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1.  Small RNA pathway genes identified by patterns of phylogenetic conservation and divergence 
Nature  2012;493(7434):694-698.
Genetic and biochemical analyses of RNA interference (RNAi) and microRNA (miRNA) pathways have revealed proteins such as Argonaute/PIWI and Dicer that process and present small RNAs to their targets. Well validated small RNA pathway cofactors, such as the Argonaute/PIWI proteins show distinctive patterns of conservation or divergence in particular animal, plant, fungal, and protist species. We compared 86 divergent eukaryotic genome sequences to discern sets of proteins that show similar phylogenetic profiles with known small RNA cofactors. A large set of additional candidate small RNA cofactors have emerged from functional genomic screens for defects in miRNA- or siRNA-mediated repression in C. elegans and D. melanogaster1,2 and from proteomic analyses of proteins co-purifying with validated small RNA pathway proteins3,4. The phylogenetic profiles of many of these candidate small RNA pathway proteins are similar to those of known small RNA cofactor proteins. We used a Bayesian approach to integrate the phylogenetic profile analysis with predictions from diverse transcriptional coregulation and proteome interaction datasets to assign a probability for each protein for a role in a small RNA pathway. Testing high-confidence candidates from this analysis for defects in RNAi silencing, we found that about half of the predicted small RNA cofactors are required for RNAi silencing. Many of the newly identified small RNA pathway proteins are orthologues of proteins implicated in RNA splicing. In support of a deep connection between the mechanism of RNA splicing and small RNA-mediated gene silencing, the presence of the Argonaute proteins and other small RNA components in the many species analysed strongly correlates with the number of introns in that species.
doi:10.1038/nature11779
PMCID: PMC3762460  PMID: 23364702
2.  piRNAs and siRNAs collaborate in Caenorhabditis elegans genome defense 
Genome Biology  2012;13(7):164.
Caenorhabditis elegans piRNAs promote genome surveillance by triggering siRNA-mediated silencing of nonself DNA in competition with licensing programs that support endogenous gene expression.
doi:10.1186/gb-2012-13-7-164
PMCID: PMC3491375  PMID: 22818087
piRNA; siRNA; Piwi; C. elegans; germline; genome surveillance; epigenetics; gene silencing; transposable elements
3.  A Conserved Upstream Motif Orchestrates Autonomous, Germline-Enriched Expression of Caenorhabditis elegans piRNAs 
PLoS Genetics  2013;9(3):e1003392.
Piwi-interacting RNAs (piRNAs) fulfill a critical, conserved role in defending the genome against foreign genetic elements. In many organisms, piRNAs appear to be derived from processing of a long, polycistronic RNA precursor. Here, we establish that each Caenorhabditis elegans piRNA represents a tiny, autonomous transcriptional unit. Remarkably, the minimal C. elegans piRNA cassette requires only a 21 nucleotide (nt) piRNA sequence and an ∼50 nt upstream motif with limited genomic context for expression. Combining computational analyses with a novel, in vivo transgenic system, we demonstrate that this upstream motif is necessary for independent expression of a germline-enriched, Piwi-dependent piRNA. We further show that a single nucleotide position within this motif directs differential germline enrichment. Accordingly, over 70% of C. elegans piRNAs are selectively expressed in male or female germline, and comparison of the genes they target suggests that these two populations have evolved independently. Together, our results indicate that C. elegans piRNA upstream motifs act as independent promoters to specify which sequences are expressed as piRNAs, how abundantly they are expressed, and in what germline. As the genome encodes well over 15,000 unique piRNA sequences, our study reveals that the number of transcriptional units encoding piRNAs rivals the number of mRNA coding genes in the C. elegans genome.
Author Summary
Across the animal kingdom, Piwi-interacting small RNAs (piRNAs) protect genome integrity and promote fertility. While the functions of piRNAs are well-characterized, far less is known about how they are generated and how their expression is regulated. In the Caenorhabditis elegans genome, a conserved sequence motif lies upstream of many piRNA loci and appears to regulate their expression. We combined computational and experimental approaches to investigate the role of this motif in the expression of C. elegans piRNAs. We discovered that >70% of piRNAs are differentially enriched in male versus female germline, and these male and female piRNAs show different upstream motifs. Using a transgenic system for expressing synthetic piRNAs in vivo, we demonstrate that variation of a single nucleotide within this motif influences piRNA germline enrichment. We further show that the conserved motif is capable of driving piRNA expression in genomic isolation. Accordingly, the genomic distribution of these motifs determines which sequences are expressed as piRNAs in C. elegans. Our results suggest that each C. elegans piRNA represents an independent transcript whose sequence, abundance, and germline enrichment are encoded by a variant upstream motif, defining a novel modality for expression of piRNAs.
doi:10.1371/journal.pgen.1003392
PMCID: PMC3597512  PMID: 23516384
4.  Pervasive and dynamic protein binding sites of the mRNA transcriptome in Saccharomyces cerevisiae 
Genome Biology  2013;14(2):R13.
