translation machinery to alter the dynamics and pathway of elongation.

Reply to Hogan: Direct evidence of RNA–protein interactions and rewiring

We agree with G. J. Hogan (1) that evolution of PUF–RNA interactions is an important problem, but disagree with his characterization of our report (2). First, we cite Hogan et al. (3) and Jiang et al. (4) seven and four times, respectively; credit them with proposing Puf3-regulon rewiring; describe Hogan’s work (3) as “elegant”; and credit it with proposing Puf4/5 duplication and divergence. We also explicitly state that our biochemical data support their earlier analyses. Second, Hogan’s (1) assertion that “the majority of results presented by the authors have been previously published” is untrue. Using crosslinking and immunopurification, we directly identify mRNAs bound in Neurospora crassa by its Puf3 (Nc\_Puf3) and Puf4/5 (Nc\_Puf4/5) proteins for the first … [↵][1]1To whom correspondence should be addressed. Email: wickens{at}biochem.wisc.edu. [1]: #xref-corresp-1-1