Publications by authors named "Sam Griffiths-Jones"

84 Publications

Single-cell visualization of mir-9a and Senseless co-expression during Drosophila melanogaster embryonic and larval peripheral nervous system development.

G3 (Bethesda) 2021 Jan;11(1)

School of Biological Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester Academic Health Science Centre, Manchester M13 9PT, UK.

The Drosophila melanogaster peripheral nervous system (PNS) comprises the sensory organs that allow the fly to detect environmental factors such as temperature and pressure. PNS development is a highly specified process where each sensilla originates from a single sensory organ precursor (SOP) cell. One of the major genetic orchestrators of PNS development is Senseless, which encodes a zinc finger transcription factor (Sens). Sens is both necessary and sufficient for SOP differentiation. Senseless expression and SOP number are regulated by the microRNA miR-9a. However, the reciprocal dynamics of Senseless and miR-9a are still obscure. By coupling single-molecule FISH with immunofluorescence, we are able to visualize transcription of the mir-9a locus and expression of Sens simultaneously. During embryogenesis, we show that the expression of mir-9a in SOP cells is rapidly lost as Senseless expression increases. However, this mutually exclusive expression pattern is not observed in the third instar imaginal wing disk, where some Senseless-expressing cells show active sites of mir-9a transcription. These data challenge and extend previous models of Senseless regulation and show complex co-expression dynamics between mir-9a and Senseless. The differences in this dynamic relationship between embryonic and larval PNS development suggest a possible switch in miR-9a function. Our work brings single-cell resolution to the understanding of dynamic regulation of PNS development by Senseless and miR-9a.
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http://dx.doi.org/10.1093/g3journal/jkaa010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7849905PMC
January 2021

Regulatory RNAs: A Universal Language for Inter-Domain Communication.

Int J Mol Sci 2020 Nov 24;21(23). Epub 2020 Nov 24.

Lydia Becker Institute of Immunology and Inflammation, Division of Infection, Immunity and Respiratory Medicine, School of Biological Sciences, Faculty of Biology Medicine and Health, University of Manchester, Manchester M13 9PT, UK.

In eukaryotes, microRNAs (miRNAs) have roles in development, homeostasis, disease and the immune response. Recent work has shown that plant and mammalian miRNAs also mediate cross-kingdom and cross-domain communications. However, these studies remain controversial and are lacking critical mechanistic explanations. Bacteria do not produce miRNAs themselves, and therefore it is unclear how these eukaryotic RNA molecules could function in the bacterial recipient. In this review, we compare and contrast the biogenesis and functions of regulatory RNAs in eukaryotes and bacteria. As a result, we discovered several conserved features and homologous components in these distinct pathways. These findings enabled us to propose novel mechanisms to explain how eukaryotic miRNAs could function in bacteria. Further understanding in this area is necessary to validate the findings of existing studies and could facilitate the use of miRNAs as novel tools for the directed remodelling of the human microbiota.
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http://dx.doi.org/10.3390/ijms21238919DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7727864PMC
November 2020

Rfam 14: expanded coverage of metagenomic, viral and microRNA families.

Nucleic Acids Res 2021 01;49(D1):D192-D200

European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, Cambridge CB10 1SD, UK.

Rfam is a database of RNA families where each of the 3444 families is represented by a multiple sequence alignment of known RNA sequences and a covariance model that can be used to search for additional members of the family. Recent developments have involved expert collaborations to improve the quality and coverage of Rfam data, focusing on microRNAs, viral and bacterial RNAs. We have completed the first phase of synchronising microRNA families in Rfam and miRBase, creating 356 new Rfam families and updating 40. We established a procedure for comprehensive annotation of viral RNA families starting with Flavivirus and Coronaviridae RNAs. We have also increased the coverage of bacterial and metagenome-based RNA families from the ZWD database. These developments have enabled a significant growth of the database, with the addition of 759 new families in Rfam 14. To facilitate further community contribution to Rfam, expert users are now able to build and submit new families using the newly developed Rfam Cloud family curation system. New Rfam website features include a new sequence similarity search powered by RNAcentral, as well as search and visualisation of families with pseudoknots. Rfam is freely available at https://rfam.org.
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http://dx.doi.org/10.1093/nar/gkaa1047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7779021PMC
January 2021

Dynamical gene regulatory networks are tuned by transcriptional autoregulation with microRNA feedback.

Sci Rep 2020 07 31;10(1):12960. Epub 2020 Jul 31.

School of Medical Sciences, Faculty of Biology Medicine and Health, The University of Manchester, Oxford Road, Manchester, M13 9PT, UK.

Concepts from dynamical systems theory, including multi-stability, oscillations, robustness and stochasticity, are critical for understanding gene regulation during cell fate decisions, inflammation and stem cell heterogeneity. However, the prevalence of the structures within gene networks that drive these dynamical behaviours, such as autoregulation or feedback by microRNAs, is unknown. We integrate transcription factor binding site (TFBS) and microRNA target data to generate a gene interaction network across 28 human tissues. This network was analysed for motifs capable of driving dynamical gene expression, including oscillations. Identified autoregulatory motifs involve 56% of transcription factors (TFs) studied. TFs that autoregulate have more interactions with microRNAs than non-autoregulatory genes and 89% of autoregulatory TFs were found in dual feedback motifs with a microRNA. Both autoregulatory and dual feedback motifs were enriched in the network. TFs that autoregulate were highly conserved between tissues. Dual feedback motifs with microRNAs were also conserved between tissues, but less so, and TFs regulate different combinations of microRNAs in a tissue-dependent manner. The study of these motifs highlights ever more genes that have complex regulatory dynamics. These data provide a resource for the identification of TFs which regulate the dynamical properties of human gene expression.
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http://dx.doi.org/10.1038/s41598-020-69791-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7395740PMC
July 2020

Silencing miR-370-3p rescues funny current and sinus node function in heart failure.

