Papers by Ravi Sachidanandam
Proceedings of the National Academy of Sciences, 2010
The discovery of regulatory small RNAs continues to reshape paradigms in both molecular biology a... more The discovery of regulatory small RNAs continues to reshape paradigms in both molecular biology and virology. Here we describe examples of influenza A virus-derived small viral RNAs (svRNAs). svRNAs are 22-27 nt in length and correspond to the 5′ end of each of the viral genomic RNA (vRNA) segments. Expression of svRNA correlates with the accumulation of vRNA and a bias in RNAdependent RNA polymerase (RdRp) activity from transcription toward genome replication. Synthesis of svRNA requires the RdRp, nucleoprotein and the nuclear export protein NS2. In addition, svRNA is detectable during replication of various influenza A virus subtypes across multiple host species and associates physically with the RdRp. We demonstrate that depletion of svRNA has a minimal impact on mRNA and complementary vRNA (cRNA) but results in a dramatic loss of vRNA in a segment-specific manner. We propose that svRNA triggers the viral switch from transcription to replication through interactions with the viral polymerase machinery. Taken together, the discovery of svRNA redefines the mechanistic switch of influenza virus transcription/replication and provides a potential target for broad-range, anti-influenza virus-based therapeutics.
Nature Methods, 2012
We introduce two large-scale resources for functional analysis of microRNA-a decoy/sponge library... more We introduce two large-scale resources for functional analysis of microRNA-a decoy/sponge library for inhibiting microRNA function and a sensor library for monitoring microRNA activity. To take advantage of the sensor library, we developed a high-throughput assay called Sensor-seq, which permits the activity of hundreds of microRNAs to be quantified simultaneously. Using this approach, we show that only the most abundant microRNAs within a cell mediate significant target suppression. Over 60% of detected microRNAs had no discernible activity, indicating that the functional 'miRNome' of a cell is considerably smaller than currently inferred from profiling studies. Moreover, some highly expressed microRNAs exhibit relatively weak activity, which in some cases correlated with a high target-to-microRNA ratio or increased nuclear localization of the microRNA. Finally, we show that the microRNA decoy library can be used for pooled loss-offunction studies. These tools provide valuable resources for studying microRNA biology and for microRNA-based therapeutics.
Nature Genetics, 2013
Lampreys are representatives of an ancient vertebrate lineage that diverged from our own ~500 mil... more Lampreys are representatives of an ancient vertebrate lineage that diverged from our own ~500 million years ago. By virtue of this deeply shared ancestry, the sea lamprey (P. marinus) genome is uniquely poised to provide insight into the ancestry of vertebrate genomes and the underlying principles of vertebrate biology. Here, we present the first lamprey whole-genome sequence and assembly. We note challenges faced owing to its high content of repetitive elements and GC bases, as well as the absence of broad-scale sequence information from closely related species. Analyses of the assembly indicate that two whole-genome duplications likely occurred before the divergence of ancestral lamprey and gnathostome lineages. Moreover, the results help define key evolutionary events within vertebrate lineages, including the origin of myelin-associated proteins and the development of appendages. The lamprey genome provides an important resource for reconstructing vertebrate origins and the evolutionary events that have shaped the genomes of extant organisms.
Genes & Development, 2009
PAZ/PIWI domain (PPD) proteins carrying small RNAs (sRNAs) function in gene and genome regulation... more PAZ/PIWI domain (PPD) proteins carrying small RNAs (sRNAs) function in gene and genome regulation. The ciliate Tetrahymena thermophila encodes numerous PPD proteins exclusively of the Piwi clade. We show that the three Tetrahymena Piwi family proteins (Twis) preferentially expressed in growing cells differ in their genetic essentiality and subcellular localization. Affinity purification of all eight distinct Twi proteins revealed unique properties of their bound sRNAs. Deep sequencing of Twi-bound and total sRNAs in strains disrupted for various silencing machinery uncovered an unanticipated diversity of 23-to 24-nt sRNA classes in growing cells, each with distinct genetic requirements for accumulation. Altogether, Twis distinguish sRNAs derived from loci of pseudogene families, three types of DNA repeats, structured RNAs, and EST-supported loci with convergent or paralogous transcripts. Most surprisingly, Twi7 binds complementary strands of unequal length, while Twi10 binds a specific permutation of the guanosine-rich telomeric repeat. These studies greatly expand the structural and functional repertoire of endogenous sRNAs and RNPs.