Background
Protein-RNA interactions are integral components of nearly every aspect of biology, including regulation of gene expression, assembly of cellular architectures, and pathogenesis of human diseases. However, studies in the past few decades have only uncovered a small fraction of the vast landscape of the protein-RNA interactome in any organism, and even less is known about the dynamics of protein-RNA interactions under changing developmental and environmental conditions.
Results
Here, we describe the gPAR-CLIP (global photoactivatable-ribonucleoside-enhanced crosslinking and immunopurification) approach for capturing regions of the untranslated, polyadenylated transcriptome bound by RNA-binding proteins (RBPs) in budding yeast. We report over 13,000 RBP crosslinking sites in untranslated regions (UTRs) covering 72% of protein-coding transcripts encoded in the genome, confirming 3' UTRs as major sites for RBP interaction. Comparative genomic analyses reveal that RBP crosslinking sites are highly conserved, and RNA folding predictions indicate that secondary structural elements are constrained by protein binding and may serve as generalizable modes of RNA recognition. Finally, 38% of 3' UTR crosslinking sites show changes in RBP occupancy upon glucose or nitrogen deprivation, with major impacts on metabolic pathways as well as mitochondrial and ribosomal gene expression.
Conclusions
Our study offers an unprecedented view of the pervasiveness and dynamics of protein-RNA interactions in vivo.
doi:10.1186/gb-2013-14-2-r13
PMCID: PMC4053964  PMID: 23409723
5.  MORC Family ATPases Required for Heterochromatin Condensation and Gene Silencing# 
Science (New York, N.Y.)  2012;336(6087):1448-1451.
Transposable elements (TEs) and DNA repeats are commonly targeted by DNA and histone methylation to achieve epigenetic gene silencing. We isolated mutations in two Arabidopsis genes, AtMORC1 and AtMORC6, which cause de-repression of DNA-methylated genes and TEs, but no losses of DNA or histone methylation. AtMORC1 and AtMORC6 are members of the conserved Microrchidia (MORC) adenosine triphosphatase (ATPase) family, predicted to catalyze alterations in chromosome superstructure. The atmorc1 and atmorc6 mutants show decondensation of pericentromeric heterochromatin, increased interaction of pericentromeric regions with the rest of the genome, and transcriptional defects that are largely restricted to loci residing in pericentromeric regions. Knockdown of the single MORC homolog in Caenorhabditis elegans also impairs transgene silencing. We propose that the MORC ATPases are conserved regulators of gene silencing in eukaryotes.
doi:10.1126/science.1221472
PMCID: PMC3376212  PMID: 22555433
6.  The Caenorhabditis elegans HEN1 Ortholog, HENN-1, Methylates and Stabilizes Select Subclasses of Germline Small RNAs 
PLoS Genetics  2012;8(4):e1002617.
Small RNAs regulate diverse biological processes by directing effector proteins called Argonautes to silence complementary mRNAs. Maturation of some classes of small RNAs involves terminal 2′-O-methylation to prevent degradation. This modification is catalyzed by members of the conserved HEN1 RNA methyltransferase family. In animals, Piwi-interacting RNAs (piRNAs) and some endogenous and exogenous small interfering RNAs (siRNAs) are methylated, whereas microRNAs are not. However, the mechanisms that determine animal HEN1 substrate specificity have yet to be fully resolved. In Caenorhabditis elegans, a HEN1 ortholog has not been studied, but there is evidence for methylation of piRNAs and some endogenous siRNAs. Here, we report that the worm HEN1 ortholog, HENN-1 (HEN of Nematode), is required for methylation of C. elegans small RNAs. Our results indicate that piRNAs are universally methylated by HENN-1. In contrast, 26G RNAs, a class of primary endogenous siRNAs, are methylated in female germline and embryo, but not in male germline. Intriguingly, the methylation pattern of 26G RNAs correlates with the expression of distinct male and female germline Argonautes. Moreover, loss of the female germline Argonaute results in loss of 26G RNA methylation altogether. These findings support a model wherein methylation status of a metazoan small RNA is dictated by the Argonaute to which it binds. Loss of henn-1 results in phenotypes that reflect destabilization of substrate small RNAs: dysregulation of target mRNAs, impaired fertility, and enhanced somatic RNAi. Additionally, the henn-1 mutant shows a weakened response to RNAi knockdown of germline genes, suggesting that HENN-1 may also function in canonical RNAi. Together, our results indicate a broad role for HENN-1 in both endogenous and exogenous gene silencing pathways and provide further insight into the mechanisms of HEN1 substrate discrimination and the diversity within the Argonaute family.