Sci Rep 2020 07 9;10(1):11279. Epub 2020 Jul 9.

Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, 2200N, Copenhagen, Denmark.

Bradyarrhythmias are an important cause of mortality in heart failure and previous studies indicate a mechanistic role for electrical remodelling of the key pacemaking ion channel HCN4 in this process. Here we show that, in a mouse model of heart failure in which there is sinus bradycardia, there is upregulation of a microRNA (miR-370-3p), downregulation of the pacemaker ion channel, HCN4, and downregulation of the corresponding ionic current, I, in the sinus node. In vitro, exogenous miR-370-3p inhibits HCN4 mRNA and causes downregulation of HCN4 protein, downregulation of I, and bradycardia in the isolated sinus node. In vivo, intraperitoneal injection of an antimiR to miR-370-3p into heart failure mice silences miR-370-3p and restores HCN4 mRNA and protein and I in the sinus node and blunts the sinus bradycardia. In addition, it partially restores ventricular function and reduces mortality. This represents a novel approach to heart failure treatment.
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http://dx.doi.org/10.1038/s41598-020-67790-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7347645PMC
July 2020

Quo vadis microRNAs?

Trends Genet 2020 07 16;36(7):461-463. Epub 2020 Apr 16.

School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK.

Since 2002, published miRNAs have been collected and named by the online repository miRBase. However, with 11 000 annual publications this has become challenging. Recently, four specialized miRNA databases were published, addressing particular needs for diverse scientific communities. This development provides major opportunities for the future of miRNA annotation and nomenclature.
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http://dx.doi.org/10.1016/j.tig.2020.03.007DOI Listing
July 2020

The Transcription Factor-microRNA Regulatory Network during hESC-chondrogenesis.

Sci Rep 2020 03 16;10(1):4744. Epub 2020 Mar 16.

Divisions of Cell Matrix Biology and Regenerative Medicine, Faculty of Biology Medicine and Health, Michael Smith Building, Oxford Road, University of Manchester, Manchester Academic Health Science Centre, Manchester, M13 9PT, UK.

Human embryonic stem cells (ESCs) offer a promising therapeutic approach for osteoarthritis (OA). The unlimited source of cells capable of differentiating to chondrocytes has potential for repairing damaged cartilage or to generate disease models via gene editing. However their use is limited by the efficiency of chondrogenic differentiation. An improved understanding of the transcriptional and post-transcriptional regulation of chondrogenesis will enable us to improve hESC chondrogenic differentiation protocols. Small RNA-seq and whole transcriptome sequencing was performed on distinct stages of hESC-directed chondrogenesis. This revealed significant changes in the expression of several microRNAs including upregulation of known cartilage associated microRNAs and those transcribed from the Hox complexes, and the downregulation of pluripotency associated microRNAs. Integration of miRomes and transcriptomes generated during hESC-directed chondrogenesis identified key functionally related clusters of co-expressed microRNAs and protein coding genes, associated with pluripotency, primitive streak, limb development and extracellular matrix. Analysis identified regulators of hESC-directed chondrogenesis such as miR-29c-3p with 10 of its established targets identified as co-regulated 'ECM organisation' genes and miR-22-3p which is highly co-expressed with ECM genes and may regulate these genes indirectly by targeting the chondrogenic regulators SP1 and HDAC4. We identified several upregulated transcription factors including HOXA9/A10/D13 involved in limb patterning and RELA, JUN and NFAT5, which have targets enriched with ECM associated genes. We have developed an unbiased approach for integrating transcriptome and miRome using protein-protein interactions, transcription factor regulation and miRNA target interactions and identified key regulatory networks prominent in hESC chondrogenesis.
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http://dx.doi.org/10.1038/s41598-020-61734-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7075910PMC
March 2020

A guide to naming human non-coding RNA genes.

EMBO J 2020 03 24;39(6):e103777. Epub 2020 Feb 24.

Department of Haematology, University of Cambridge School of Clinical Medicine, Cambridge, UK.

Research on non-coding RNA (ncRNA) is a rapidly expanding field. Providing an official gene symbol and name to ncRNA genes brings order to otherwise potential chaos as it allows unambiguous communication about each gene. The HUGO Gene Nomenclature Committee (HGNC, www.genenames.org) is the only group with the authority to approve symbols for human genes. The HGNC works with specialist advisors for different classes of ncRNA to ensure that ncRNA nomenclature is accurate and informative, where possible. Here, we review each major class of ncRNA that is currently annotated in the human genome and describe how each class is assigned a standardised nomenclature.
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http://dx.doi.org/10.15252/embj.2019103777DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7073466PMC
March 2020

Enhanced genome assembly and a new official gene set for Tribolium castaneum.

BMC Genomics 2020 Jan 14;21(1):47. Epub 2020 Jan 14.

Georg-August-Universität Göttingen, Göttingen, Germany.

Background: The red flour beetle Tribolium castaneum has emerged as an important model organism for the study of gene function in development and physiology, for ecological and evolutionary genomics, for pest control and a plethora of other topics. RNA interference (RNAi), transgenesis and genome editing are well established and the resources for genome-wide RNAi screening have become available in this model. All these techniques depend on a high quality genome assembly and precise gene models. However, the first version of the genome assembly was generated by Sanger sequencing, and with a small set of RNA sequence data limiting annotation quality.

