Jakub Orzechowski Westholm

Vive la différence: biogenesis and evolution of microRNAs in plants and animals

MicroRNAs are pervasive in both plants and animals, but many aspects of their biogenesis, function and evolution differ. We reveal how these differences contribute to characteristic features of microRNA evolution in the two kingdoms.

Genome-wide Analysis of Drosophila Circular RNAs Reveals Their Structural and Sequence Properties and Age-Dependent Neural Accumulation

Circularization was recently recognized to broadly expand transcriptome complexity. Here, we exploit massive Drosophila total RNA-sequencing data, >5 billion paired-end reads from >100 libraries covering diverse developmental stages, tissues, and cultured cells, to rigorously annotate >2,500 fruit fly circular RNAs. These mostly derive from back-splicing of protein-coding genes and lack poly(A) tails, and the circularization of hundreds of genes is conserved across multiple Drosophila species. We elucidate structural and sequence properties of Drosophila circular RNAs, which exhibit commonalities and distinctions from mammalian circles. Notably, Drosophila circular RNAs harbor >1,000 well-conserved canonical miRNA seed matches, especially within coding regions, and coding conserved miRNA sites reside preferentially within circularized exons. Finally, we analyze the developmental and tissue specificity of circular RNAs and note their preferred derivation from neural genes and enhanced accumulation in neural tissues. Interestingly, circular isoforms increase substantially relative to linear isoforms during CNS aging and constitute an aging biomarker.

Mirtrons: microRNA biogenesis via splicing

A well-defined mechanism governs the maturation of most microRNAs (miRNAs) in animals, via stepwise cleavage of precursor hairpin transcripts by the Drosha and Dicer RNase III enzymes. Recently, several alternative miRNA biogenesis pathways were elucidated, the most prominent of which substitutes Drosha cleavage with splicing. Such short hairpin introns are known as mirtrons, and their study has uncovered related pathways that combine splicing with other ribonucleolytic machinery to yield Dicer substrates for miRNA biogenesis. In this review, we consider the mechanisms of splicing-mediated miRNA biogenesis, computational strategies for mirtron discovery, and the evolutionary implications of the existence of multiple miRNA biogenesis pathways. Altogether, the features of mirtron pathways illustrate unexpected flexibility in combining RNA processing pathways, and highlight how multiple functions can be encoded by individual transcripts.

Transcriptional profiling reveals a critical role for tyrosine phosphatase VE-PTP in regulation of VEGFR2 activity and endothelial cell morphogenesis

To define molecular events accompanying formation of the 3‐dimensional (3D) vascular tube, we have characterized gene expression during vascular endothelial growth factor (VEGF)‐induced tubular morphogenesis of endothelial cells. Microarray analyses were performed comparing gene induction in growth‐ arrested, tube‐forming endothelial cells harvested from 3D collagen cultures to that in proliferating endothelial cells cultured on fibronectin. Differentially expressed genes were clustered and analyzed for specific endothelial expression through publicly available datasets. We validated the contribution of one of the identified genes, vascular endothelial protein tyrosine phosphatase (VE‐PTP), to endothelial morphogenesis. Silencing of VE‐PTP expression was accompanied by increased VEGFreceptor‐2 (VEGFR2) tyrosine phosphorylation and activation of downstream signaling pathways. The increased VEGFR2 activity promoted endothelial cell cycle progression, overcoming the G0/G1 arrest associated with organization into tubular structures in the 3D cultures. Proximity ligation showed close association between VEGFR2 and VE‐PTP in resting cells. Activation of VEGFR2 by VEGF led to rapid loss of association, which was resumed with time in parallel with decreased receptor activity. In conclusion, we have identified genes, which may serve critical functions in formation of the vascular tube. One of these, VE‐PTP, regulates VEGFR2 activity thereby modulating the VEGF‐response during angiogenesis.— Mellberg, S., Dimberg, A., Bahram, F., Hayashi, M., Rennel, E., Ameur, A., Westholm, J. O., Larsson, E., Lindahl, P., Cross, M. J., Claesson‐Welsh, L. Transcriptional profiling reveals a critical role for tyrosine phosphatase VE‐PTP in regulation of VEGFR2 activity and endothelial cell morphogenesis. FASEBJ. 23, 1490–1502 (2009)

