Mining ice in genomes: comparative genomics of integrative elements in prokaryotic genomes

Tese de mestrado. Biologia (Biologia Molecular e Genética). Universidade de Lisboa, Faculdade de Ciências, 2010Elementos integrativos e conjugativos (ICEs) são um grupo muito diverso de elementos genéticos móveis, que se caracterizam por partilharem características de fagos e plasmídios. Como fagos temperados, os ICES integram-se no genoma do hospedeiro, estando dependentes deste para a sua replicação; como plasmídios, são transmitidos para outras células através de conjugação. Estes elementos são portanto responsáveis por transferência horizontal de genes (HGT) em procariotas. A sua estrutura é composta por três módulos: manutenção, transmissão e regulação. O módulo de manutenção codifica a recombinase, a proteína responsável pela integração dos ICES no genoma do hospedeiro. O módulo de disseminação inclui o sistema conjugativo, tipicamente um sistema de secreção do tipo IV (T4SS). O módulo de regulação é composto por genes que regulam a transferência dos elementos. No entanto, apesar do estudo dos ICE se revestir de enorme importância clínica, uma vez que transmitem características como resistência a antibióticos, produção de factores de virulência ou mesmo produção de biofilmes, pouco se sabe ainda acerca do seu conteúdo génico, tamanho, e que mecanismos de integração e conjugação utilizam. Este é o primeiro estudo que identifica ICEs em todos os genomas procarióticos sequenciados. Nos 1055 genomas disponíveis, identificámos e caracterizámos a distribuição de 315 ICEs. Uma das teorias mais aceites acerca do papel dos ICEs na THG especula que estes terão um papel dominante em Firmicutes, mas que em Proteobactérias são plasmídios conjugativos os verdadeiros responsáveis pela transferência horizontal de genes. Utilizando dados de um estudo prévio do nosso laboratório que caracterizou a mobilidade em plasmídios, verificámos que esta relação não parece ser verdadeira. Uma vez que este se trata de um estudo pioneiro, os resultados por nós obtidos podem abrir novas portas na investigação de ICEs.Integrative conjugative elements (ICEs) are a diverse group of mobile genetic elements characterized by their dual phage and plasmid behaviour. Like temperate phages, ICEs can integrate into the host chromosome and replicate with it, and like plasmids they are transferred by conjugation. These elements contribute to horizontal gene transfer (HGT) in prokaryotes, and are responsible for the transmission of traits such as antibiotic resistance, virulence factors and biofilm formation. Its core structure can be divided in three modules: maintenance, dissemination and regulation. The maintenance module encodes a recombinase, which is responsible for ICEs integration into host replicons. The dissemination module includes the conjugating system, typically IV secretion system (T4SS). The regulation module comprises the genes that regulate ICEs transfer. The studies on ICEs are very recent and therefore the knowledge about their cargo content, their size and how they conjugate and integrate into the host genome is still reduced. Therefore, studying these elements is of vital importance. This is the first large-scale study that identifies integrative conjugative elements in all the sequenced prokaryotic genomes. In the 1055 available genomes, we identified and characterized the distribution of 315 ICEs. We were also able to identify T4SS systems not involved in conjugation, and their distribution was compared to those of ICEs. We used data from a previous work of our laboratory, which characterised plasmid mobility, in order to compare the T4SS systems involved in the conjugation of ICEs and conjugative plasmids. We were able to contradict the mainstream idea of ICE being the major contributors to HGT in Firmicutes, whereas that role was played by conjugative plasmids in Proteobacteria. Because this is a pioneer study, the obtained results may open new avenues of reasearch in this field

Mirnovo: genome-free prediction of microRNAs from small RNA sequencing data and single-cells using decision forests.

The discovery of microRNAs (miRNAs) remains an important problem, particularly given the growth of high-throughput sequencing, cell sorting and single cell biology. While a large number of miRNAs have already been annotated, there may well be large numbers of miRNAs that are expressed in very particular cell types and remain elusive. Sequencing allows us to quickly and accurately identify the expression of known miRNAs from small RNA-Seq data. The biogenesis of miRNAs leads to very specific characteristics observed in their sequences. In brief, miRNAs usually have a well-defined 5' end and a more flexible 3' end with the possibility of 3' tailing events, such as uridylation. Previous approaches to the prediction of novel miRNAs usually involve the analysis of structural features of miRNA precursor hairpin sequences obtained from genome sequence. We surmised that it may be possible to identify miRNAs by using these biogenesis features observed directly from sequenced reads, solely or in addition to structural analysis from genome data. To this end, we have developed mirnovo, a machine learning based algorithm, which is able to identify known and novel miRNAs in animals and plants directly from small RNA-Seq data, with or without a reference genome. This method performs comparably to existing tools, however is simpler to use with reduced run time. Its performance and accuracy has been tested on multiple datasets, including species with poorly assembled genomes, RNaseIII (Drosha and/or Dicer) deficient samples and single cells (at both embryonic and adult stage)

MiR-277/4989 regulate transcriptional landscape during juvenile to adult transition in the parasitic helminth Schistosoma mansoni.

