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1.  miR-124 Regulates Diverse Aspects of Rhythmic Behavior in Drosophila 
The Journal of Neuroscience  2016;36(12):3414-3421.
Circadian clocks enable organisms to anticipate and adapt to fluctuating environmental conditions. Despite substantial knowledge of central clock machineries, we have less understanding of how the central clock's behavioral outputs are regulated. Here, we identify Drosophila miR-124 as a critical regulator of diurnal activity. During normal light/dark cycles, mir-124 mutants exhibit profoundly abnormal locomotor activity profiles, including loss of anticipatory capacities at morning and evening transitions. Moreover, mir-124 mutants exhibited striking behavioral alterations in constant darkness (DD), including a temporal advance in peak activity. Nevertheless, anatomical and functional tests demonstrate a normal circadian pacemaker in mir-124 mutants, indicating this miRNA regulates clock output. Among the extensive miR-124 target network, heterozygosity for targets in the BMP pathway substantially corrected the evening activity phase shift in DD. Thus, excess BMP signaling drives specific circadian behavioral output defects in mir-124 knock-outs.
SIGNIFICANCE STATEMENT Circadian clocks control rhythmic behaviors of most life-forms. Despite extensive knowledge of the central clock, there is less understanding of how its behavioral outputs are regulated. Here, we identify a conserved neural microRNA as a critical regulator of diurnal behavior. We find Drosophila mir-124 mutants exhibit robust activity abnormalities during normal light/dark cycles and during constant darkness. Nevertheless, as the central pacemaker is functional in these mutants, miR-124 regulates clock output. We provide mechanistic insight by showing deregulation of miR-124 targets in BMP signaling drives specific mir-124 defects. In summary, Drosophila mir-124 mutants reveal post-transcriptional control of circadian activities, and impact of BMP signaling in behavioral output.
doi:10.1523/JNEUROSCI.3287-15.2016
PMCID: PMC4804003  PMID: 27013671
activity mode; activity phase; BMP signaling; circadian rhythm; Drosophila; microRNA
2.  Hox miRNA regulation within the Drosophila Bithorax Complex: patterning behavior 
Mechanisms of development  2015;138(0 2):151-159.
The study of Drosophila Hox genes, located in the Antennapedia complex (ANT-C) and Bithorax complex (BX-C), have provided fundamental insights into mechanisms of how the segments of the animal body plan are specified. Notably, even though the analysis of the BX-C formally began over a century ago, surprises continue to emerge regarding its regulation and function. Even simply the gene content of the BX-C has been regularly revised in past years, especially with regard to non-coding RNAs (ncRNAs), including microRNAs. In this perspective, we review the history of studies of non-coding transcription in the BX-C, and highlight recent studies of its miRNAs that provide new insights into their tissue-specific roles in Hox gene regulation. In particular, we have demonstrated unexpected importance of endogenous BX-C miRNAs to restrict the spatial accumulation of Hox proteins and their TALE cofactors in the ventral nerve cord, and link this to aberrant neural differentiation and reproductive behavior. These findings open new directions on studying Hox miRNA function, and we speculate that further understanding of their roles in insect models may provide new leads for studying the enigmatic biological functions of analogous miRNAs located in vertebrate Hox clusters.
doi:10.1016/j.mod.2015.08.006
PMCID: PMC4673027  PMID: 26311219
3.  Selective suppression of the splicing-mediated microRNA pathway by the terminal uridyltransferase Tailor 
Molecular cell  2015;59(2):217-228.
Summary
Several terminal uridyltransferases (TUTases) are known to modulate small RNA biogenesis and/or function via diverse mechanisms. Here, we demonstrate that Drosophila splicing-derived pre-miRNAs (mirtrons) are efficiently modified by the previously uncharacterized TUTase, Tailor. Tailor is necessary and sufficient for mirtron hairpin uridylation, and this modification inhibits mirtron biogenesis. Genomewide analyses demonstrate that mirtrons are dominant Tailor substrates, and three features contribute to substrate specificity. First, reprogramming experiments show Tailor preferentially identifies splicing-derived miRNAs. Second, in vitro tests indicate Tailor prefers substrate hairpins over mature miRNAs. Third, Tailor exhibit sequence preference for 3′-terminal AG, a defining mirtron characteristic. Our work supports that Tailor preferentially suppresses biogenesis of mirtrons, an evolutionarily adventitious pre-miRNA substrate class. Moreover, we detect preferential activity of Tailor on 3′-G canonical pre-miRNAs, and specific depletion of such loci from the pool of conserved miRNAs. Thus, Tailor activity may have had collateral impact on shaping populations of canonical miRNAs.
