Diverse modes of evolutionary emergence and flux of conserved microRNA clusters

Many animal miRNA loci reside in genomic clusters that generate multicistronic primary-miRNA transcripts. While clusters that contain copies of the same miRNA hairpin are clearly products of local duplications, the evolutionary provenance of clusters with disparate members is less clear. Recently, it was proposed that essentially all such clusters in Drosophila derived from de novo formation of miRNA-like hairpins within existing miRNA transcripts, and that the maintenance of multiple miRNAs in such clusters was due to evolutionary hitchhiking on a major cluster member. However, this model seems at odds with the fact that many such miRNA clusters are composed of well-conserved miRNAs. In an effort to trace the birth and expansion of miRNA clusters that are presently well-conserved across Drosophilids, we analyzed a broad swath of metazoan species, with particular emphasis on arthropod evolution. Beyond duplication and de novo birth, we highlight a diversity of modes that contribute to miRNA evolution, including neofunctionalization of miRNA copies, fissioning of locally duplicated miRNA clusters, miRNA deletion, and miRNA cluster expansion via the acquisition and/or neofunctionalization of miRNA copies from elsewhere in the genome. In particular, we suggest that miRNA clustering by acquisition represents an expedient strategy to bring cohorts of target genes under coordinate control by miRNAs that had already been individually selected for regulatory impact on the transcriptome

Challenges in funding and developing genomic software: roots and remedies

The computer software used for genomic analysis has become a crucial component of the infrastructure for life sciences. However, genomic software is still typically developed in an ad hoc manner, with inadequate funding, and by academic researchers not trained in software development, at substantial costs to the research community. I examine the roots of the incongruity between the importance of and the degree of investment in genomic software, and I suggest several potential remedies for current problems. As genomics continues to grow, new strategies for funding and developing the software that powers the field will become increasingly essential

The impact of age, biogenesis, and genomic clustering on Drosophila microRNA evolution

The molecular evolutionary signatures of miRNAs inform our understanding of their emergence, biogenesis, and function. The known signatures of miRNA evolution have derived mostly from the analysis of deeply conserved, canonical loci. In this study, we examine the impact of age, biogenesis pathway, and genomic arrangement on the evolutionary properties of Drosophila miRNAs. Crucial to the accuracy of our results was our curation of high-quality miRNA alignments, which included nearly 150 corrections to ortholog calls and nucleotide sequences of the global 12-way Drosophilid alignments currently available. Using these data, we studied primary sequence conservation, normalized free-energy values, and types of structure-preserving substitutions. We expand upon common miRNA evolutionary patterns that reflect fundamental features of miRNAs that are under functional selection. We observe that melanogaster-subgroup-specific miRNAs, although recently emerged and rapidly evolving, nonetheless exhibit evolutionary signatures that are similar to well-conserved miRNAs and distinct from other structured noncoding RNAs and bulk conserved non-miRNA hairpins. This provides evidence that even young miRNAs may be selected for regulatory activities. More strikingly, we observe that mirtrons and clustered miRNAs both exhibit distinct evolutionary properties relative to solo, well-conserved miRNAs, even after controlling for sequence depth. These studies highlight the previously unappreciated impact of biogenesis strategy and genomic location on the evolutionary dynamics of miRNAs, and affirm that miRNAs do not evolve as a unitary class

Adaptive evolution of testis-specific, recently evolved, clustered miRNAs in Drosophila

The propensity of animal miRNAs to regulate targets bearing modest complementarity, most notably via pairing with miRNA positions approximately 2-8 (the "seed"), is believed to drive major aspects of miRNA evolution. First, minimal targeting requirements have allowed most conserved miRNAs to acquire large target cohorts, thus imposing strong selection on miRNAs to maintain their seed sequences. Second, the modest pairing needed for repression suggests that evolutionarily nascent miRNAs may generally induce net detrimental, rather than beneficial, regulatory effects. Hence, levels and activities of newly emerged miRNAs are expected to be limited to preserve the status quo of gene expression. In this study, we unexpectedly show that Drosophila testes specifically express a substantial miRNA population that contravenes these tenets. We find that multiple genomic clusters of testis-restricted miRNAs harbor recently evolved miRNAs, whose experimentally verified orthologs exhibit divergent sequences, even within seed regions. Moreover, this class of miRNAs exhibits higher expression and greater phenotypic capacities in transgenic misexpression assays than do non-testis-restricted miRNAs of similar evolutionary age. These observations suggest that these testis-restricted miRNAs may be evolving adaptively, and several methods of evolutionary analysis provide strong support for this notion. Consistent with this, proof-of-principle tests show that orthologous miRNAs with divergent seeds can distinguish target sensors in a species-cognate manner. Finally, we observe that testis-restricted miRNA clusters exhibit extraordinary dynamics of miRNA gene flux in other Drosophila species. Altogether, our findings reveal a surprising tissue-directed influence of miRNA evolution, involving a distinct mode of miRNA function connected to adaptive gene regulation in the testis