Schistosomes are parasitic helminths that cause schistosomiasis, a disease affecting circa 200 million people, primarily in underprivileged regions of the world. Schistosoma mansoni is the most experimentally tractable schistosome species due to its ease of propagation in the laboratory and the high quality of its genome assembly and annotation. Although there is growing interest in microRNAs (miRNAs) in trematodes, little is known about the role these molecules play in the context of developmental processes. We use the completely unaware "miRNA-blind" bioinformatics tool Sylamer to analyse the 3'-UTRs of transcripts differentially expressed between the juvenile and adult stages. We show that the miR-277/4989 family target sequence is the only one significantly enriched in the transition from juvenile to adult worms. Further, we describe a novel miRNA, sma-miR-4989 showing that its proximal genomic location to sma-miR-277 suggests that they form a miRNA cluster, and we propose hairpin folds for both miRNAs compatible with the miRNA pathway. In addition, we found that expression of sma-miR-277/4989 miRNAs are up-regulated in adults while their predicted targets are characterised by significant down-regulation in paired adult worms but remain largely undisturbed in immature "virgin" females. Finally, we show that sma-miR-4989 is expressed in tegumental cells located proximal to the oesophagus gland and also distributed throughout the male worms' body. Our results indicate that sma-miR-277/4989 might play a dominant role in post-transcriptional regulation during development of juvenile worms and suggest an important role in the sexual development of female schistosomes

The repertoire of ICE in prokaryotes underscores the unity, diversity, and ubiquity of conjugation

Horizontal gene transfer shapes the genomes of prokaryotes by allowing rapid acquisition of novel adaptive functions. Conjugation allows the broadest range and the highest gene transfer input per transfer event. While conjugative plasmids have been studied for decades, the number and diversity of integrative conjugative elements (ICE) in prokaryotes remained unknown. We defined a large set of protein profiles of the conjugation machinery to scan over 1,000 genomes of prokaryotes. We found 682 putative conjugative systems among all major phylogenetic clades and showed that ICEs are the most abundant conjugative elements in prokaryotes. Nearly half of the genomes contain a type IV secretion system (T4SS), with larger genomes encoding more conjugative systems. Surprisingly, almost half of the chromosomal T4SS lack co-localized relaxases and, consequently, might be devoted to protein transport instead of conjugation. This class of elements is preponderant among small genomes, is less commonly associated with integrases, and is rarer in plasmids. ICEs and conjugative plasmids in proteobacteria have different preferences for each type of T4SS, but all types exist in both chromosomes and plasmids. Mobilizable elements outnumber self-conjugative elements in both ICEs and plasmids, which suggests an extensive use of T4SS in trans. Our evolutionary analysis indicates that switch of plasmids to and from ICEs were frequent and that extant elements began to differentiate only relatively recently. According to the present results, ICEs are the most abundant conjugative elements in practically all prokaryotic clades and might be far more frequently domesticated into non-conjugative protein transport systems than previously thought. While conjugative plasmids and ICEs have different means of genomic stabilization, their mechanisms of mobility by conjugation show strikingly conserved patterns, arguing for a unitary view of conjugation in shaping the genomes of prokaryotes by horizontal gene transfer

Correction: MiR-277/4989 regulate transcriptional landscape during juvenile to adult transition in the parasitic helminth Schistosoma mansoni.

[This corrects the article DOI: 10.1371/journal.pntd.0005559.]

Sma-miR-277 family predicted targets downregulated in developing female parasites.

<p>Fold change expression (Log₂) of high confidence targets of sma-miR-277 family during the development of male and female worms in two conditions: paired (solid line red) and unpaired (dashed green). Black lines represent the mean expression of genes in paired (solid black line) and unpaired (dashed black line) worms.</p

High confidence targets of the sma-miR-277 family identified with a combined approach (Sylamer, miRanda and TargetScan with conservation).

<p>F = female; M = male.</p

MiRNA target prediction based on both miRNA-unaware and miRNA-guided approaches.

<p>(A) Sylamer enrichment landscape plots for 7mers in male (top) and female (bottom) expression data. The x-axis represents a list of transcripts, ranked from more expressed in juveniles to more expressed in adults. The y-axis represents the significance values acquired for each 7mer at each position in the ranked list of transcripts. Coloured boxes represent the fraction of transcripts significantly (adjusted p-value < 0.01) differentially expressed between juvenile and adult worm as found using DESeq2. These transcripts were subsequently filtered based on the presence of the 7mers TGCATTT or GCATTTA as found by Sylamer. The resulting sets are referred to as Male and Female Sylamer genes. (B) Venn Diagram showing the intersection of Male and Female Sylamer genes with schistosome-conserved miRNA targets as found using TargetScan with conservation + miRanda. The overlap represents transcripts with highly conserved sma-miR-277 target sites across the three <i>Schistosome</i> spp (S. <i>mansoni</i>, <i>S</i>. <i>haematobium</i> and S. <i>japonicum</i>) that are also significantly down regulated during worm development.</p