Genome Res, 2007
Many human diseases, including Fanconi anemia, hemophilia B, neurofibromatosis, and phenylketonur... more Many human diseases, including Fanconi anemia, hemophilia B, neurofibromatosis, and phenylketonuria, can be caused by 5Ј-splice-site (5Јss) mutations that are not predicted to disrupt splicing, according to position weight matrices. By using comparative genomics, we identify pairwise dependencies between 5Јss nucleotides as a conserved feature of the entire set of 5Јss. These dependencies are also conserved in human-mouse pairs of orthologous 5Јss. Many disease-associated 5Јss mutations disrupt these dependencies, as can some human SNPs that appear to alter splicing. The consistency of the evidence signifies the relevance of this approach and suggests that 5Јss SNPs play a role in complex diseases.
Molecular cell, Jan 6, 2015
PIWI-interacting RNAs (piRNAs) guide PIWI proteins to suppress transposons in the cytoplasm and n... more PIWI-interacting RNAs (piRNAs) guide PIWI proteins to suppress transposons in the cytoplasm and nucleus of animal germ cells, but how silencing in the two compartments is coordinated is not known. Here we demonstrate that endonucleolytic slicing of a transcript by the cytosolic mouse PIWI protein MILI acts as a trigger to initiate its further 5'→3' processing into non-overlapping fragments. These fragments accumulate as new piRNAs within both cytosolic MILI and the nuclear MIWI2. We also identify Exonuclease domain-containing 1 (EXD1) as a partner of the MIWI2 piRNA biogenesis factor TDRD12. EXD1 homodimers are inactive as a nuclease but function as an RNA adaptor within a PET (PIWI-EXD1-Tdrd12) complex. Loss of Exd1 reduces sequences generated by MILI slicing, impacts biogenesis of MIWI2 piRNAs, and de-represses LINE1 retrotransposons. Thus, piRNA biogenesis triggered by PIWI slicing, and promoted by EXD1, ensures that the same guides instruct PIWI proteins in the nucleus a...
Pacific Symposium on Biocomputing Pacific Symposium on Biocomputing, Feb 1, 2008
The advent of large-scale sequencing has opened up new areas of research, such as the study of Pi... more The advent of large-scale sequencing has opened up new areas of research, such as the study of Piwi-interacting small RNAs (piRNAs). piRNAs are longer than miRNAs, close to 30 nucleotides in length, involved in various functions, such as the suppression of transposons in germline 3,4,5 . Since a large number of them (many tens of thousands) are generated from a wide range of positions in the genome, large-scale sequencing is the only way to study them. The key to understanding their genesis and biological roles is efficient analysis, which is complicated by the large volumes of sequence data. Taking account of the underlying biology is also important. We describe here novel analyses techniques and tools applied to small RNAs from germ cells in D. melanogaster, that allowed us to infer mechanism and biological function.
Genes & development, Jan 15, 2015
PIWI clade Argonaute proteins silence transposon expression in animal gonads. Their target specif... more PIWI clade Argonaute proteins silence transposon expression in animal gonads. Their target specificity is defined by bound ∼23- to 30-nucleotide (nt) PIWI-interacting RNAs (piRNAs) that are processed from single-stranded precursor transcripts via two distinct pathways. Primary piRNAs are defined by the endonuclease Zucchini, while biogenesis of secondary piRNAs depends on piRNA-guided transcript cleavage and results in piRNA amplification. Here, we analyze the interdependencies between these piRNA biogenesis pathways in developing Drosophila ovaries. We show that secondary piRNA-guided target slicing is the predominant mechanism that specifies transcripts—including those from piRNA clusters—as primary piRNA precursors and defines the spectrum of Piwi-bound piRNAs in germline cells. Post-transcriptional silencing in the cytoplasm therefore enforces nuclear transcriptional target silencing, which ensures the tight suppression of transposons during oogenesis. As target slicing also def...
