Single-nucleotide control of tRNA folding cooperativity under near-cellular conditions

RNA folding is often studied by renaturing full-length RNA in vitro and tracking folding transitions. However, the intracellular transcript folds as it emerges from the RNA polymerase. Here, we investigate the folding pathways and stability of numerous late-transcriptional intermediates of yeast and Escherichia coli transfer RNAs (tRNAs). Transfer RNA is a highly regulated functional RNA that undergoes multiple steps of posttranscriptional processing and is found in very different lengths during its lifetime in the cell. The precursor transcript is extended on both the 5′ and 3′ ends of the cloverleaf core, and these extensions get trimmed before addition of the 3′-CCA and aminoacylation. We studied the thermodynamics and structures of the precursor tRNA and of late-transcriptional intermediates of the cloverleaf structure. We examined RNA folding at both the secondary and tertiary structural levels using multiple biochemical and biophysical approaches. Our findings suggest that perhaps nature has selected for a single-base addition to control folding to the functional 3D structure. In near-cellular conditions, yeast tRNAPhe and E. coli tRNAAla transcripts fold in a single, cooperative transition only when nearly all of the nucleotides in the cloverleaf are transcribed by indirectly enhancing folding cooperativity. Furthermore, native extensions on the 5′ and 3′ ends do not interfere with cooperative core folding. This highly controlled cooperative folding has implications for recognition of tRNA by processing and modification enzymes and quality control of tRNA in cells.

Regulation of RNA Polymerase I Transcription in Development, Disease, and Aging

Ribosome biogenesis is a complex and highly energy-demanding process that requires the concerted action of all three nuclear RNA polymerases (Pol I–III) in eukaryotes. The three largest ribosomal RNAs (rRNAs) originate from a precursor transcript (pre-rRNA) that is encoded by multicopy genes located in the nucleolus. Transcription of these rRNA genes (rDNA) by Pol I is the key regulation step in ribosome production and is tightly controlled by an intricate network of signaling pathways and epigenetic mechanisms. In this article, we give an overview of the composition of the basal Pol I machinery and rDNA chromatin. We discuss rRNA gene regulation in response to environmental signals and developmental cues and focus on perturbations occurring in diseases linked to either excessive or limited rRNA levels. Finally, we discuss the emerging view that rDNA integrity and activity may be involved in the aging process.

Evolutionary analysis implicates RNA polymerase II pausing and chromatin structure in nematode piRNA biogenesis

Piwi-interacting RNAs (piRNAs) control transposable elements widely across metazoans but have rapidly evolving biogenesis pathways. In Caenorhabditis elegans, almost all piRNA loci are found within two 3Mb clusters on Chromosome IV. Each piRNA locus possesses an upstream motif that recruits RNA polymerase II to produce a ∼28 nt precursor transcript. Here, we use comparative epigenomics across nematodes to gain insight into piRNA biogenesis. We show that the piRNA upstream motif is derived from core promoter elements controlling snRNA biogenesis. We describe two alternative modes of piRNA organisation in nematodes: in C. elegans and closely related nematodes, piRNAs are clustered within repressive H3K27me3 chromatin, whilst in other species, typified by Pristionchus pacificus, piRNAs are distributed genome-wide within introns of actively transcribed genes. In both groups, piRNA production depends on downstream sequence signals associated with RNA polymerase II pausing, which synergise with the chromatin environment to control piRNA precursor transcription.

MatPred: Computational Identification of Mature MicroRNAs within Novel Pre-MicroRNAs

Background.MicroRNAs (miRNAs) are short noncoding RNAs integral for regulating gene expression at the posttranscriptional level. However, experimental methods often fall short in finding miRNAs expressed at low levels or in specific tissues. While several computational methods have been developed for predicting the localization of mature miRNAs within the precursor transcript, the prediction accuracy requires significant improvement.Methodology/Principal Findings.Here, we present MatPred, which predicts mature miRNA candidates within novel pre-miRNA transcripts. In addition to the relative locus of the mature miRNA within the pre-miRNA hairpin loop and minimum free energy, we innovatively integrated features that describe the nucleotide-specific RNA secondary structure characteristics. In total, 94 features were extracted from the mature miRNA loci and flanking regions. The model was trained based on a radial basis function kernel/support vector machine (RBF/SVM). Our method can predict precise locations of mature miRNAs, as affirmed by experimentally verified human pre-miRNAs or pre-miRNAs candidates, thus achieving a significant advantage over existing methods.Conclusions.MatPred is a highly effective method for identifying mature miRNAs within novel pre-miRNA transcripts. Our model significantly outperformed three other widely used existing methods. Such processing prediction methods may provide important insight into miRNA biogenesis.