Graphical Abstract
doi:10.1016/j.molcel.2015.05.034
PMCID: PMC4517475  PMID: 26145174
4.  Alteration of miRNA activity via context-specific modifications of Argonaute proteins 
Trends in cell biology  2014;24(9):546-553.
microRNAs (miRNAs) are enclosed within Argonaute proteins, the downstream effectors of small RNA-mediated gene silencing. As miRNAs mediate extensive networks of post-transcriptional control, cells have evolved multiple strategies to control their activity with precision. A growing theme of recent years regards how post-translational modifications of Argonaute proteins, such as prolyl-hydroxylation, phosphorylation, ubiquitination, and poly-ADP-ribosylation, alter miRNA activity at global or specific levels. In this review, we discuss recent advances on Argonaute modifications in mammalian cells, and emphasize how such alterations modulate small RNA function to coordinate appropriate downstream cellular responses. These findings provide a framework to understand how Argonaute protein modifications are linked to reorganization of post-transcriptional regulatory networks, enabling dynamic responses to diverse external stimuli and changing environmental conditions.
doi:10.1016/j.tcb.2014.04.008
PMCID: PMC4149831  PMID: 24865524
Argonaute; microRNA; hydroxylation; phosphorylation; ubiquitination; PARylation
5.  Homeotic function of Drosophila Bithorax-Complex miRNAs mediates fertility by restricting multiple Hox genes and TALE cofactors in the central nervous system 
Developmental cell  2014;29(6):635-648.
The Drosophila Bithorax-Complex (BX-C) Hox cluster contains a bidirectionally-transcribed miRNA locus, and a deletion mutant (∆mir) lays no eggs and is completely sterile. We show these miRNAs are expressed and active in distinct spatial registers along the anterior-posterior axis in the central nervous system. ∆mir larvae derepress a network of direct homeobox gene targets in the posterior ventral nerve cord (VNC), including BX-C genes and their TALE cofactors. These are phenotypically critical targets, since sterility of ∆mir mutants was substantially rescued by heterozygosity of these genes. The posterior VNC contains Ilp7+ oviduct motoneurons, whose innervation and morphology are defective in ∆mir females, and substantially rescued by heterozygosity of ∆mir targets, especially within the BX-C. Collectively, we reveal (1) critical roles for Hox miRNAs that determine segment-specific expression of homeotic genes, which are not masked by transcriptional regulation, and (2) that BX-C miRNAs are essential for neural patterning and reproductive behavior.
doi:10.1016/j.devcel.2014.04.023
PMCID: PMC4111139  PMID: 24909902
6.  Adaptive Regulation of Testis Gene Expression and Control of Male Fertility by the Drosophila Harpin RNA Pathway 
Molecular cell  2014;57(1):165-178.
SUMMARY
Although endogenous siRNAs (endo-siRNAs) have been described in many species, still little is known about their endogenous utility. Here, we show that Drosophila hairpin RNAs (hpRNAs) generate an endo-siRNA class with predominant expression in testes. Although hpRNAs are universally recently evolved, we identify highly complementary protein-coding targets for all hpRNAs. Importantly, we find broad evidence for evolutionary divergences that preferentially maintain compensatory pairing between hpRNAs and targets, serving as first evidence for adaptive selection for siRNA-mediated target regulation in metazoans. We demonstrate organismal impact of hpRNA activity, since knockout of hpRNA1 derepresses its target ATP synthase-β in testes and compromises spermatogenesis and male fertility. Moreover, we reveal surprising male-specific impact of RNAi factors on germ cell development and fertility, consistent with testis-directed function of the hpRNA pathway. Finally, the collected hpRNA loci chronicle an evolutionary timeline that reflects their origins from prospective target genes, mirroring a strategy described for plant miRNAs.
doi:10.1016/j.molcel.2014.11.025
PMCID: PMC4289472  PMID: 25544562
7.  Synchronous right hepatectomy and cesarean section in a pregnant lady with hepatocellular carcinoma 
INTRODUCTION
Cancer in pregnancy is rare and hepatocellular carcinoma (HCC) during pregnancy is even rarer. Due to limited experience, management of these patients remains challenging.
