Recurrent rewiring and emergence of RNA regulatory networks.

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.

Architecture and dynamics of overlapped RNA regulatory networks.

A single protein can bind and regulate many mRNAs. Multiple proteins with similar specificities often bind and control overlapping sets of mRNAs. Yet little is known about the architecture or dynamics of overlapped networks. We focused on three proteins with similar structures and related RNA-binding specificities-Puf3p, Puf4p, and Puf5p of S. cerevisiae Using RNA Tagging, we identified a "super-network" comprised of four subnetworks: Puf3p, Puf4p, and Puf5p subnetworks, and one controlled by both Puf4p and Puf5p. The architecture of individual subnetworks, and thus the super-network, is determined by competition among particular PUF proteins to bind mRNAs, their affinities for binding elements, and the abundances of the proteins. The super-network responds dramatically: The remaining network can either expand or contract. These strikingly opposite outcomes are determined by an interplay between the relative abundance of the RNAs and proteins, and their affinities for one another. The diverse interplay between overlapping RNA-protein networks provides versatile opportunities for regulation and evolution.

Protein-RNA networks revealed through covalent RNA marks.

Protein-RNA networks are ubiquitous and central in biological control. We present an approach termed RNA Tagging that enables the user to identify protein-RNA interactions in vivo by analyzing purified cellular RNA, without protein purification or cross-linking. An RNA-binding protein of interest is fused to an enzyme that adds uridines to the end of RNA. RNA targets bound by the chimeric protein in vivo are covalently marked with uridines and subsequently identified from extracted RNA via high-throughput sequencing. We used this approach to identify hundreds of RNAs bound by a Saccharomyces cerevisiae PUF protein, Puf3p. The results showed that although RNA-binding proteins productively bind specific RNAs to control their function, they also 'sample' RNAs without exerting a regulatory effect. We used the method to uncover hundreds of new and likely regulated targets for a protein without canonical RNA-binding domains, Bfr1p. RNA Tagging is well suited to detect and analyze protein-RNA networks in vivo.

Divergence of Pumilio/fem-3 mRNA binding factor (PUF) protein specificity through variations in an RNA-binding pocket.

mRNA control networks depend on recognition of specific RNA sequences. Pumilio-fem-3 mRNA binding factor (PUF) RNA-binding proteins achieve that specificity through variations on a conserved scaffold. Saccharomyces cerevisiae Puf3p achieves specificity through an additional binding pocket for a cytosine base upstream of the core RNA recognition site. Here we demonstrate that this chemically simple adaptation is prevalent and contributes to the diversity of RNA specificities among PUF proteins. Bioinformatics analysis shows that mRNAs associated with Caenorhabditis elegans fem-3 mRNA binding factor (FBF)-2 in vivo contain an upstream cytosine required for biological regulation. Crystal structures of FBF-2 and C. elegans PUF-6 reveal binding pockets structurally similar to that of Puf3p, whereas sequence alignments predict a pocket in PUF-11. For Puf3p, FBF-2, PUF-6, and PUF-11, the upstream pockets and a cytosine are required for maximal binding to RNA, but the quantitative impact on binding affinity varies. Furthermore, the position of the upstream cytosine relative to the core PUF recognition site can differ, which in the case of FBF-2 originally masked the identification of this consensus sequence feature. Importantly, other PUF proteins lack the pocket and so do not discriminate upstream bases. A structure-based alignment reveals that these proteins lack key residues that would contact the cytosine, and in some instances, they also present amino acid side chains that interfere with binding. Loss of the pocket requires only substitution of one serine, as appears to have occurred during the evolution of certain fungal species.

N6-adenosine methylation controls the translation of insulin mRNA.

Control of insulin mRNA translation is crucial for energy homeostasis, but the mechanisms remain largely unknown. We discovered that insulin mRNAs across invertebrates, vertebrates and mammals feature the modified base N6-methyladenosine (m6A). In flies, this RNA modification enhances insulin mRNA translation by promoting the association of the transcript with polysomes. Depleting m6A in Drosophila melanogaster insulin 2 mRNA (dilp2) directly through specific 3' untranslated region (UTR) mutations, or indirectly by mutating the m6A writer Mettl3, decreases dilp2 protein production, leading to aberrant energy homeostasis and diabetic-like phenotypes. Together, our findings reveal adenosine mRNA methylation as a key regulator of insulin protein synthesis with notable implications for energy balance and metabolic disease.

Nutrigenomic regulation of sensory plasticity.

Diet profoundly influences brain physiology, but how metabolic information is transmuted into neural activity and behavior changes remains elusive. Here, we show that the metabolic enzyme O-GlcNAc Transferase (OGT) moonlights on the chromatin of the D. melanogaster gustatory neurons to instruct changes in chromatin accessibility and transcription that underlie sensory adaptations to a high-sugar diet. OGT works synergistically with the Mitogen Activated Kinase/Extracellular signal Regulated Kinase (MAPK/ERK) rolled and its effector stripe (also known as EGR2 or Krox20) to integrate activity information. OGT also cooperates with the epigenetic silencer Polycomb Repressive Complex 2.1 (PRC2.1) to decrease chromatin accessibility and repress transcription in the high-sugar diet. This integration of nutritional and activity information changes the taste neurons' responses to sugar and the flies' ability to sense sweetness. Our findings reveal how nutrigenomic signaling generates neural activity and behavior in response to dietary changes in the sensory neurons.

New insights into the blood-stage transcriptome of Plasmodium falciparum using RNA-Seq.

