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1.  Evolution of animal Piwi-interacting RNAs and prokaryotic CRISPRs 
Briefings in Functional Genomics  2012;11(4):277-288.
Piwi-interacting RNAs (piRNAs) and CRISPR RNAs (crRNAs) are two recently discovered classes of small noncoding RNA that are found in animals and prokaryotes, respectively. Both of these novel RNA species function as components of adaptive immune systems that protect their hosts from foreign nucleic acids—piRNAs repress transposable elements in animal germlines, whereas crRNAs protect their bacterial hosts from phage and plasmids. The piRNA and CRISPR systems are nonhomologous but rather have independently evolved into logically similar defense mechanisms based on the specificity of targeting via nucleic acid base complementarity. Here we review what is known about the piRNA and CRISPR systems with a focus on comparing their evolutionary properties. In particular, we highlight the importance of several factors on the pattern of piRNA and CRISPR evolution, including the population genetic environment, the role of alternate defense systems and the mechanisms of acquisition of new piRNAs and CRISPRs.
PMCID: PMC3398257  PMID: 22539610
piRNA; CRISPR; co-evolution; transposable elements; phage; plasmids
2.  Spectacle: fast chromatin state annotation using spectral learning 
Genome Biology  2015;16(1):33.
Epigenomic data from ENCODE can be used to associate specific combinations of chromatin marks with regulatory elements in the human genome. Hidden Markov models and the expectation-maximization (EM) algorithm are often used to analyze epigenomic data. However, the EM algorithm can have overfitting problems in data sets where the chromatin states show high class-imbalance and it is often slow to converge. Here we use spectral learning instead of EM and find that our software Spectacle overcame these problems. Furthermore, Spectacle is able to find enhancer subtypes not found by ChromHMM but strongly enriched in GWAS SNPs. Spectacle is available at
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-015-0598-0) contains supplementary material, which is available to authorized users.
PMCID: PMC4355146  PMID: 25786205
3.  Variation in piRNA and Transposable Element Content in Strains of Drosophila melanogaster 
Genome Biology and Evolution  2014;6(10):2786-2798.
Transposable elements (TEs) are one of the most important features of genome architecture, so their evolution and relationship with host defense mechanisms have been topics of intense study, especially in model systems such as Drosophila melanogaster. Recently, a novel small RNA-based defense mechanism in animals called the Piwi-interacting RNA (piRNA) pathway was discovered to form an adaptive defense mechanism against TEs. To investigate the relationship between piRNA and TE content between strains of a species, we sequenced piRNAs from 16 inbred lines of D. melanogaster from the Drosophila Genetic Reference Panel. Instead of a global correlation of piRNA expression and TE content, we found evidence for a host response through de novo piRNA production from novel TE insertions. Although approximately 20% of novel TE insertions induced de novo piRNA production, the abundance of de novo piRNAs was low and did not markedly affect the global pool of ovarian piRNAs. Our results provide new insights into the evolution of TEs and the piRNA system in an important model organism.
PMCID: PMC4224344  PMID: 25267446
piRNA; transposable elements; Drosophila melanogaster; de novo piRNA production
4.  MixMir: microRNA motif discovery from gene expression data using mixed linear models 
Nucleic Acids Research  2014;42(17):e135.
microRNAs (miRNAs) are a class of ∼22nt non-coding RNAs that potentially regulate over 60% of human protein-coding genes. miRNA activity is highly specific, differing between cell types, developmental stages and environmental conditions, so the identification of active miRNAs in a given sample is of great interest. Here we present a novel computational approach for analyzing both mRNA sequence and gene expression data, called MixMir. Our method corrects for 3’ UTR background sequence similarity between transcripts, which is known to correlate with mRNA transcript abundance. We demonstrate that after accounting for kmer sequence similarities in 3’ UTRs, a statistical linear model based on motif presence/absence can effectively discover active miRNAs in a sample. MixMir utilizes fast software implementations for solving mixed linear models, which are widely used in genome-wide association studies (GWASs). Essentially we use 3’ UTR sequence similarity in place of population cryptic relatedness in the GWAS problem. Compared to similar methods such as miReduce, Sylamer and cWords, we found that MixMir performed better at discovering true miRNA motifs in three mouse Dicer-knockout experiments from different tissues, two of which were collected by our group. We confirmed these results on protein and mRNA expression data obtained from miRNA transfection experiments in human cell lines. MixMir can be freely downloaded from
PMCID: PMC4176157  PMID: 25081207
5.  A comprehensive analysis of piRNAs from adult human testis and their relationship with genes and mobile elements 
BMC Genomics  2014;15(1):545.
Piwi-interacting RNAs (piRNAs) are a recently discovered class of small non-coding RNAs whose best-understood function is to repress mobile element (ME) activity in animal germline. To date, nearly all piRNA studies have been conducted in model organisms and little is known about piRNA diversity, target specificity and biological function in human.
Here we performed high-throughput sequencing of piRNAs from three human adult testis samples. We found that more than 81% of the ~17 million putative piRNAs mapped to ~6,000 piRNA-producing genomic clusters using a relaxed definition of clusters. A set of human protein-coding genes produces a relatively large amount of putative piRNAs from their 3’UTRs, and are significantly enriched for certain biological processes, suggestive of non-random sampling by the piRNA biogenesis machinery. Up to 16% of putative piRNAs mapped to a few hundred annotated long non-coding RNA (lncRNA) genes, suggesting that some lncRNA genes can act as piRNA precursors. Among major ME families, young families of LTR and endogenous retroviruses have a greater association with putative piRNAs than other MEs. In addition, piRNAs preferentially mapped to specific regions in the consensus sequences of several ME (sub)families and some piRNA mapping peaks showed patterns consistent with the “ping-pong” cycle of piRNA targeting and amplification.