Gene regulation by CcpA and catabolite repression explored by RNA-Seq in Streptococcus mutans

A bacterial transcriptome of the primary etiological agent of human dental caries, Streptococcus mutans, is described here using deep RNA sequencing. Differential expression profiles of the transcriptome in the context of carbohydrate source, and of the presence or absence of the catabolite control protein CcpA, revealed good agreement with previously-published DNA microarrays. In addition, RNA-seq considerably expanded the repertoire of DNA sequences that showed statistically-significant changes in expression as a function of the presence of CcpA and growth carbohydrate. Novel mRNAs and small RNAs were identified, some of which were differentially expressed in conditions tested in this study, suggesting that the function of the S. mutans CcpA protein and the influence of carbohydrate sources has a more substantial impact on gene regulation than previously appreciated. Likewise, the data reveal that the mechanisms underlying prioritization of carbohydrate utilization are more diverse than what is currently understood. Collectively, this study demonstrates the validity of RNA-seq as a potentially more-powerful alternative to DNA microarrays in studying gene regulation in S. mutans because of the capacity of this approach to yield a more precise landscape of transcriptomic changes in response to specific mutations and growth conditions

Accurate reconstruction of insertion-deletion histories by statistical phylogenetics

The Multiple Sequence Alignment (MSA) is a computational abstraction that represents a partial summary either of indel history, or of structural similarity. Taking the former view (indel history), it is possible to use formal automata theory to generalize the phylogenetic likelihood framework for finite substitution models (Dayhoff's probability matrices and Felsenstein's pruning algorithm) to arbitrary-length sequences. In this paper, we report results of a simulation-based benchmark of several methods for reconstruction of indel history. The methods tested include a relatively new algorithm for statistical marginalization of MSAs that sums over a stochastically-sampled ensemble of the most probable evolutionary histories. For mammalian evolutionary parameters on several different trees, the single most likely history sampled by our algorithm appears less biased than histories reconstructed by other MSA methods. The algorithm can also be used for alignment-free inference, where the MSA is explicitly summed out of the analysis. As an illustration of our method, we discuss reconstruction of the evolutionary histories of human protein-coding genes.Comment: 28 pages, 15 figures. arXiv admin note: text overlap with arXiv:1103.434

Parking functions, labeled trees and DCJ sorting scenarios

In genome rearrangement theory, one of the elusive questions raised in recent years is the enumeration of rearrangement scenarios between two genomes. This problem is related to the uniform generation of rearrangement scenarios, and the derivation of tests of statistical significance of the properties of these scenarios. Here we give an exact formula for the number of double-cut-and-join (DCJ) rearrangement scenarios of co-tailed genomes. We also construct effective bijections between the set of scenarios that sort a cycle and well studied combinatorial objects such as parking functions and labeled trees.Comment: 12 pages, 3 figure

GAGA Factor Maintains Nucleosome-Free Regions and Has a Role in RNA Polymerase II Recruitment to Promoters

Previous studies have shown that GAGA Factor (GAF) is enriched on promoters with paused RNA Polymerase II (Pol II), but its genome-wide function and mechanism of action remain largely uncharacterized. We assayed the levels of transcriptionally-engaged polymerase using global run-on sequencing (GRO-seq) in control and GAF-RNAi Drosophila S2 cells and found promoter-proximal polymerase was significantly reduced on a large subset of paused promoters where GAF occupancy was reduced by knock down. These promoters show a dramatic increase in nucleosome occupancy upon GAF depletion. These results, in conjunction with previous studies showing that GAF directly interacts with nucleosome remodelers, strongly support a model where GAF directs nucleosome displacement at the promoter and thereby allows the entry Pol II to the promoter and pause sites. This action of GAF on nucleosomes is at least partially independent of paused Pol II because intergenic GAF binding sites with little or no Pol II also show GAF-dependent nucleosome displacement. In addition, the insulator factor BEAF, the BEAF-interacting protein Chriz, and the transcription factor M1BP are strikingly enriched on those GAF-associated genes where pausing is unaffected by knock down, suggesting insulators or the alternative promoter-associated factor M1BP protect a subset of GAF-bound paused genes from GAF knock-down effects. Thus, GAF binding at promoters can lead to the local displacement of nucleosomes, but this activity can be restricted or compensated for when insulator protein or M1BP complexes also reside at GAF bound promoters