Results: Here, we present an improved genome assembly (Tcas5.2) and an enhanced genome annotation resulting in a new official gene set (OGS3) for Tribolium castaneum, which significantly increase the quality of the genomic resources. By adding large-distance jumping library DNA sequencing to join scaffolds and fill small gaps, the gaps in the genome assembly were reduced and the N50 increased to 4753kbp. The precision of the gene models was enhanced by the use of a large body of RNA-Seq reads of different life history stages and tissue types, leading to the discovery of 1452 novel gene sequences. We also added new features such as alternative splicing, well defined UTRs and microRNA target predictions. For quality control, 399 gene models were evaluated by manual inspection. The current gene set was submitted to Genbank and accepted as a RefSeq genome by NCBI.

Conclusions: The new genome assembly (Tcas5.2) and the official gene set (OGS3) provide enhanced genomic resources for genetic work in Tribolium castaneum. The much improved information on transcription start sites supports transgenic and gene editing approaches. Further, novel types of information such as splice variants and microRNA target genes open additional possibilities for analysis.
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http://dx.doi.org/10.1186/s12864-019-6394-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6961396PMC
January 2020

Restoring the constitutional alignment with a restrictive kinematic protocol improves quantitative soft-tissue balance in total knee arthroplasty: a randomized controlled trial.

Bone Joint J 2020 Jan;102-B(1):117-124

Orthopaedic Surgery, Ingham Institute for Applied Medical Research, South Western Sydney Clinical School, University of New South Wales, Sydney, Australia.

Aims: It is unknown whether kinematic alignment (KA) objectively improves knee balance in total knee arthroplasty (TKA), despite this being the biomechanical rationale for its use. This study aimed to determine whether restoring the constitutional alignment using a restrictive KA protocol resulted in better quantitative knee balance than mechanical alignment (MA).

Methods: We conducted a randomized superiority trial comparing patients undergoing TKA assigned to KA within a restrictive safe zone or MA. Optimal knee balance was defined as an intercompartmental pressure difference (ICPD) of 15 psi or less using a pressure sensor. The primary endpoint was the mean intraoperative ICPD at 10° of flexion prior to knee balancing. Secondary outcomes included balance at 45° and 90°, requirements for balancing procedures, and presence of tibiofemoral lift-off.

Results: A total of 63 patients (70 knees) were randomized to KA and 62 patients (68 knees) to MA. Mean ICPD at 10° flexion in the KA group was 11.7 psi (SD 13.1) compared with 32.0 psi in the MA group (SD 28.9), with a mean difference in ICPD between KA and MA of 20.3 psi (p < 0.001). Mean ICPD in the KA group was significantly lower than in the MA group at 45° and 90°, respectively (25.2 psi MA vs 14.8 psi KA, p = 0.004; 19.1 psi MA vs 11.7 psi KA, p < 0.002, respectively). Overall, participants in the KA group were more likely to achieve optimal knee balance (80% vs 35%; p < 0.001). Bone recuts to achieve knee balance were more likely to be required in the MA group (49% vs 9%; p < 0.001). More participants in the MA group had tibiofemoral lift-off (43% vs 13%; p < 0.001).

Conclusion: This study provides persuasive evidence that restoring the constitutional alignment with KA in TKA results in a statistically significant improvement in quantitative knee balance, and further supports this technique as a viable alternative to MA. Cite this article: 2020;102-B(1):117-124.
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http://dx.doi.org/10.1302/0301-620X.102B1.BJJ-2019-0674.R2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6974544PMC
January 2020

miRBase: from microRNA sequences to function.

Nucleic Acids Res 2019 01;47(D1):D155-D162

School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester M13 9PT, UK.

miRBase catalogs, names and distributes microRNA gene sequences. The latest release of miRBase (v22) contains microRNA sequences from 271 organisms: 38 589 hairpin precursors and 48 860 mature microRNAs. We describe improvements to the database and website to provide more information about the quality of microRNA gene annotations, and the cellular functions of their products. We have collected 1493 small RNA deep sequencing datasets and mapped a total of 5.5 billion reads to microRNA sequences. The read mapping patterns provide strong support for the validity of between 20% and 65% of microRNA annotations in different well-studied animal genomes, and evidence for the removal of >200 sequences from the database. To improve the availability of microRNA functional information, we are disseminating Gene Ontology terms annotated against miRBase sequences. We have also used a text-mining approach to search for microRNA gene names in the full-text of open access articles. Over 500 000 sentences from 18 542 papers contain microRNA names. We score these sentences for functional information and link them with 12 519 microRNA entries. The sentences themselves, and word clouds built from them, provide effective summaries of the functional information about specific microRNAs. miRBase is publicly and freely available at http://mirbase.org/.
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http://dx.doi.org/10.1093/nar/gky1141DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6323917PMC
January 2019

Large-scale profiling of noncoding RNA function in yeast.

PLoS Genet 2018 03 12;14(3):e1007253. Epub 2018 Mar 12.

Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, United Kingdom.

Noncoding RNAs (ncRNAs) are emerging as key regulators of cellular function. We have exploited the recently developed barcoded ncRNA gene deletion strain collections in the yeast Saccharomyces cerevisiae to investigate the numerous ncRNAs in yeast with no known function. The ncRNA deletion collection contains deletions of tRNAs, snoRNAs, snRNAs, stable unannotated transcripts (SUTs), cryptic unstable transcripts (CUTs) and other annotated ncRNAs encompassing 532 different individual ncRNA deletions. We have profiled the fitness of the diploid heterozygous ncRNA deletion strain collection in six conditions using batch and continuous liquid culture, as well as the haploid ncRNA deletion strain collections arrayed individually onto solid rich media. These analyses revealed many novel environmental-specific haplo-insufficient and haplo-proficient phenotypes providing key information on the importance of each specific ncRNA in every condition. Co-fitness analysis using fitness data from the heterozygous ncRNA deletion strain collection identified two ncRNA groups required for growth during heat stress and nutrient deprivation. The extensive fitness data for each ncRNA deletion strain has been compiled into an easy to navigate database called Yeast ncRNA Analysis (YNCA). By expanding the original ncRNA deletion strain collection we identified four novel essential ncRNAs; SUT527, SUT075, SUT367 and SUT259/691. We defined the effects of each new essential ncRNA on adjacent gene expression in the heterozygote background identifying both repression and induction of nearby genes. Additionally, we discovered a function for SUT527 in the expression, 3' end formation and localization of SEC4, an essential protein coding mRNA. Finally, using plasmid complementation we rescued the SUT075 lethal phenotype revealing that this ncRNA acts in trans. Overall, our findings provide important new insights into the function of ncRNAs.
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http://dx.doi.org/10.1371/journal.pgen.1007253DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5864082PMC
March 2018

Small RNAs: Big Impact on Plant Development.