PRESENTATION OF CASE
A 33-year old pregnant lady presented with HCC at 28 weeks of gestation. She underwent synchronous cesarean section and right hepatectomy at 32 weeks of gestation. The post-operative course was uneventful. She was discharged home on day 10 after surgery. Histolopathology confirmed HCC. The surgical resection margins were clear. At a follow-up of 3 months after surgery, the mother was disease free and the infant was well.
DISCUSSION
HCC during pregnancy is extremely rare. The experience in its management and outcomes are lacking. In managing any patient diagnosed with a malignant neoplasm in pregnancy, both the mother and the fetus have to be considered.
CONCLUSION
With adequate preoperative assessment and a good management strategy, good results can be obtained for both the mother and the baby for a pregnant patient with HCC.
doi:10.1016/j.ijscr.2012.10.014
PMCID: PMC3537950  PMID: 23159911
Hepatocellular carcinoma; Hepatectomy; Pregnancy; Cesarean section
8.  Genomewide analysis of Drosophila circular RNAs reveals their structural and sequence properties and age-dependent neural accumulation 
Cell reports  2014;9(5):1966-1980.
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 >2500 fruitfly circular RNAs. These mostly derive from back-splicing of protein-coding genes and lack poly(A) tails, and 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 >1000 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 dramatically relative to linear isoforms during CNS aging, and constitute a novel aging biomarker.
doi:10.1016/j.celrep.2014.10.062
PMCID: PMC4279448  PMID: 25544350
9.  The Hippo pathway regulates hematopoiesis in Drosophila melanogaster 
Current biology : CB  2014;24(22):2673-2680.
SUMMARY
The Salvador-Warts-Hippo (Hippo) pathway is an evolutionarily conserved regulator of organ growth and cell fate. It performs these functions in epithelial and neural tissues of both insects and mammals, and in mammalian organs such as the liver and heart. Despite rapid advances in Hippo pathway research, a definitive role for this pathway in hematopoiesis has remained enigmatic. The hematopoietic compartments of Drosophila melanogaster and mammals possess several conserved features [1, 2]. D. melanogaster possess three types of hematopoietic cells that most closely resemble mammalian myeloid cells: plasmatocytes (macrophage-like cells), crystal cells (involved in wound healing) and lamellocytes (which encapsulate parasites). The proteins that control differentiation of these cells also control important blood lineage decisions in mammals [3–10]. Here, we define the Hippo pathway as a key mediator of hematopoiesis by showing that it controls differentiation and proliferation of the two major types of D. melanogaster blood cells, plasmatocytes and crystal cells. In animals lacking the downstream Hippo pathway kinase Warts, lymph gland cells overproliferated, differentiated prematurely and often adopted a mixed lineage fate. The Hippo pathway regulated crystal cell numbers by both cell autonomous and non-cell autonomous mechanisms. Yorkie and its partner transcription factor Scalloped were found to regulate transcription of the Runx family transcription factor, Lozenge, which is a key regulator of crystal cell fate. Further, Yorkie or Scalloped hyperactivation induced ectopic crystal cells in a non-cell autonomous, and Notch pathway-dependent fashion.
doi:10.1016/j.cub.2014.10.031
PMCID: PMC4269548  PMID: 25454587
10.  A Genome-Wide Survey of Sexually Dimorphic Expression of Drosophila miRNAs Identifies the Steroid Hormone-Induced miRNA let-7 as a Regulator of Sexual Identity 
Genetics  2014;198(2):647-668.