Genomics Data, 2015
CD24 is an anchored cell surface marker that is highly expressed in cancer cells (Lee et al., 200... more CD24 is an anchored cell surface marker that is highly expressed in cancer cells (Lee et al., 2009) and its expression is associated with poorer outcome of cancer patients (Kristiansen et al., 2003). Phenotype comparison between two subpopulations derived from the Mvt1 cell line, CD24(-) cells (with no CD24 cell surface expression) and the CD24(+) cells, identified high tumorigenic capacity for the CD24(+) cells. In order to reveal the transcripts that support the CD24(+) aggressive and invasive phenotype we compared the gene profiles of these two subpopulations. mRNA profiles of CD24(-) and CD24(+) cells were generated by deep sequencing, in triplicate, using an Illumina HiSeq 2500. Here we provide a detailed description of the mRNA-seq analysis from our recent study (Rostoker et al., 2015). The mRNA-seq data have been deposited in the NCBI GEO database (accession number GSE68746).
Cell Reports, 2015
In developing male germ cells, prospermatogonia, two Piwi proteins, MILI and MIWI2, use Piwi-inte... more In developing male germ cells, prospermatogonia, two Piwi proteins, MILI and MIWI2, use Piwi-interacting RNA (piRNA) guides to repress transposable element (TE) expression and ensure genome stability and proper gametogenesis. In addition to their roles in post-transcriptional TE repression, both proteins are required for DNA methylation of TE sequences. Here, we analyzed the effect of Miwi2 deficiency on piRNA biogenesis and transposon repression. Miwi2 deficiency had only a minor impact on piRNA biogenesis; however, the piRNA profile of Miwi2-knockout mice indicated overexpression of several LINE1 TE families that led to activation of the ping-pong piRNA cycle. Furthermore, we found that MILI and MIWI2 have distinct functions in TE repression in the nucleus. MILI is responsible for DNA methylation of a larger subset of TE families than MIWI2 is, suggesting that the proteins have independent roles in establishing DNA methylation patterns.
BMC Cancer, 2015
Alternate transcripts from a single gene locus greatly enhance the combinatorial flexibility of t... more Alternate transcripts from a single gene locus greatly enhance the combinatorial flexibility of the human transcriptome. Different patterns of exon usage have been observed when comparing normal tissue to cancers, suggesting that variant transcripts may play a role in the tumor phenotype. Ribonucleic acid-sequencing (RNA-seq) data from breast cancer samples was used to identify an intronic start variant transcript of Acyl-CoA oxidase 2, ACOX2 (ACOX2-i9). Difference in expression between Estrogen Receptor (ER) positive and ER negative patients was assessed by the Wilcoxon rank sum test, and the findings validated in The Cancer Genome Atlas (TCGA) breast cancer dataset (BRCA). ACOX2-i9 expression was also assessed in cell lines using both quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and Western blot analysis. Knock down by short hairpin RNA (shRNA) and colony formation assays were used to determine whether ACOX2-i9 expression would influence cellular fitness. The effect of ACOX2-i9 expression on patient survival was assessed by the Kaplan-Meier survival function, and association to clinical parameters was analyzed using a Fisher exact test. The expression and translation of ACOX2-i9 into a 25 kDa protein was demonstrated in HepG2 cells as well as in several breast cancer cell lines. shRNA knock down of the ACOX2-i9 variant resulted in decreased cell viability of T47D and MDA-MB 436 cells. Moreover, expression of ACOX2-i9 was shown to be estrogen regulated, being induced by propyl pyrazoletriol and inhibited by tamoxifen and fulvestrant in ER+ T47D and Mcf-7 cells, but not in the ER- MDA-MB 436 cell line. This variant transcript showed expression predominantly in ER-positive breast tumors as assessed in our initial set of 53 breast cancers and further validated in 87 tumor/normal pairs from the TCGA breast cancer dataset, and expression was associated with better outcome in ER positive patients. ACOX2-i9 is specifically enriched in ER+ breast cancers where expression of the variant is associated with improved outcome. These data identify variant ACOX2 as a potential novel therapeutic biomarker in ER+ breast tumors.
Cell Reports, 2015
PIWI proteins and PIWI-interacting RNAs (piRNAs) mediate repression of transposons in the animal ... more PIWI proteins and PIWI-interacting RNAs (piRNAs) mediate repression of transposons in the animal gonads. Primary processing converts long single-stranded RNAs into ∼30-nt piRNAs, but their entry into the biogenesis pathway is unknown. Here, we demonstrate that an RNA element at the 5' end of a piRNA cluster-which we termed piRNA trigger sequence (PTS)-can induce primary processing of any downstream sequence. We propose that such signals are triggers for the generation of the original pool of piRNAs. We also demonstrate that endonucleolytic cleavage of a transcript by a cytosolic PIWI results in its entry into primary processing, which triggers the generation of non-overlapping, contiguous primary piRNAs in the 3' direction from the target transcript. These piRNAs are loaded into a nuclear PIWI, thereby linking cytoplasmic post-transcriptional silencing to nuclear transcriptional repression.