Trends Plant Sci 2017 12 12;22(12):1056-1068. Epub 2017 Oct 12.

Faculty of Biology, Medicine, and Health, University of Manchester, Oxford Road, Manchester M13 9PT, UK. Electronic address:

While the role of proteins in determining cell identity has been extensively studied, the contribution of small noncoding RNA molecules such as miRNAs and siRNAs has been also recognised. miRNAs bind to complementary sites in target mRNA molecules to trigger the degradation or translational inhibition of those targets. Recent studies have revealed that miRNAs play pivotal roles in key developmental processes such as patterning of the embryo, meristem, leaf, and flower. Furthermore, these miRNAs have been recruited throughout plant evolution into pathways that create diverse plant organ forms and shapes. This review focuses on the roles of miRNAs in establishing plant cell identity during key plant development processes and creating morphological diversity during plant evolution.
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http://dx.doi.org/10.1016/j.tplants.2017.09.009DOI Listing
December 2017

Abundant expression of somatic transposon-derived piRNAs throughout Tribolium castaneum embryogenesis.

Genome Biol 2017 09 26;18(1):184. Epub 2017 Sep 26.

Faculty of Biology, Medicine and Health, University of Manchester, Michael Smith Building, Oxford Road, Manchester, M13 9PT, UK.

Background: Piwi-interacting RNAs (piRNAs) are a class of short (~26-31-nucleotide) non-protein-coding RNAs expressed in the metazoan germline. The piRNA pathway in arthropods is best understood in the ovary of Drosophila melanogaster, where it acts to silence active transposable elements (TEs). Maternal loading of piRNAs in oocytes is further required for the inheritance of piRNA-mediated transposon defence. However, our understanding of the diversity, evolution and function of the piRNA complement beyond drosophilids is limited. The red flour beetle, Tribolium castaneum, is an emerging model organism separated from Drosophila by ~ 350 million years of evolution that displays a number of features ancestral to arthropods, including short germ embryogenesis. Here, we characterize the maternally deposited and zygotically expressed small RNA and mRNA complements throughout T. castaneum embryogenesis.

Results: We find that beetle oocytes and embryos of all stages are abundant in heterogeneous ~ 28-nucleotide RNAs. These small RNAs originate from discrete genomic loci enriched in TE sequences and display the molecular signatures of transposon-derived piRNAs. In addition to the maternally loaded primary piRNAs, Tribolium embryos produce secondary piRNAs by the cleavage of zygotically activated TE transcripts via the ping-pong mechanism. The two Tribolium piRNA pathway effector proteins, Tc-Piwi/Aub and Tc-Ago3, are also expressed throughout the soma of early embryos.

Conclusions: Our results show that the piRNA pathway in Tribolium is not restricted to the germline, but also operates in the embryo and may act to antagonize zygotically activated transposons. Taken together, these data highlight a functional divergence of the piRNA pathway between insects.
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http://dx.doi.org/10.1186/s13059-017-1304-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5613491PMC
September 2017

The house spider genome reveals an ancient whole-genome duplication during arachnid evolution.

BMC Biol 2017 07 31;15(1):62. Epub 2017 Jul 31.

Department of Biological and Medical Sciences, Oxford Brookes University, Gipsy Lane, Oxford, OX3 0BP, UK.

Background: The duplication of genes can occur through various mechanisms and is thought to make a major contribution to the evolutionary diversification of organisms. There is increasing evidence for a large-scale duplication of genes in some chelicerate lineages including two rounds of whole genome duplication (WGD) in horseshoe crabs. To investigate this further, we sequenced and analyzed the genome of the common house spider Parasteatoda tepidariorum.

Results: We found pervasive duplication of both coding and non-coding genes in this spider, including two clusters of Hox genes. Analysis of synteny conservation across the P. tepidariorum genome suggests that there has been an ancient WGD in spiders. Comparison with the genomes of other chelicerates, including that of the newly sequenced bark scorpion Centruroides sculpturatus, suggests that this event occurred in the common ancestor of spiders and scorpions, and is probably independent of the WGDs in horseshoe crabs. Furthermore, characterization of the sequence and expression of the Hox paralogs in P. tepidariorum suggests that many have been subject to neo-functionalization and/or sub-functionalization since their duplication.

Conclusions: Our results reveal that spiders and scorpions are likely the descendants of a polyploid ancestor that lived more than 450 MYA. Given the extensive morphological diversity and ecological adaptations found among these animals, rivaling those of vertebrates, our study of the ancient WGD event in Arachnopulmonata provides a new comparative platform to explore common and divergent evolutionary outcomes of polyploidization events across eukaryotes.
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http://dx.doi.org/10.1186/s12915-017-0399-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5535294PMC
July 2017

RNAcentral: a comprehensive database of non-coding RNA sequences.

Nucleic Acids Res 2017 01 28;45(D1):D128-D134. Epub 2016 Oct 28.