MiRNAs bear an increasing number of functions throughout development and in the aging adult. Here we address their role in establishing sexually dimorphic traits and sexual identity in male and female Drosophila. Our survey of miRNA populations in each sex identifies sets of miRNAs differentially expressed in male and female tissues across various stages of development. The pervasive sex-biased expression of miRNAs generally increases with the complexity and sexual dimorphism of tissues, gonads revealing the most striking biases. We find that the male-specific regulation of the X chromosome is relevant to miRNA expression on two levels. First, in the male gonad, testis-biased miRNAs tend to reside on the X chromosome. Second, in the soma, X-linked miRNAs do not systematically rely on dosage compensation. We set out to address the importance of a sex-biased expression of miRNAs in establishing sexually dimorphic traits. Our study of the conserved let-7-C miRNA cluster controlled by the sex-biased hormone ecdysone places let-7 as a primary modulator of the sex-determination hierarchy. Flies with modified let-7 levels present doublesex-related phenotypes and express sex-determination genes normally restricted to the opposite sex. In testes and ovaries, alterations of the ecdysone-induced let-7 result in aberrant gonadal somatic cell behavior and non-cell-autonomous defects in early germline differentiation. Gonadal defects as well as aberrant expression of sex-determination genes persist in aging adults under hormonal control. Together, our findings place ecdysone and let-7 as modulators of a somatic systemic signal that helps establish and sustain sexual identity in males and females and differentiation in gonads. This work establishes the foundation for a role of miRNAs in sexual dimorphism and demonstrates that similar to vertebrate hormonal control of cellular sexual identity exists in Drosophila.
doi:10.1534/genetics.114.169268
PMCID: PMC4196619  PMID: 25081570
miRNA; sex determination; ecdysteroid; gonad; development; Drosophila; genetics of sex
11.  A Deathly DNAse activity for Dicer 
Developmental cell  2010;18(5):692-694.
Recently reporting in Science, Nakagawa et al. describe an unexpected role for Dicer in chromosome fragmentation during apoptosis in C. elegans. They find that cleavage of DCR-1 by the caspase CED-3 redirects its regulatory activity, by destroying its dsRNAse activity while activating an intrinsic DNAse activity.
doi:10.1016/j.devcel.2010.05.004
PMCID: PMC4583219  PMID: 20493803
12.  A transgenic resource for conditional competitive inhibition of conserved Drosophila microRNAs 
Nature communications  2015;6:7279.
Although the impact of microRNAs (miRNAs) in development and disease is well established, understanding the function of individual miRNAs remains challenging. Development of competitive inhibitor molecules such as miRNA sponges has allowed the community to address individual miRNA function in vivo. However, the application of these loss-of-function strategies has been limited. Here we offer a comprehensive library of 141 conditional miRNA sponges targeting well-conserved miRNAs in Drosophila. Ubiquitous miRNA sponge delivery and consequent systemic miRNA inhibition uncovers a relatively small number of miRNA families underlying viability and gross morphogenesis, with false discovery rates in the 4–8% range. In contrast, tissue-specific silencing of muscle-enriched miRNAs reveals a surprisingly large number of novel miRNA contributions to the maintenance of adult indirect flight muscle structure and function. A strong correlation between miRNA abundance and physiological relevance is not observed, underscoring the importance of unbiased screens when assessing the contributions of miRNAs to complex biological processes.
doi:10.1038/ncomms8279
PMCID: PMC4471878  PMID: 26081261
13.  A transgenic resource for conditional competitive inhibition of conserved Drosophila microRNAs 
Nature Communications  2015;6:7279.
Although the impact of microRNAs (miRNAs) in development and disease is well established, understanding the function of individual miRNAs remains challenging. Development of competitive inhibitor molecules such as miRNA sponges has allowed the community to address individual miRNA function in vivo. However, the application of these loss-of-function strategies has been limited. Here we offer a comprehensive library of 141 conditional miRNA sponges targeting well-conserved miRNAs in Drosophila. Ubiquitous miRNA sponge delivery and consequent systemic miRNA inhibition uncovers a relatively small number of miRNA families underlying viability and gross morphogenesis, with false discovery rates in the 4–8% range. In contrast, tissue-specific silencing of muscle-enriched miRNAs reveals a surprisingly large number of novel miRNA contributions to the maintenance of adult indirect flight muscle structure and function. A strong correlation between miRNA abundance and physiological relevance is not observed, underscoring the importance of unbiased screens when assessing the contributions of miRNAs to complex biological processes.