Computational and experimental identification of mirtrons in Drosophila melanogaster and Caenorhabditis elegans

Mirtrons are intronic hairpin substrates of the dicing machinery that generate functional microRNAs. In this study, we describe experimental assays that defined the essential requirements for entry of introns into the mirtron pathway. These data informed a bioinformatic screen that effectively identified functional mirtrons from the Drosophila melanogaster transcriptome. These included 17 known and six confident novel mirtrons among the top 51 candidates, and additional candidates had limited read evidence in available small RNA data. Our computational model also proved effective on Caenorhabditis elegans, for which the identification of 14 cloned mirtrons among the top 22 candidates more than tripled the number of validated mirtrons in this species. A few low-scoring introns generated mirtron-like read patterns from atypical RNA structures, but their paucity suggests that relatively few such loci were not captured by our model. Unexpectedly, we uncovered examples of clustered mirtrons in both fly and worm genomes, including a <8-kb region in C. elegans harboring eight distinct mirtrons. Altogether, we demonstrate that discovery of functional mirtrons, unlike canonical miRNAs, is amenable to computational methods independent of evolutionary constraint.

Combinatorial control of gene expression by the three yeast repressors Mig1, Mig2 and Mig3

BackgroundExpression of a large number of yeast genes is repressed by glucose. The zinc finger protein Mig1 is the main effector in glucose repression, but yeast also has two related proteins: Mig2 and Mig3. We have used microarrays to study global gene expression in all possible combinations of mig1, mig2 and mig3 deletion mutants.ResultsMig1 and Mig2 repress a largely overlapping set of genes on 2% glucose. Genes that are upregulated in a mig1 mig2 double mutant were grouped according to the contribution of Mig2. Most of them show partially redundant repression, with Mig1 being the major repressor, but some genes show complete redundancy, and some are repressed only by Mig1. Several redundantly repressed genes are involved in phosphate metabolism. The promoters of these genes are enriched for Pho4 sites, a novel GGGAGG motif, and a variant Mig1 site which is absent from genes repressed only by Mig1. Genes repressed only by Mig1 on 2% glucose include the hexose transporter gene HXT4, but Mig2 contributes to HXT4 repression on 10% glucose. HXT6 is one of the few genes that are more strongly repressed by Mig2. Mig3 does not seem to overlap in function with Mig1 and Mig2. Instead, Mig3 downregulates the SIR2 gene encoding a histone deacetylase involved in gene silencing and the control of aging.ConclusionMig2 fine-tunes glucose repression by targeting a subset of the Mig1-repressed genes, and by responding to higher glucose concentrations. Mig3 does not target the same genes as Mig1 and Mig2, but instead downregulates the SIR2 gene.

Diversity of miRNAs, siRNAs, and piRNAs across 25 Drosophila cell lines

We expanded the knowledge base for Drosophila cell line transcriptomes by deeply sequencing their small RNAs. In total, we analyzed more than 1 billion raw reads from 53 libraries across 25 cell lines. We verify reproducibility of biological replicate data sets, determine common and distinct aspects of miRNA expression across cell lines, and infer the global impact of miRNAs on cell line transcriptomes. We next characterize their commonalities and differences in endo-siRNA populations. Interestingly, most cell lines exhibit enhanced TE-siRNA production relative to tissues, suggesting this as a common aspect of cell immortalization. We also broadly extend annotations of cis-NAT-siRNA loci, identifying ones with common expression across diverse cells and tissues, as well as cell-restricted loci. Finally, we characterize small RNAs in a set of ovary-derived cell lines, including somatic cells (OSS and OSC) and a mixed germline/somatic cell population (fGS/OSS) that exhibits ping-pong piRNA signatures. Collectively, the ovary data reveal new genic piRNA loci, including unusual configurations of piRNA-generating regions. Together with the companion analysis of mRNAs described in a previous study, these small RNA data provide comprehensive information on the transcriptional landscape of diverse Drosophila cell lines. These data should encourage broader usage of fly cell lines, beyond the few that are presently in common usage.