RNAcentral is a database of non-coding RNA (ncRNA) sequences that aggregates data from specialised ncRNA resources and provides a single entry point for accessing ncRNA sequences of all ncRNA types from all organisms. Since its launch in 2014, RNAcentral has integrated twelve new resources, taking the total number of collaborating database to 22, and began importing new types of data, such as modified nucleotides from MODOMICS and PDB. We created new species-specific identifiers that refer to unique RNA sequences within a context of single species. The website has been subject to continuous improvements focusing on text and sequence similarity searches as well as genome browsing functionality. All RNAcentral data is provided for free and is available for browsing, bulk downloads, and programmatic access at http://rnacentral.org/.
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http://dx.doi.org/10.1093/nar/gkw1008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5210518PMC
January 2017

Mutations in SNORD118 cause the cerebral microangiopathy leukoencephalopathy with calcifications and cysts.

Nat Genet 2016 10 29;48(10):1185-92. Epub 2016 Aug 29.

Faculty of Biology, Medicine and Health, School of Biological Sciences, Division of Evolution and Genomic Sciences, University of Manchester, Manchester, UK.

Although ribosomes are ubiquitous and essential for life, recent data indicate that monogenic causes of ribosomal dysfunction can confer a remarkable degree of specificity in terms of human disease phenotype. Box C/D small nucleolar RNAs (snoRNAs) are evolutionarily conserved non-protein-coding RNAs involved in ribosome biogenesis. Here we show that biallelic mutations in the gene SNORD118, encoding the box C/D snoRNA U8, cause the cerebral microangiopathy leukoencephalopathy with calcifications and cysts (LCC), presenting at any age from early childhood to late adulthood. These mutations affect U8 expression, processing and protein binding and thus implicate U8 as essential in cerebral vascular homeostasis.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5045717PMC
http://dx.doi.org/10.1038/ng.3661DOI Listing
October 2016

Pervasive microRNA Duplication in Chelicerates: Insights from the Embryonic microRNA Repertoire of the Spider Parasteatoda tepidariorum.

Genome Biol Evol 2016 08 3;8(7):2133-44. Epub 2016 Aug 3.

Department of Biological and Medical Sciences, Oxford Brookes University, Gipsy Lane, Oxford, OX3 0BP, United Kingdom

MicroRNAs are small (∼22 nt) noncoding RNAs that repress translation and therefore regulate the production of proteins from specific target mRNAs. microRNAs have been found to function in diverse aspects of gene regulation within animal development and many other processes. Among invertebrates, both conserved and novel, lineage specific, microRNAs have been extensively studied predominantly in holometabolous insects such as Drosophila melanogaster However little is known about microRNA repertoires in other arthropod lineages such as the chelicerates. To understand the evolution of microRNAs in this poorly sampled subphylum, we characterized the microRNA repertoire expressed during embryogenesis of the common house spider Parasteatoda tepidariorum We identified a total of 148 microRNAs in P. tepidariorum representing 66 families. Approximately half of these microRNA families are conserved in other metazoans, while the remainder are specific to this spider. Of the 35 conserved microRNAs families 15 had at least two copies in the P. tepidariorum genome. A BLAST-based approach revealed a similar pattern of duplication in other spiders and a scorpion, but not among other chelicerates and arthropods, with the exception of a horseshoe crab. Among the duplicated microRNAs we found examples of lineage-specific tandem duplications, and the duplication of entire microRNA clusters in three spiders, a scorpion, and in a horseshoe crab. Furthermore, we found that paralogs of many P. tepidariorum microRNA families exhibit arm switching, which suggests that duplication was often followed by sub- or neofunctionalization. Our work shows that understanding the evolution of microRNAs in the chelicerates has great potential to provide insights into the process of microRNA duplication and divergence and the evolution of animal development.
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http://dx.doi.org/10.1093/gbe/evw143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4987109PMC
August 2016

Novel Intronic RNA Structures Contribute to Maintenance of Phenotype in Saccharomyces cerevisiae.

Genetics 2016 07 18;203(3):1469-81. Epub 2016 May 18.

Faculty of Life Sciences, University of Manchester, M13 9PT, United Kingdom

The Saccharomyces cerevisiae genome has undergone extensive intron loss during its evolutionary history. It has been suggested that the few remaining introns (in only 5% of protein-coding genes) are retained because of their impact on function under stress conditions. Here, we explore the possibility that novel noncoding RNA structures (ncRNAs) are embedded within intronic sequences and are contributing to phenotype and intron retention in yeast. We employed de novo RNA structure prediction tools to screen intronic sequences in S. cerevisiae and 36 other fungi. We identified and validated 19 new intronic RNAs via RNA sequencing (RNA-seq) and RT-PCR. Contrary to the common belief that excised introns are rapidly degraded, we found that, in six cases, the excised introns were maintained intact in the cells. In another two cases we showed that the ncRNAs were further processed from their introns. RNA-seq analysis confirmed that introns in ribosomal protein genes are more highly expressed when they contain predicted RNA structures. We deleted the novel intronic RNA structure within the GLC7 intron and showed that this region, rather than the intron itself, is responsible for the cell's ability to respond to salt stress. We also showed a direct association between the in cis presence of the intronic RNA and GLC7 expression. Overall, these data support the notion that some introns may have been maintained in the genome because they harbor functional RNA structures.
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http://dx.doi.org/10.1534/genetics.115.185363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4937481PMC
July 2016

MicroRNA evolution, expression, and function during short germband development in Tribolium castaneum.

Genome Res 2016 Jan 30;26(1):85-96. Epub 2015 Oct 30.

Faculty of Life Sciences, University of Manchester, Manchester, M13 9PT, United Kingdom.