Transgenic RNA ‘sponges' that inhibit specific microRNAs are valuable tools for functional studies. Fulga et al. present a library of conditional microRNA sponges in Drosophila, and reveal widespread involvement of microRNAs in the maintenance of adult muscle structure and function.
doi:10.1038/ncomms8279
PMCID: PMC4471878  PMID: 26081261
14.  The evolution and functional diversification of animal microRNA genes 
Cell research  2008;18(10):985-996.
microRNAs (miRNAs) are an abundant class of ~22 nucleotide (nt) regulatory RNAs that are pervasive in higher eukaryotic genomes. In order to fully understand their prominence in genomes, it is necessary to elucidate the molecular mechanisms that can diversify miRNA activities. In this review, we describe some of the many strategies that allow novel miRNA functions to emerge, with particular emphasis on how miRNA genes evolve in animals. These mechanisms include changes in their sequence, processing, or expression pattern; acquisition of miRNA* functionality or antisense processing; and de novo gene birth. The facility and versatility of miRNAs to evolve and change likely underlies how they have become dominant constituents of higher genomes.
doi:10.1038/cr.2008.278
PMCID: PMC2712117  PMID: 18711447
microRNA; evolution; subfunctionalization; neofunctionalization
15.  Analysis of Nearly One Thousand Mammalian Mirtrons Reveals Novel Features of Dicer Substrates 
PLoS Computational Biology  2015;11(9):e1004441.
Mirtrons are microRNA (miRNA) substrates that utilize the splicing machinery to bypass the necessity of Drosha cleavage for their biogenesis. Expanding our recent efforts for mammalian mirtron annotation, we use meta-analysis of aggregate datasets to identify ~500 novel mouse and human introns that confidently generate diced small RNA duplexes. These comprise nearly 1000 total loci distributed in four splicing-mediated biogenesis subclasses, with 5'-tailed mirtrons as, by far, the dominant subtype. Thus, mirtrons surprisingly comprise a substantial fraction of endogenous Dicer substrates in mammalian genomes. Although mirtron-derived small RNAs exhibit overall expression correlation with their host mRNAs, we observe a subset with substantial differences that suggest regulated processing or accumulation. We identify characteristic sequence, length, and structural features of mirtron loci that distinguish them from bulk introns, and find that mirtrons preferentially emerge from genes with larger numbers of introns. While mirtrons generate miRNA-class regulatory RNAs, we also find that mirtrons exhibit many features that distinguish them from canonical miRNAs. We observe that conventional mirtron hairpins are substantially longer than Drosha-generated pre-miRNAs, indicating that the characteristic length of canonical pre-miRNAs is not a general feature of Dicer substrate hairpins. In addition, mammalian mirtrons exhibit unique patterns of ordered 5' and 3' heterogeneity, which reveal hidden complexity in miRNA processing pathways. These include broad 3'-uridylation of mirtron hairpins, atypically heterogeneous 5' termini that may result from exonucleolytic processing, and occasionally robust decapitation of the 5' guanine (G) of mirtron-5p species defined by splicing. Altogether, this study reveals that this extensive class of non-canonical miRNA bears a multitude of characteristic properties, many of which raise general mechanistic questions regarding the processing of endogenous hairpin transcripts.
Author Summary
Bulk miRNAs in most animal cells derive from cleavage of hairpin precursor transcripts by the Drosha and Dicer RNase III enzymes. A variety of non-canonical miRNA biogenesis strategies are known, including "mirtrons" for which splicing substitutes for Drosha. However, as non-canonical miRNAs usually account for a minor fraction of total miRNA reads, the alternate pathways are often considered of minor impact. In this study, we describe meta-analysis of >1000 mouse and human small RNA datasets, and surprisingly, find evidence of nearly 1000 mirtron using stringent criteria, half of which were novel annotations. Therefore, mirtrons comprise a substantial fraction of total Dicer substrates. We use this catalog to perform diverse sequence and structural analyses of mirtron loci that differentiate them from bulk introns and also to perform diverse comparisons of mirtrons and canonical miRNAs. Surprisingly, we find that many seemingly fundamental features of miRNA genes, which have been well-studied over the years, are not actually general features of miRNAs, but specifically those of hairpins that transit the Drosha-Dicer pathway. Instead, the nearly 1000 splicing-derived miRNAs reveal expanded and/or distinct properties encompassed by endogenous Dicer substrates in mammalian cells. These features reveal hidden impacts that shape miRNA processing, function, and evolution.