Molecular Cloning and Pharmacological Characterization of Two Novel GnRH Receptors in the Lamprey (Petromyzon marinus)

This paper reports the identification, expression, binding kinetics, and functional studies of two novel type III lamprey GnRH receptors (lGnRH-R-2 and lGnRH-R-3) in the sea lamprey, a basal vertebrate. These novel GnRH receptors share the structural features and amino acid motifs common to other known gnathostome GnRH receptors. The ligand specificity and activation of intracellular signaling studies showed ligands lGnRH-II and -III induced an inositol phosphate (IP) response at lGnRH-R-2 and lGnRH-R-3, whereas the ligand lGnRH-I did not stimulate an IP response. lGnRH-II was a more potent activator of lGnRH-R-3 than lGnRH-III. Stimulation of lGnRH-R-2 and lGnRH-R-3 testing all three lGnRH ligands did not elicit a cAMP response. lGnRH-R-2 has a higher binding affinity in response to lGnRH-III than lGnRH-II, whereas lGnRH-R-3 has a higher binding affinity in response to lGnRH-II than IGnRH-III. lGnRH-R-2 precursor transcript was detected in a wide variety of tissues including the pituitary whereas lGnRH-R-3 precursor transcript was not as widely expressed and primarily expressed in the brain and eye of male and female lampreys. From our phylogenetic analysis, we propose that lGnRH-R-1 evolved from a common ancestor of all vertebrate GnRH receptors and lGnRH-R-2 and lGnRH-R-3 likely occurred due to a gene duplication within the lamprey lineage. In summary, we propose from our findings of receptor subtypes in the sea lamprey that the evolutionary recruitment of specific pituitary GnRH receptor subtypes for particular physiological functions seen in later evolved vertebrates was an ancestral character that first arose in a basal vertebrate.

miR-451 regulates zebrafish erythroid maturation in vivo via its target gata2

We demonstrate that in zebrafish, the microRNA miR-451 plays a crucial role in promoting erythroid maturation, in part via its target transcript gata2. Zebrafish miR-144 and miR-451 are processed from a single precursor transcript selectively expressed in erythrocytes. In contrast to other hematopoietic mutants, the zebrafish mutant meunier (mnr) showed intact erythroid specification but diminished miR-144/451 expression. Although erythropoiesis initiated normally in mnr, erythrocyte maturation was morphologically retarded. Morpholino knockdown of miR-451 increased erythrocyte immaturity in wild-type embryos, and miR-451 RNA duplexes partially rescued erythroid maturation in mnr, demonstrating a requirement and role for miR-451 in erythrocyte maturation. mnr provided a selectively miR-144/451-deficient background, facilitating studies to discern miRNA function and validate candidate targets. Among computer-predicted miR-451 targets potentially mediating these biologic effects, the pro-stem cell transcription factor gata2 was an attractive candidate. In vivo reporter assays validated the predicted miR-451/gata2-3′UTR interaction, gata2 down-regulation was delayed in miR-451-knockdown and mnr embryos, and gata2 knockdown partially restored erythroid maturation in mnr, collectively confirming gata2 down-regulation as pivotal for miR-451-driven erythroid maturation. These studies define a new genetic pathway promoting erythroid maturation (mnr/miR-451/gata2) and provide a rare example of partial rescue of a mutant phenotype solely by miRNA overexpression.

Processing of eukaryotic pre-rRNA: the role of the transcribed spacers

The 17–18S, 5.8S, and 25–28S rRNA species of eukaryotic cells are produced by a series of nucleolytic reactions that liberate the mature rRNAs from the large primary precursor transcript synthesized by RNA polymerase I. Whereas the order of the cleavage reactions has long been established, until recently little information was available on their molecular details, such as the nature of the proteins, including the nucleolytic enzymes, involved and the signals directing the processing machinery to the correct sites. This situation is now rapidly changing, in particular where yeast is concerned. The use of recently developed systems for in vivo mutational analysis of yeast rDNA has considerably enhanced our knowledge of cis-acting structural features within the pre-rRNA, in particular the transcribed spacer sequences, that are critical for correct and efficient removal of these spacers. The same systems also allow a link to be forged between trans-acting processing factors and these cis-acting elements. In this paper, we will focus predominantly on the nature and role of the cis-acting processing elements as identified in the transcribed spacer regions of Saccharomyces cerevisiae pre-rRNA.Key words: ribosome, processing, precursor rRNA, eukaryote, transcribed spacer.