MicroRNAs are well-established players in the development of multicellular animals. Most of our understanding of microRNA function in arthropod development comes from studies in Drosophila. Despite their advantages as model systems, the long germband embryogenesis of fruit flies is an evolutionary derived state restricted to several holometabolous insect lineages. MicroRNA evolution and expression across development in animals exhibiting the ancestral and more widespread short germband mode of embryogenesis has not been characterized. We sequenced small RNA libraries of oocytes and successive intervals covering the embryonic development of the short germband model organism, Tribolium castaneum. We analyzed the evolution and temporal expression of the microRNA complement and sequenced libraries of total RNA to investigate the relationships with microRNA target expression. We show microRNA maternal loading and sequence-specific 3' end nontemplate oligoadenylation of maternally deposited microRNAs that is conserved between Tribolium and Drosophila. We further uncover large clusters encoding multiple paralogs from several Tribolium-specific microRNA families expressed during a narrow interval of time immediately after the activation of zygotic transcription. These novel microRNAs, together with several early expressed conserved microRNAs, target a significant number of maternally deposited transcripts. Comparison with Drosophila shows that microRNA-mediated maternal transcript targeting is a conserved process in insects, but the number and sequences of microRNAs involved have diverged. The expression of fast-evolving and species-specific microRNAs in the early blastoderm of T. castaneum is consistent with previous findings in Drosophila and shows that the unique permissiveness for microRNA innovation at this stage is a conserved phenomenon.
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http://dx.doi.org/10.1101/gr.193367.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4691753PMC
January 2016

Conservation and losses of non-coding RNAs in avian genomes.

PLoS One 2015 30;10(3):e0121797. Epub 2015 Mar 30.

Bioinformatics Group, Department of Computer Science; and Interdisciplinary Center for Bioinformatics, University of Leipzig, Härtelstrasse 16-18, D-04107 Leipzig, Germany; Max Planck Institute for Mathematics in the Sciences, Inselstraße 22, D-04103 Leipzig, Germany; Fraunhofer Institute for Cell Therapy and Immunology, Perlickstrasse 1, D-04103 Leipzig, Germany; Department of Theoretical Chemistry of the University of Vienna, Währingerstrasse 17, A-1090 Vienna, Austria; Center for RNA in Technology and Health, Univ. Copenhagen, Grønnegårdsvej 3, Frederiksberg C, Denmark; Santa Fe Institute, 1399 Hyde Park Road, Santa Fe NM 87501, USA; German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig, Germany.

Here we present the results of a large-scale bioinformatics annotation of non-coding RNA loci in 48 avian genomes. Our approach uses probabilistic models of hand-curated families from the Rfam database to infer conserved RNA families within each avian genome. We supplement these annotations with predictions from the tRNA annotation tool, tRNAscan-SE and microRNAs from miRBase. We identify 34 lncRNA-associated loci that are conserved between birds and mammals and validate 12 of these in chicken. We report several intriguing cases where a reported mammalian lncRNA, but not its function, is conserved. We also demonstrate extensive conservation of classical ncRNAs (e.g., tRNAs) and more recently discovered ncRNAs (e.g., snoRNAs and miRNAs) in birds. Furthermore, we describe numerous "losses" of several RNA families, and attribute these to either genuine loss, divergence or missing data. In particular, we show that many of these losses are due to the challenges associated with assembling avian microchromosomes. These combined results illustrate the utility of applying homology-based methods for annotating novel vertebrate genomes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0121797PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4378963PMC
April 2016

Bias in microRNA functional enrichment analysis.

Bioinformatics 2015 May 20;31(10):1592-8. Epub 2015 Jan 20.

Faculty of Medical and Human Sciences, and Faculty of Life Sciences, University of Manchester, UK.

Motivation: Many studies have investigated the differential expression of microRNAs (miRNAs) in disease states and between different treatments, tissues and developmental stages. Given a list of perturbed miRNAs, it is common to predict the shared pathways on which they act. The standard test for functional enrichment typically yields dozens of significantly enriched functional categories, many of which appear frequently in the analysis of apparently unrelated diseases and conditions.

Results: We show that the most commonly used functional enrichment test is inappropriate for the analysis of sets of genes targeted by miRNAs. The hypergeometric distribution used by the standard method consistently results in significant P-values for functional enrichment for targets of randomly selected miRNAs, reflecting an underlying bias in the predicted gene targets of miRNAs as a whole. We developed an algorithm to measure enrichment using an empirical sampling approach, and applied this in a reanalysis of the gene ontology classes of targets of miRNA lists from 44 published studies. The vast majority of the miRNA target sets were not significantly enriched in any functional category after correction for bias. We therefore argue against continued use of the standard functional enrichment method for miRNA targets.
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http://dx.doi.org/10.1093/bioinformatics/btv023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4426843PMC
May 2015

The first myriapod genome sequence reveals conservative arthropod gene content and genome organisation in the centipede Strigamia maritima.