doi:10.1371/journal.pcbi.1004441
PMCID: PMC4556696  PMID: 26325366
17.  Exploiting Drosophila genetics to understand microRNA function and regulation 
Although a great deal is known about the identity, biogenesis, and targeting capacity of microRNAs (miRNAs) in animal cells, far less is known about their functional requirements at the organismal level. Much remains to be understood about the necessity of miRNAs for overt phenotypes, the identity of critical miRNA targets, and the control of miRNA transcription. In this review, we provide an overview of genetic strategies to study miRNAs in the Drosophila system, including loss- and gain-of-function techniques, genetic interaction strategies, and transgenic reporters of miRNA expression and activity. As we illustrate the usage of these techniques in intact Drosophila, we see certain recurrent themes for miRNA functions, including energy homeostasis, apoptosis suppression, growth control, and regulation of core cell signaling pathways. Overall, we hope that this exposition of Drosophila genetic techniques, well-known to the legions of fly geneticists and used to study all genes, can inform the general miRNA community that focuses on other biochemical, molecular, computational, and structural avenues. Clearly, it is the combination of these myriad techniques that has accelerated miRNA research to its extraordinary pace.
doi:10.1016/B978-0-12-387038-4.00008-2
PMCID: PMC4505732  PMID: 22365740
18.  A signaling-induced switch in Dicer localization and function 
Developmental cell  2014;31(5):523-524.
In this issue of Developmental Cell, Drake and colleagues (2014) report that Ras signaling results in Dicer phosphorylation, which induces its nuclear localization and modulates its function. This regulatory strategy, conserved in mammals, allows dynamic control of miRNA function required for C. elegans germline development and oogenesis.
doi:10.1016/j.devcel.2014.11.032
PMCID: PMC4505742  PMID: 25490263
19.  Maternal mRNA deadenylation and decay by the piRNA pathway in the early Drosophila embryo 
Nature  2010;467(7319):1128-1132.
doi:10.1038/nature09465
PMCID: PMC4505748  PMID: 20953170
CCR4 deadenylase; mRNA decay; nanos; piRNA; RNA silencing; Translational control
20.  Alternative polyadenylation in the nervous system: to what lengths will 3′ UTR extensions take us? 
Alternative cleavage and polyadenylation (APA) can diversify coding and non-coding regions, but has particular impact on increasing 3′ UTR diversity. Through the gain or loss of regulatory elements such as RNA binding protein and microRNA sites, APA can influence transcript stability, localization, and translational efficiency. Strikingly, the central nervous systems of invertebrate and vertebrate species express a broad range of transcript isoforms bearing extended 3′ UTRs. The molecular mechanism that permits proximal 3′ end bypass in neurons is mysterious, and only beginning to be elucidated. This landscape of neural 3′ UTR extensions, many reaching unprecedented lengths, may help service the unique post-transcriptional regulatory needs of neurons. A combination of approaches, including transcriptome-wide profiling, genetic screening to identify APA factors, biochemical dissection of alternative 3′ end formation, and manipulation of individual neural APA targets, will be necessary to gain fuller perspectives on the mechanism and biology of neural-specific 3′ UTR lengthening.
doi:10.1002/bies.201300174
PMCID: PMC4503322  PMID: 24903459
21.  Multiple In Vivo Biological Processes Are Mediated by Functionally Redundant Activities of Drosophila mir-279 and mir-996 
PLoS Genetics  2015;11(6):e1005245.