Author Summary
Small RNAs serve as sentinels of the genome, policing activity of selfish genetic elements, modulating chromatin dynamics, and fine-tuning gene expression. Nowhere is this more important than in the germline, where endogenous small interfering RNAs (endo-siRNAs) and Piwi-interacting RNAs (piRNAs) promote formation of functional gametes and ensure viable, fertile progeny. Small RNAs act primarily by associating with effector proteins called Argonautes to direct repression of complementary mRNAs. HEN1 methyltransferases, which methylate small RNAs, play a critical role in accumulation of these silencing signals. In this study, we report that the 26G RNAs, a class of C. elegans endo-siRNAs, are differentially methylated in male and female germlines. 26G RNAs derived from the two germlines are virtually indistinguishable, except that they associate with evolutionarily divergent Argonautes. Our data support a model wherein the methylation status and, consequently, stability of a small RNA are determined by the associated Argonaute. Therefore, selective expression of Argonautes that permit or prohibit methylation may represent a new mechanism for regulating small RNA turnover. As we observe this phenomenon in the germline, it may be particularly pertinent for directing inheritance of small RNAs, which can carry information not encoded in progeny DNA that is essential for continued transgenerational genome surveillance.
doi:10.1371/journal.pgen.1002617
PMCID: PMC3330095  PMID: 22548001
7.  Integrative Analysis of the Caenorhabditis elegans Genome by the modENCODE Project 
Gerstein, Mark B. | Lu, Zhi John | Van Nostrand, Eric L. | Cheng, Chao | Arshinoff, Bradley I. | Liu, Tao | Yip, Kevin Y. | Robilotto, Rebecca | Rechtsteiner, Andreas | Ikegami, Kohta | Alves, Pedro | Chateigner, Aurelien | Perry, Marc | Morris, Mitzi | Auerbach, Raymond K. | Feng, Xin | Leng, Jing | Vielle, Anne | Niu, Wei | Rhrissorrakrai, Kahn | Agarwal, Ashish | Alexander, Roger P. | Barber, Galt | Brdlik, Cathleen M. | Brennan, Jennifer | Brouillet, Jeremy Jean | Carr, Adrian | Cheung, Ming-Sin | Clawson, Hiram | Contrino, Sergio | Dannenberg, Luke O. | Dernburg, Abby F. | Desai, Arshad | Dick, Lindsay | Dosé, Andréa C. | Du, Jiang | Egelhofer, Thea | Ercan, Sevinc | Euskirchen, Ghia | Ewing, Brent | Feingold, Elise A. | Gassmann, Reto | Good, Peter J. | Green, Phil | Gullier, Francois | Gutwein, Michelle | Guyer, Mark S. | Habegger, Lukas | Han, Ting | Henikoff, Jorja G. | Henz, Stefan R. | Hinrichs, Angie | Holster, Heather | Hyman, Tony | Iniguez, A. Leo | Janette, Judith | Jensen, Morten | Kato, Masaomi | Kent, W. James | Kephart, Ellen | Khivansara, Vishal | Khurana, Ekta | Kim, John K. | Kolasinska-Zwierz, Paulina | Lai, Eric C. | Latorre, Isabel | Leahey, Amber | Lewis, Suzanna | Lloyd, Paul | Lochovsky, Lucas | Lowdon, Rebecca F. | Lubling, Yaniv | Lyne, Rachel | MacCoss, Michael | Mackowiak, Sebastian D. | Mangone, Marco | McKay, Sheldon | Mecenas, Desirea | Merrihew, Gennifer | Miller, David M. | Muroyama, Andrew | Murray, John I. | Ooi, Siew-Loon | Pham, Hoang | Phippen, Taryn | Preston, Elicia A. | Rajewsky, Nikolaus | Rätsch, Gunnar | Rosenbaum, Heidi | Rozowsky, Joel | Rutherford, Kim | Ruzanov, Peter | Sarov, Mihail | Sasidharan, Rajkumar | Sboner, Andrea | Scheid, Paul | Segal, Eran | Shin, Hyunjin | Shou, Chong | Slack, Frank J. | Slightam, Cindie | Smith, Richard | Spencer, William C. | Stinson, E. O. | Taing, Scott | Takasaki, Teruaki | Vafeados, Dionne | Voronina, Ksenia | Wang, Guilin | Washington, Nicole L. | Whittle, Christina M. | Wu, Beijing | Yan, Koon-Kiu | Zeller, Georg | Zha, Zheng | Zhong, Mei | Zhou, Xingliang | Ahringer, Julie | Strome, Susan | Gunsalus, Kristin C. | Micklem, Gos | Liu, X. Shirley | Reinke, Valerie | Kim, Stuart K. | Hillier, LaDeana W. | Henikoff, Steven | Piano, Fabio | Snyder, Michael | Stein, Lincoln | Lieb, Jason D. | Waterston, Robert H.