Recent advances in high-throughput sequencing present a new opportunity to deeply probe an organism's transcriptome. In this study, we used Illumina-based massively parallel sequencing to gain new insight into the transcriptome (RNA-Seq) of the human malaria parasite, Plasmodium falciparum. Using data collected at seven time points during the intraerythrocytic developmental cycle, we (i) detect novel gene transcripts; (ii) correct hundreds of gene models; (iii) propose alternative splicing events; and (iv) predict 5' and 3' untranslated regions. Approximately 70% of the unique sequencing reads map to previously annotated protein-coding genes. The RNA-Seq results greatly improve existing annotation of the P. falciparum genome with over 10% of gene models modified. Our data confirm 75% of predicted splice sites and identify 202 new splice sites, including 84 previously uncharacterized alternative splicing events. We also discovered 107 novel transcripts and expression of 38 pseudogenes, with many demonstrating differential expression across the developmental time series. Our RNA-Seq results correlate well with DNA microarray analysis performed in parallel on the same samples, and provide improved resolution over the microarray-based method. These data reveal new features of the P. falciparum transcriptional landscape and significantly advance our understanding of the parasite's red blood cell-stage transcriptome.

Clonally variant gene families in Plasmodium falciparum share a common activation factor.

The genome of the malaria parasite Plasmodium falciparum contains several multicopy gene families, including var, rifin, stevor and Pfmc-2TM. These gene families undergo expression switching and appear to play a role in antigenic variation. It has recently been shown that forcing parasites to express high copy numbers of transcriptionally active, episomal var promoters led to gradual downregulation and eventual silencing of the entire var gene family, suggesting that a limiting titratable factor plays a role in var gene activation. Through similar experiments using rifin, stevor or Pfmc-2TM episomal promoters we show that promoter titration can be used as a general method to downregulate multicopy gene families in P. falciparum. Additionally, we show that promoter titration with var, rifin, stevor or Pfmc-2TM episomal promoters results in downregulation of expression not only of the family to which the episomal promoter belongs, but also members of the other gene families, suggesting that the var-specific titratable factor previously described is shared by all four families. Further, transcriptionally active promoters from different families colocalize within the same subnuclear expression site, indicating that the role that nuclear architecture plays in var gene regulation also likely applies to the other multicopy gene families of P. falciparum.

Rapid metabolic shifts occur during the transition between hunger and satiety in Drosophila melanogaster.

Metabolites are active controllers of cellular physiology, but their role in complex behaviors is less clear. Here we report metabolic changes that occur during the transition between hunger and satiety in Drosophila melanogaster. To analyze these data in the context of fruit fly metabolic networks, we developed Flyscape, an open-access tool. We show that in response to eating, metabolic profiles change in quick, but distinct ways in the heads and bodies. Consumption of a high sugar diet dulls the metabolic and behavioral differences between the fasted and fed state, and reshapes the way nutrients are utilized upon eating. Specifically, we found that high dietary sugar increases TCA cycle activity, alters neurochemicals, and depletes 1-carbon metabolism and brain health metabolites N-acetyl-aspartate and kynurenine. Together, our work identifies the metabolic transitions that occur during hunger and satiation, and provides a platform to study the role of metabolites and diet in complex behavior.

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.

Co-inhibition of Plasmodium falciparum S-adenosylmethionine decarboxylase/ornithine decarboxylase reveals perturbation-specific compensatory mechanisms by transcriptome, proteome, and metabolome analyses.

Polyamines are ubiquitous components of all living cells, and their depletion usually causes cytostasis, a strategy employed for treatment of West African trypanosomiasis. To evaluate polyamine depletion as an anti-malarial strategy, cytostasis caused by the co-inhibition of S-adenosylmethionine decarboxylase/ornithine decarboxylase in Plasmodium falciparum was studied with a comprehensive transcriptome, proteome, and metabolome investigation. Highly synchronized cultures were sampled just before and during cytostasis, and a novel zero time point definition was used to enable interpretation of results in lieu of the developmentally regulated control of gene expression in P. falciparum. Transcriptome analysis revealed the occurrence of a generalized transcriptional arrest just prior to the growth arrest due to polyamine depletion. However, the abundance of 538 transcripts was differentially affected and included three perturbation-specific compensatory transcriptional responses as follows: the increased abundance of the transcripts for lysine decarboxylase and ornithine aminotransferase and the decreased abundance of that for S-adenosylmethionine synthetase. Moreover, the latter two compensatory mechanisms were confirmed on both protein and metabolite levels confirming their biological relevance. In contrast with previous reports, the results provide evidence that P. falciparum responds to alleviate the detrimental effects of polyamine depletion via regulation of its transcriptome and subsequently the proteome and metabolome.

Host-parasite interactions revealed by Plasmodium falciparum metabolomics.

Intracellular pathogens have devised mechanisms to exploit their host cells to ensure their survival and replication. The malaria parasite Plasmodium falciparum relies on an exchange of metabolites with the host for proliferation. Here we describe a mass spectrometry-based metabolomic analysis of the parasite throughout its 48 hr intraerythrocytic developmental cycle. Our results reveal a general modulation of metabolite levels by the parasite, with numerous metabolites varying in phase with the developmental cycle. Others differed from uninfected cells irrespective of the developmental stage. Among these was extracellular arginine, which was specifically converted to ornithine by the parasite. To identify the biochemical basis for this effect, we disrupted the plasmodium arginase gene in the rodent malaria model P. berghei. These parasites were viable but did not convert arginine to ornithine. Our results suggest that systemic arginine depletion by the parasite may be a factor in human malarial hypoargininemia associated with cerebral malaria pathogenesis.