While most miRNA knockouts exhibit only subtle defects, a handful of miRNAs are profoundly required for development or physiology. A particularly compelling locus is Drosophila mir-279, which was reported as essential to restrict the emergence of CO2-sensing neurons, to maintain circadian rhythm, and to regulate ovarian border cells. The mir-996 locus is located near mir-279 and bears a similar seed, but they otherwise have distinct, conserved, non-seed sequences, suggesting their evolutionary maintenance for separate functions. We generated single and double deletion mutants of the mir-279 and mir-996 hairpins, and cursory analysis suggested that miR-996 was dispensable. However, discrepancies in the strength of individual mir-279 deletion alleles led us to uncover that all extant mir-279 mutants are deficient for mature miR-996, even though they retain its genomic locus. We therefore engineered a panel of genomic rescue transgenes into the double deletion background, allowing a pure assessment of miR-279 and miR-996 requirements. Surprisingly, detailed analyses of viability, olfactory neuron specification, and circadian rhythm indicate that miR-279 is completely dispensable. Instead, an endogenous supply of either mir-279 or mir-996 suffices for normal development and behavior. Sensor tests of nine key miR-279/996 targets showed their similar regulatory capacities, although transgenic gain-of-function experiments indicate partially distinct activities of these miRNAs that may underlie that co-maintenance in genomes. Altogether, we elucidate the unexpected genetics of this critical miRNA operon, and provide a foundation for their further study. More importantly, these studies demonstrate that multiple, vital, loss-of-function phenotypes can be rescued by endogenous expression of divergent seed family members, highlighting the importance of this miRNA region for in vivo function.
Author Summary
Amongst the small number of miRNA knockouts that exhibit substantially overt phenotypes, mutants of Drosophila mir-279 are notable. Previous studies have uncovered its essential requirements in a range of developmental and behavioral assays. Surprisingly, we find that the phenotypes attributed to mir-279 deletions depend on the unanticipated loss of expression of the downstream locus mir-996, whose genomic locus is retained in extant mir-279 mutants. These miRNAs share their seed regions but are divergent elsewhere in the mature sequences. We use precise genetic engineering to show that a single endogenous copy of either mir-279 or mir-996 can fully rescue viability, olfactory neuron, and circadian rhythm defects of double deletion animals. These data and genetic reagents set a new foundation for developmental and behavioral studies of this critical miRNA locus. More generally, these data demonstrate that multiple loss-of-function phenotypes can be rescued by endogenous expression of divergent seed family members, highlighting the importance and potentially sufficiency of this region for in vivo function.
doi:10.1371/journal.pgen.1005245
PMCID: PMC4456407  PMID: 26042831
22.  Diversity and dynamics of the Drosophila transcriptome 
Nature  2014;512(7515):393-399.
Animal transcriptomes are dynamic, each cell type, tissue and organ system expressing an ensemble of transcript isoforms that give rise to substantial diversity. We identified new genes, transcripts, and proteins using poly(A)+ RNA sequence from Drosophila melanogaster cultured cell lines, dissected organ systems, and environmental perturbations. We found a small set of mostly neural-specific genes has the potential to encode thousands of transcripts each through extensive alternative promoter usage and RNA splicing. The magnitudes of splicing changes are larger between tissues than between developmental stages, and most sex-specific splicing is gonad-specific. Gonads express hundreds of previously unknown coding and long noncoding RNAs (lncRNAs) some of which are antisense to protein-coding genes and produce short regulatory RNAs. Furthermore, previously identified pervasive intergenic transcription occurs primarily within newly identified introns. The fly transcriptome is substantially more complex than previously recognized arising from combinatorial usage of promoters, splice sites, and polyadenylation sites.
doi:10.1038/nature12962
PMCID: PMC4152413  PMID: 24670639
23.  Dicer-independent, Ago2-mediated microRNA biogenesis in vertebrates 
Cell Cycle  2010;9(22):4455-4460.