Authors:
Ariel D Chipman David E K Ferrier Carlo Brena Jiaxin Qu Daniel S T Hughes Reinhard Schröder Montserrat Torres-Oliva Nadia Znassi Huaiyang Jiang Francisca C Almeida Claudio R Alonso Zivkos Apostolou Peshtewani Aqrawi Wallace Arthur Jennifer C J Barna Kerstin P Blankenburg Daniela Brites Salvador Capella-Gutiérrez Marcus Coyle Peter K Dearden Louis Du Pasquier Elizabeth J Duncan Dieter Ebert Cornelius Eibner Galina Erikson Peter D Evans Cassandra G Extavour Liezl Francisco Toni Gabaldón William J Gillis Elizabeth A Goodwin-Horn Jack E Green Sam Griffiths-Jones Cornelis J P Grimmelikhuijzen Sai Gubbala Roderic Guigó Yi Han Frank Hauser Paul Havlak Luke Hayden Sophie Helbing Michael Holder Jerome H L Hui Julia P Hunn Vera S Hunnekuhl LaRonda Jackson Mehwish Javaid Shalini N Jhangiani Francis M Jiggins Tamsin E Jones Tobias S Kaiser Divya Kalra Nathan J Kenny Viktoriya Korchina Christie L Kovar F Bernhard Kraus François Lapraz Sandra L Lee Jie Lv Christigale Mandapat Gerard Manning Marco Mariotti Robert Mata Tittu Mathew Tobias Neumann Irene Newsham Dinh N Ngo Maria Ninova Geoffrey Okwuonu Fiona Ongeri William J Palmer Shobha Patil Pedro Patraquim Christopher Pham Ling-Ling Pu Nicholas H Putman Catherine Rabouille Olivia Mendivil Ramos Adelaide C Rhodes Helen E Robertson Hugh M Robertson Matthew Ronshaugen Julio Rozas Nehad Saada Alejandro Sánchez-Gracia Steven E Scherer Andrew M Schurko Kenneth W Siggens DeNard Simmons Anna Stief Eckart Stolle Maximilian J Telford Kristin Tessmar-Raible Rebecca Thornton Maurijn van der Zee Arndt von Haeseler James M Williams Judith H Willis Yuanqing Wu Xiaoyan Zou Daniel Lawson Donna M Muzny Kim C Worley Richard A Gibbs Michael Akam Stephen Richards

PLoS Biol 2014 Nov 25;12(11):e1002005. Epub 2014 Nov 25.

Human Genome Sequencing Center, Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, United States of America.

Myriapods (e.g., centipedes and millipedes) display a simple homonomous body plan relative to other arthropods. All members of the class are terrestrial, but they attained terrestriality independently of insects. Myriapoda is the only arthropod class not represented by a sequenced genome. We present an analysis of the genome of the centipede Strigamia maritima. It retains a compact genome that has undergone less gene loss and shuffling than previously sequenced arthropods, and many orthologues of genes conserved from the bilaterian ancestor that have been lost in insects. Our analysis locates many genes in conserved macro-synteny contexts, and many small-scale examples of gene clustering. We describe several examples where S. maritima shows different solutions from insects to similar problems. The insect olfactory receptor gene family is absent from S. maritima, and olfaction in air is likely effected by expansion of other receptor gene families. For some genes S. maritima has evolved paralogues to generate coding sequence diversity, where insects use alternate splicing. This is most striking for the Dscam gene, which in Drosophila generates more than 100,000 alternate splice forms, but in S. maritima is encoded by over 100 paralogues. We see an intriguing linkage between the absence of any known photosensory proteins in a blind organism and the additional absence of canonical circadian clock genes. The phylogenetic position of myriapods allows us to identify where in arthropod phylogeny several particular molecular mechanisms and traits emerged. For example, we conclude that juvenile hormone signalling evolved with the emergence of the exoskeleton in the arthropods and that RR-1 containing cuticle proteins evolved in the lineage leading to Mandibulata. We also identify when various gene expansions and losses occurred. The genome of S. maritima offers us a unique glimpse into the ancestral arthropod genome, while also displaying many adaptations to its specific life history.
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http://dx.doi.org/10.1371/journal.pbio.1002005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4244043PMC
November 2014

Intron evolution in Saccharomycetaceae.

Genome Biol Evol 2014 Sep;6(9):2543-56

Introns in protein-coding genes are very rare in hemiascomycetous yeast genomes. It has been suggested that these species have experienced extensive intron loss during their evolution from the postulated intron-rich fungal ancestor. However, no intron-devoidy east species have been identified and some of the introns remaining within the genomes of intron-poor species, such as Saccharomyces cerevisiae, appear to be beneficial during growth under stress conditions. In order to reveal the pattern of intron retention within intron-poor yeast species and better understand the mechanisms of intron evolution, we generated a comprehensive set of 250 orthologous introns in the 20 species that comprise the Saccharomycetaceae, by analyzing RNA deep-sequencing data and alignments of intron-containing genes. Analysis of these intron sets shows that intron loss is at least two orders of magnitude more frequent than intron gain. Fine mapping of intron positions shows that intron sliding is rare, and that introns are almost always removed without changing the primary sequence of the encoded protein. The latter finding is consistent with the prevailing view that homologous recombination between reverse-transcribed mature mRNAs and the corresponding genomic locus is the primary mechanism of intron loss. However, we also find evidence that loss of a small number of introns is mediated by micro-homology, and that the number of intron losses is diminished in yeast species that have lost the microhomology end joining and nonhomologous end joining machinery.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4202332PMC
http://dx.doi.org/10.1093/gbe/evu196DOI Listing
September 2014

RNAcentral: an international database of ncRNA sequences.

Nucleic Acids Res 2015 01 28;43(Database issue):D123-9. Epub 2014 Oct 28.

The field of non-coding RNA biology has been hampered by the lack of availability of a comprehensive, up-to-date collection of accessioned RNA sequences. Here we present the first release of RNAcentral, a database that collates and integrates information from an international consortium of established RNA sequence databases. The initial release contains over 8.1 million sequences, including representatives of all major functional classes. A web portal (http://rnacentral.org) provides free access to data, search functionality, cross-references, source code and an integrated genome browser for selected species.
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http://dx.doi.org/10.1093/nar/gku991DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4384043PMC
January 2015

Conserved temporal patterns of microRNA expression in Drosophila support a developmental hourglass model.

Genome Biol Evol 2014 Aug 27;6(9):2459-67. Epub 2014 Aug 27.

Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom

The spatiotemporal control of gene expression is crucial for the successful completion of animal development. The evolutionary constraints on development are particularly strong for the mid-embryonic stage when body segments are specified, as evidenced by a high degree of morphological and protein-coding gene conservation during this period--a phenomenon known as the developmental hourglass. The discovery of microRNA-mediated gene control revealed an entirely new layer of complexity of the molecular networks that orchestrate development. However, the constraints on microRNA developmental expression and evolution, and the implications for animal evolution are less well understood. To systematically explore the conservation of microRNAs during development, we carried out a genome-wide comparative study of microRNA expression levels throughout the ontogenesis of two divergent fruit flies, Drosophila melanogaster and D. virilis. We show that orthologous microRNAs display highly similar temporal profiles regardless of their mutation rates, suggesting that the timely expression of microRNA genes can be more constrained than their sequence. Furthermore, transitions between key developmental events in the different species are accompanied by conserved shifts in microRNA expression profiles, with the mid-embryonic period between gastrulation and segmentation characterized by the highest similarity of microRNA expression. The conservation of microRNA expression therefore displays an hourglass pattern similar to that observed for protein-coding genes.
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http://dx.doi.org/10.1093/gbe/evu183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4202322PMC
August 2014

Target repression induced by endogenous microRNAs: large differences, small effects.

PLoS One 2014 20;9(8):e104286. Epub 2014 Aug 20.

Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom.

MicroRNAs are small RNAs that regulate protein levels. It is commonly assumed that the expression level of a microRNA is directly correlated with its repressive activity - that is, highly expressed microRNAs will repress their target mRNAs more. Here we investigate the quantitative relationship between endogenous microRNA expression and repression for 32 mature microRNAs in Drosophila melanogaster S2 cells. In general, we find that more abundant microRNAs repress their targets to a greater degree. However, the relationship between expression and repression is nonlinear, such that a 10-fold greater microRNA concentration produces only a 10% increase in target repression. The expression/repression relationship is the same for both dominant guide microRNAs and minor mature products (so-called passenger strands/microRNA* sequences). However, we find examples of microRNAs whose cellular concentrations differ by several orders of magnitude, yet induce similar repression of target mRNAs. Likewise, microRNAs with similar expression can have very different repressive abilities. We show that the association of microRNAs with Argonaute proteins does not explain this variation in repression. The observed relationship is consistent with the limiting step in target repression being the association of the microRNA/RISC complex with the target site. These findings argue that modest changes in cellular microRNA concentration will have minor effects on repression of targets.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0104286PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4139194PMC
May 2015

Natural antisense transcripts and long non-coding RNA in Neurospora crassa.

PLoS One 2014 12;9(3):e91353. Epub 2014 Mar 12.

The Michael Smith Building, Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.

The prevalence of long non-coding RNAs (lncRNA) and natural antisense transcripts (NATs) has been reported in a variety of organisms. While a consensus has yet to be reached on their global importance, an increasing number of examples have been shown to be functional, regulating gene expression at the transcriptional and post-transcriptional level. Here, we use RNA sequencing data from the ABI SOLiD platform to identify lncRNA and NATs obtained from samples of the filamentous fungus Neurospora crassa grown under different light and temperature conditions. We identify 939 novel lncRNAs, of which 477 are antisense to annotated genes. Across the whole dataset, the extent of overlap between sense and antisense transcripts is large: 371 sense/antisense transcripts are complementary over 500 nts or more and 236 overlap by more than 1000 nts. Most prevalent are 3' end overlaps between convergently transcribed sense/antisense pairs, but examples of divergently transcribed pairs and nested transcripts are also present. We confirm the expression of a subset of sense/antisense transcript pairs by qPCR. We examine the size, types of overlap and expression levels under the different environmental stimuli of light and temperature, and identify 11 lncRNAs that are up-regulated in response to light. We also find differences in transcript length and the position of introns between protein-coding transcripts that have antisense expression and transcripts with no antisense expression. These results demonstrate the ability of N. crassa lncRNAs and NATs to be regulated by different environmental stimuli and provide the scope for further investigation into the function of NATs.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0091353PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3951366PMC
May 2015

Fast-evolving microRNAs are highly expressed in the early embryo of Drosophila virilis.

RNA 2014 Mar 21;20(3):360-72. Epub 2014 Jan 21.

MicroRNAs are short non-protein-coding RNAs that regulate gene expression at the post-transcriptional level and are essential for the embryonic development of multicellular animals. Comparative genome-scale analyses have revealed that metazoan evolution is accompanied by the continuous acquisition of novel microRNA genes. This suggests that novel microRNAs may promote innovation and diversity in development. We determined the evolutionary origins of extant Drosophila microRNAs and estimated the sequence divergence between the 130 orthologous microRNAs in Drosophila melanogaster and Drosophila virilis, separated by 63 million years of evolution. We then generated small RNA sequencing data sets covering D. virilis development and explored the relationship between microRNA conservation and expression in a developmental context. We find that late embryonic, larval, and adult stages are dominated by conserved microRNAs. This pattern, however, does not hold for the early embryo, where rapidly evolving microRNAs are uniquely present at high levels in both species. The group of fast-evolving microRNAs that are highly expressed in the early embryo belong to two Drosophilid lineage-specific clusters: mir-310 ∼ 313 and mir-309 ∼ 6. These clusters have particularly complex evolutionary histories of duplication, gain, and loss. Our analyses suggest that the early embryo is a more permissive environment for microRNA changes and innovations. Fast-evolving microRNAs, therefore, have the opportunity to become preferentially integrated in early developmental processes, and may impact the evolution of development. The relationship between microRNA conservation and expression throughout the development of Drosophila differs from that previously observed for protein-coding genes.
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http://dx.doi.org/10.1261/rna.041657.113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3923130PMC
March 2014