Science (New York, N.Y.)  2010;330(6012):1775-1787.
We systematically generated large-scale data sets to improve genome annotation for the nematode Caenorhabditis elegans, a key model organism. These data sets include transcriptome profiling across a developmental time course, genome-wide identification of transcription factor–binding sites, and maps of chromatin organization. From this, we created more complete and accurate gene models, including alternative splice forms and candidate noncoding RNAs. We constructed hierarchical networks of transcription factor–binding and microRNA interactions and discovered chromosomal locations bound by an unusually large number of transcription factors. Different patterns of chromatin composition and histone modification were revealed between chromosome arms and centers, with similarly prominent differences between autosomes and the X chromosome. Integrating data types, we built statistical models relating chromatin, transcription factor binding, and gene expression. Overall, our analyses ascribed putative functions to most of the conserved genome.
doi:10.1126/science.1196914
PMCID: PMC3142569  PMID: 21177976
8.  The Landscape of C. elegans 3′UTRs 
Science (New York, N.Y.)  2010;329(5990):432-435.
Three-prime untranslated regions (3′UTRs) of metazoan messenger RNAs (mRNAs) contain numerous regulatory elements, yet remain largely uncharacterized. Using polyA capture, 3′ rapid amplification of complementary DNA (cDNA) ends, full-length cDNAs, and RNA-seq, we defined ∼26,000 distinct 3′UTRs in Caenorhabditis elegans for ∼85% of the 18,328 experimentally supported protein-coding genes and revised ∼40% of gene models. Alternative 3′UTR isoforms are frequent, often differentially expressed during development. Average 3′UTR length decreases with animal age. Surprisingly, no polyadenylation signal (PAS) was detected for 13% of polyadenylation sites, predominantly among shorter alternative isoforms. Trans-spliced (versus non–trans-spliced) mRNAs possess longer 3′UTRs and frequently contain no PAS or variant PAS. We identified conserved 3′UTR motifs, isoform-specific predicted microRNA target sites, and polyadenylation of most histone genes. Our data reveal a rich complexity of 3′UTRs, both genome-wide and throughout development.
doi:10.1126/science.1191244
PMCID: PMC3142571  PMID: 20522740
9.  The microRNA miR-1 regulates a MEF-2 dependent retrograde signal at neuromuscular junctions 
Cell  2008;133(5):903-915.
Summary
We show that miR-1, a conserved muscle specific microRNA, regulates aspects of both pre- and post-synaptic function at C. elegans neuromuscular junctions. miR-1 regulates the expression level of two nicotinic acetylcholine receptor (nAChR) subunits (UNC-29 and UNC-63), thereby altering muscle sensitivity to acetylcholine (ACh). miR-1 also regulates the muscle transcription factor MEF-2, which results in altered pre-synaptic ACh secretion, suggesting that MEF-2 activity in muscles controls a retrograde signal. The effect of the MEF-2-dependent retrograde signal on secretion is mediated by the synaptic vesicle protein RAB-3. Finally, acute activation of levamisole-sensitive nAChRs stimulates MEF-2-dependent transcriptional responses, and induces the MEF-2-dependent retrograde signal. We propose that miR-1 refines synaptic function by coupling changes in muscle activity to changes in pre-synaptic function.
doi:10.1016/j.cell.2008.04.035
PMCID: PMC2553566  PMID: 18510933
microRNA; miR-1; synapse; nAChR; nicotinic receptors; acetylcholine; MEF2; MEF-2; RAB-3; Rab3; retrograde; synaptic transmission; C. elegans

Results 1-9 (9)