A canonical biogenesis pathway involving sequential cleavage by the Drosha and Dicer RNAse III enzymes governs the maturation of most animal microRNAs. However, there exist a variety of alternative miRNA biogenesis pathways, most of which bypass Drosha processing. Recently, three groups described for the first time a vertebrate microRNA pathway that bypasses Dicer cleavage. This mechanism was characterized with respect to the highly conserved vertebrate gene mir-451, for which Drosha processing yields a short (42 nucleotide) hairpin that is directly loaded into Ago2, the sole vertebrate “Slicer” Argonaute. Ago2-mediated cleavage of this hairpin yields a 30 nucleotide intermediate, whose 3′ end is resected to generate the dominantly cloned ∼23 nucleotide mature miR-451. Knowledge of this pathway provides an unprecedented tool with which to express microRNAs and small interfering RNAs in Dicer mutant cells. More generally, the mir-451 backbone constitutes a new platform for gene silencing that complements existing shRNA technology.
doi:10.4161/cc.9.22.13958
PMCID: PMC3048044  PMID: 21088485
mir-451; Ago2; Slicer; Dicer-independent; erythropoiesis
24.  Bi-functional cross-linking reagents efficiently capture protein-DNA complexes in Drosophila embryos 
Fly  2013;8(1):43-51.
Chromatin immunoprecipitation (ChIP) is widely used for mapping DNA-protein interactions across eukaryotic genomes in cells, tissues or even whole organisms. Critical to this procedure is the efficient cross-linking of chromatin-associated proteins to DNA sequences that are in close proximity. Since the mid-nineties formaldehyde fixation has been the method of choice. However, some protein-DNA complexes cannot be successfully captured for ChIP using formaldehyde. One such formaldehyde refractory complex is the developmentally regulated insulator factor, Elba. Here we describe a new embryo fixation procedure using the bi-functional cross-linking reagents DSG (disuccinimidyl glutarate) and DSP (dithiobis[succinimidyl propionate). We show that unlike standard formaldehyde fixation protocols, it is possible to capture Elba association with insulator elements in 2–5 h embryos using this new cross-linking procedure. We show that this new cross-linking procedure can also be applied to localize nuclear proteins that are amenable to ChIP using standard formaldehyde cross-linking protocols, and that in the cases tested the enrichment was generally superior to that achieved using formaldehyde cross-linking.
doi:10.4161/fly.26805
PMCID: PMC3974894  PMID: 24135698
chromatin immunoprecipitation; ChIP; formadelhyde; bi-functional cross-linkers; insulators; DSG DSP; DNA binding; Elba; Insensitive
25.  Associating liver partition and portal vein ligation for a patient with hepatocellular carcinoma with a background of hepatitis B related fibrotic liver 
Highlights
•Associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) has rarely been reported for patients with hepatocellular carcinoma (HCC).•ALPPS is feasible even in the context of HCC with a background of chronic hepatitis B related liver fibrosis.•The hypertrophy rate of the liver remnant was slower and the time needed between the two stages of the operation was longer.
INTRODUCTION
Associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) has recently been developed for patients with predicted insufficient future liver remnant volumes to induce more rapid hepatic hypertrophy and increase resectability. In the medical literature, the use of ALPPS in hepatocellular carcinoma (HCC) has rarely been reported.
PRESENTATION OF CASE
We reported the use of ALPPS in a patient with primarily unresectable HCC arising from a background of hepatitis B related liver fibrosis. Preoperative computed tomography (CT) showed 2 large conglomerated tumors measuring 16 cm × 10.5 cm in liver segments 5, 6, 7 and 8, and at least 3 satellite nodules with the largest one measuring 3 cm around the main tumor and another 4 cm tumor in segment 4. Right trisectionectomy after ALPPS was successfully performed. He was discharged from hospital on postoperative day 13 after the second operation. Follow-up CT scan at 6 weeks after the second operation showed further hypertrophy of the liver remnant and no liver recurrence.
DISCUSSION
Our case showed that this novel strategy is feasible even in the context of a background of chronic hepatitis B related liver fibrosis, although the hypertrophy rate was a little bit slower and the time needed was longer.
CONCLUSION
ALPPS is also feasible in liver fibrosis. It gives hope to patients with HCC who previously were considered as having unresectable diseases. More studies are needed to further evaluate the effectiveness and oncological outcomes of ALPPS from these patients.
doi:10.1016/j.ijscr.2014.11.008
PMCID: PMC4275789  PMID: 25460479
ALPPS; PPVE; Trisectionectomy; Future liver remnant; Hepatocellular carcinoma; Hepatitis B

Results 1-25 (80)