Current Protocols in Bioinformatics, 2002
Encyclopedia of Life Sciences, 2001
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BMC genomics, 2005
Independent identification of genes in different organisms and assays has led to a multitude of n... more Independent identification of genes in different organisms and assays has led to a multitude of names for each gene. This balkanization makes it difficult to use gene names to locate genomic resources, homologs in other species and relevant publications. We solve the naming problem by collecting data from a variety of sources and building a name-translation database. We have also built a table of homologs across several model organisms: H. sapiens, M. musculus, R. norvegicus, D. melanogaster, C. elegans, S. cerevisiae, S. pombe and A. thaliana. This allows GeneSeer to draw phylogenetic trees and identify the closest homologs. This, in turn, allows the use of names from one species to identify homologous genes in another species. A website http://geneseer.cshl.org/ is connected to the database to allow user-friendly access to our tools and external genomic resources using familiar gene names. GeneSeer allows access to gene information through common names and can map sequences to nam...
Physical review. B, Condensed matter, Jan 15, 1994
ABSTRACT Orientational ordering transitions in C70 are studied by constructing a Landau free ener... more ABSTRACT Orientational ordering transitions in C70 are studied by constructing a Landau free energy in terms of order parameters describing long-range orientational order. This theory predicts that the transition from the orientationally disordered state into a partially ordered state, where the long axes of the molecules are parallel to one another, is discontinuous with an elastic distortion. Order parameters describing the lower temperature transition, where spinning about the long axis becomes hindered, are also discussed.
Physical review. B, Condensed matter, Jan 15, 1992
ABSTRACT We determine the allowed structures for orientationally ordered icosahedral molecules on... more ABSTRACT We determine the allowed structures for orientationally ordered icosahedral molecules on a fcc lattice such that there are four molecules per simple-cubic unit cell. The allowed space groups are Pm3, Pn3, and Pa3. In the latter two, an angle of rotation assumes a value not fixed by symmetry. The locations of all 240 atoms in the unit cell as deduced from the powder x-ray data of Heiney et al. are tabulated. We discuss a number of minima in the free energy which correspond to the observed Pa3 structure of solid C60. We introduce orientational order parameters which lead to a Landau free energy, from which we predict that the orientational transition is discontinuous.
Physical review. B, Condensed matter, Jan 15, 1994
ABSTRACT A symmetry analysis of the 2a phase recently observed in some samples of C60 is presente... more ABSTRACT A symmetry analysis of the 2a phase recently observed in some samples of C60 is presented. This phase is described by a unit cell with eight molecules in inequivalent orientations. We first show that if this structure is assumed to be exactly cubic, there are only three allowed space groups, none of which corresponds to the PA3¯ arrangement of threefold axes previously established for C60 by several groups. Our calculated powder diffraction spectra for these space groups are not consistent with existing experimental data. Second, if the symmetry of the PA3¯ structure is lowered by a doubling of the unit cell, we show that the resulting structure is trigonal, space group R3¯. We calculate powder diffraction spectra for this scenario and thereby place upper limits on both the angular distortion and the trigonal lattice distortion. Third, since the microscopic origin of this distortion probably involves defects of some presently unknown type, we consider a phenomenological scenario for the origin of this trigonal distortion. Within this scenario, we study the symmetry of the interactions needed to explain this structure. We start by giving an analysis of the structural distortion within harmonic lattice dynamics. However, to obtain the correct (R3¯) symmetry structure we were forced to study the cubic coupling between zone-corner librons and macroscopic strains. In this way we relate the development of R3¯ symmetry from the PA3¯ structure in terms of a phenomenological model of lattice dynamics. Fourth, we extend the above arguments to construct a Landau theory for the hypothesized PA3¯-->R3¯ phase transition, which occurs as a function of the concentration of the presumed defects. The resulting free energy has no cubic terms (so the transition can be continuous) but has five fourth-order invariants.
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Papers by Ravi Sachidanandam