Neurophysiological Defects and Neuronal Gene Deregulation in Drosophila mir-124 Mutants

miR-124 is conserved in sequence and neuronal expression across the animal kingdom and is predicted to have hundreds of mRNA targets. Diverse defects in neural development and function were reported from miR-124 antisense studies in vertebrates, but a nematode knockout of mir-124 surprisingly lacked detectable phenotypes. To provide genetic insight from Drosophila, we deleted its single mir-124 locus and found that it is dispensable for gross aspects of neural specification and differentiation. On the other hand, we detected a variety of mutant phenotypes that were rescuable by a mir-124 genomic transgene, including short lifespan, increased dendrite variation, impaired larval locomotion, and aberrant synaptic release at the NMJ. These phenotypes reflect extensive requirements of miR-124 even under optimal culture conditions. Comparison of the transcriptomes of cells from wild-type and mir-124 mutant animals, purified on the basis of mir-124 promoter activity, revealed broad upregulation of direct miR-124 targets. However, in contrast to the proposed mutual exclusion model for miR-124 function, its functional targets were relatively highly expressed in miR-124–expressing cells and were not enriched in genes annotated with epidermal expression. A notable aspect of the direct miR-124 network was coordinate targeting of five positive components in the retrograde BMP signaling pathway, whose activation in neurons increases synaptic release at the NMJ, similar to mir-124 mutants. Derepression of the direct miR-124 target network also had many secondary effects, including over-activity of other post-transcriptional repressors and a net incomplete transition from a neuroblast to a neuronal gene expression signature. Altogether, these studies demonstrate complex consequences of miR-124 loss on neural gene expression and neurophysiology.

Gis1 and Rph1 Regulate Glycerol and Acetate Metabolism in Glucose Depleted Yeast Cells

Aging in organisms as diverse as yeast, nematodes, and mammals is delayed by caloric restriction, an effect mediated by the nutrient sensing TOR, RAS/cAMP, and AKT/Sch9 pathways. The transcription factor Gis1 functions downstream of these pathways in extending the lifespan of nutrient restricted yeast cells, but the mechanisms involved are still poorly understood. We have used gene expression microarrays to study the targets of Gis1 and the related protein Rph1 in different growth phases. Our results show that Gis1 and Rph1 act both as repressors and activators, on overlapping sets of genes as well as on distinct targets. Interestingly, both the activities and the target specificities of Gis1 and Rph1 depend on the growth phase. Thus, both proteins are associated with repression during exponential growth, targeting genes with STRE or PDS motifs in their promoters. After the diauxic shift, both become involved in activation, with Gis1 acting primarily on genes with PDS motifs, and Rph1 on genes with STRE motifs. Significantly, Gis1 and Rph1 control a number of genes involved in acetate and glycerol formation, metabolites that have been implicated in aging. Furthermore, several genes involved in acetyl-CoA metabolism are downregulated by Gis1.

Intertwined pathways for Argonaute-mediated microRNA biogenesis in Drosophila

Although Dicer is essential for general microRNA (miRNA) biogenesis, vertebrate mir-451 is Dicer independent. Instead, its short pre-miRNA hairpin is ‘sliced’ by Ago2, then 3′-resected into mature miRNAs. Here, we show that Drosophila cells and animals generate functional small RNAs from mir-451-type precursors. However, their bulk maturation arrests as Ago-cleaved pre-miRNAs, which mostly associate with the RNAi effector AGO2. Routing of pre-mir-451 hairpins to the miRNA effector AGO1 was inhibited by Dicer-1 and its partner Loqs. Loss of these miRNA factors promoted association of pre-mir-451 with AGO1, which sliced them and permitted maturation into ∼23–26 nt products. The difference was due to the 3′ modification of single-stranded species in AGO2 by Hen1 methyltransferase, whose depletion permitted 3′ trimming of Ago-cleaved pre-miRNAs in AGO2. Surprisingly, Nibbler, a 3′–5′ exoribonuclease that trims ‘long’ mature miRNAs in AGO1, antagonized miR-451 processing. We used an in vitro reconstitution assay to identify a soluble, EDTA-sensitive activity that resects sliced pre-miRNAs in AGO1 complexes. Finally, we use deep sequencing to show that depletion of dicer-1 increases the diversity of small RNAs in AGO1, including some candidate mir-451-like loci. Altogether, we document unexpected aspects of miRNA biogenesis and Ago sorting, and provide insights into maturation of Argonaute-cleaved miRNA substrates.