Overall our data provide a comprehensive analysis and improved annotation of human piRNAs in adult human testes and shed new light into the relationship of piRNAs with protein-coding genes, lncRNAs, and mobile genetic elements in human.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-545) contains supplementary material, which is available to authorized users.
PMCID: PMC4094622  PMID: 24981367
Human piRNA; piRNA cluster; Protein coding gene; Mobile element; High-throughput sequencing
6.  Evidence for the biogenesis of more than 1,000 novel human microRNAs 
Genome Biology  2014;15(4):R57.
MicroRNAs (miRNAs) are established regulators of development, cell identity and disease. Although nearly two thousand human miRNA genes are known and new ones are continuously discovered, no attempt has been made to gauge the total miRNA content of the human genome.
Employing an innovative computational method on massively pooled small RNA sequencing data, we report 2,469 novel human miRNA candidates of which 1,098 are validated by in-house and published experiments. Almost 300 candidates are robustly expressed in a neuronal cell system and are regulated during differentiation or when biogenesis factors Dicer, Drosha, DGCR8 or Ago2 are silenced. To improve expression profiling, we devised a quantitative miRNA capture system. In a kidney cell system, 400 candidates interact with DGCR8 at transcript positions that suggest miRNA hairpin recognition, and 1,000 of the new miRNA candidates interact with Ago1 or Ago2, indicating that they are directly bound by miRNA effector proteins. From kidney cell CLASH experiments, in which miRNA-target pairs are ligated and sequenced, we observe hundreds of interactions between novel miRNAs and mRNA targets. The novel miRNA candidates are specifically but lowly expressed, raising the possibility that not all may be functional. Interestingly, the majority are evolutionarily young and overrepresented in the human brain.
In summary, we present evidence that the complement of human miRNA genes is substantially larger than anticipated, and that more are likely to be discovered in the future as more tissues and experimental conditions are sequenced to greater depth.
PMCID: PMC4054668  PMID: 24708865
7.  Modes of Action of ADP-Ribosylated Elongation Factor 2 in Inhibiting the Polypeptide Elongation Cycle: A Modeling Study 
PLoS ONE  2013;8(7):e66446.
Despite the fact that ADP-ribosylation of eukaryotic elongation factor 2 (EF2) leads to inhibition of protein synthesis, the mechanism by which ADP-ribosylated EF2 (ADPR•EF2) causes this inhibition remains controversial. Here, we applied modeling approaches to investigate the consequences of various modes of ADPR•EF2 inhibitory actions on the two coupled processes, the polypeptide chain elongation and ADP-ribosylation of EF2. Modeling of experimental data indicates that ADPR•EF2 fully blocks the late-phase translocation of tRNAs; but the impairment in the translocation upstream process, mainly the GTP-dependent factor binding with the pretranslocation ribosome and/or the guanine nucleotide exchange in EF2, is responsible for the overall inhibition kinetics. The reduced ADPR•EF2-ribosome association spares the ribosome to bind and shield native EF2 against toxin attack, thereby deferring the inhibition of protein synthesis inhibition and inactivation of EF2. Minimum association with the ribosome also keeps ADPR•EF2 in an accessible state for toxins to catalyze the reverse reaction when nicotinamide becomes available. Our work underscores the importance of unveiling the interactions between ADPR•EF2 and the ribosome, and argues against that toxins inhibit protein synthesis through converting native EF2 to a competitive inhibitor to actively disable the ribosome.
PMCID: PMC3704607  PMID: 23861744
8.  Selective Constraint on Copy Number Variation in Human Piwi-Interacting RNA Loci 
PLoS ONE  2012;7(10):e46611.
Piwi-interacting RNAs (piRNAs) are a recently discovered class of small non-coding RNA found in animals. PiRNAs are primarily expressed in the germline where their best understood function is to repress transposable elements. Unlike previous studies that investigated the evolution of piRNA-generating loci at the level of nucleotide substitutions, here we studied the evolution of piRNA-generating loci at the level of copy number variation (i.e. duplications and deletions) using genome-wide copy number variation data from three human populations. Our analysis shows that at the level of copy number variation there is strong selective constraint and a very high mutation rate in human piRNA-generating loci. Our results differ from a model of positive selection on copy number variation in piRNA-generating loci previously proposed in rodents. We discuss possible reasons for this difference based on the transposable element insertion histories in the rodent and primate lineages.
PMCID: PMC3464240  PMID: 23056369
9.  High Definition Spectral Domain Optical Coherence Tomography Findings in Three Patients with Solar Retinopathy and Review of the Literature 
To describe ocular findings in 3 cases of solar retinopathy using high definition, spectral domain optical coherence tomography (SD-OCT) and review the literature for optical coherence tomography (OCT) characteristics associated with worse vision.
Case series and retrospective review of clinical features and Spectralis SD-OCT (Heidelberg Engineering, Vista, California, United States of America). A literature review of OCT findings in cases of solar retinopathy reported on MEDLINE was also performed and analyzed.
Six eyes of 3 patients with solar retinopathy revealed significant foveal pathology. Visual acuity ranged from Snellen 20/30 to 20/50. High definition SD-OCT demonstrated defects at the level of the inner and outer segment junction of the photoreceptors as well as in the inner high reflective layer. There was a significant correlation between chronic disruption of the inner photoreceptor junction with worse vision based on the current case series and literature review.
Screening patients with exposure to central foveal damage from solar retinopathy with high definition SD-OCT improves diagnosis and assessment of photoreceptor damage and vision loss.
PMCID: PMC3394112  PMID: 22798967
Solar retinopathy; fovea; spectral domain optical coherence tomography; Spectralis SD-OCT.

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