Small RNA Regulation in Drosophila

Lead Research Organisation: University of Oxford

Abstract

The surface of the earth is populated by living things with extraordinary different appearances. However, they all made of cells and these units of life share the same set of organelles (‘small organs’) for their most basic functions. These small organs are present in almost every cell in our bodies and if they do not work properly, we will get sick or even die. The Liu group is interested in a few newly-found small organs within the cell and try to study how they work in the fruit fly, one of the most studied living things on our planet. Learning more about how these small organs work in fruit flies will help us to understand how they work in human and to find a better way to cure related disease.

Technical Summary

Intracellular compartments such as organelles are essential for a cell’s function. RNA occurs in every compartment in a cell. Research in the Liu group focuses on three fundamental questions relating to RNA, for which we seek an in vivo understanding. We choose the fruitfly Drosophila melanogaster as our prime model system because of its genetic tractability and the depth of genomic data. Studies in Drosophila have provided a key to vertebrate development and human biology. First, we will study the mechanisms by which the synthesis of CTP, one of the critical precursors of RNA and DNA, is compartmentalized within a cell. We have recently discovered that CTP synthase is compartmentalized in a novel evolutionarily conserved organelle, the cytoophidium. Compartmentation is essential for the localization of biological processes within a eukaryotic cell. CTP synthase has been an attractive target for developing agents against cancer, virus and parasites. We will investigate how CTP synthase is assembled into the cytoophidium and how the cytoophidium is linked to cancer biology. Second, we are interested in the biological roles of long noncoding RNAs in Drosophila. While much knowledge has been gained on the functionality of protein-coding genes, we know very little about the mechanisms by which noncoding RNAs function in a fly. We will analyse the spatial and temporal expression of long noncoding RNAs during Drosophila development and will investigate the underlying mechanisms of how they function in vivo. Finally, we will investigate how an RNP assembly and RNA splicing factor SMN and the abundance of U bodies are regulated during development. SMN, a major constituent of U bodies which contain snRNPs, is the determining factor for SMA. The study of SMN and the U body thus holds the key to identifying the cellular mechanism of SMA. We are particularly excited to investigate the role of SMN in the maintenance of stem cells and their pluripotency.

Publications

10 25 50
 
Title Cell Picture Show 
Description Ji-Long Liu's images on cytoophidia, recent-discovered subcellular structures, have been selected by Cell Picture Show - Cell Curiosities, 29 August 2013. 
Type Of Art Image 
Year Produced 2013 
Impact greater awareness of the research on cytoophidia 
URL http://www.cell.com/pictureshow/cell-curiosities
 
Title Journal Cover - Frontier in Biology 04/15 
Description Journal cover of Frontier in Biology (Issue 4, August 2015) 
Type Of Art Artwork 
Year Produced 2015 
Impact Greater awareness of our work on SMN. 
URL http://link.springer.com/journal/11515/10/4/page/1
 
Title Journal Cover - JCS 10/15 
Description Journal Cover of the Journal of Cell Science (1 Oct 2015). 
Type Of Art Image 
Year Produced 2015 
Impact Greater awareness of our work on cytoophidia. 
URL http://jcs.biologists.org/content/128/19.cover-expansion
 
Title Journal Cover - JGG 05/15) 
Description Cover image for the Journal of Genetics and Genomics (May 2015). 
Type Of Art Image 
Year Produced 2015 
Impact Greater awareness of our work on cytoophidia. 
URL http://www.sciencedirect.com/science/journal/16738527/42/5
 
Title Journal Cover - JGG 06/16 
Description Cover image for the Journal of Genetics and Genomics (June 2016) 
Type Of Art Image 
Year Produced 2016 
Impact Greater awareness of our work on cytoophidium 
URL http://www.sciencedirect.com/science/journal/16738527/43/6
 
Title Journal cover- JGG 01/14 
Description Cover image for the Journal of Genetics and Genomics (Jan 2014) 
Type Of Art Image 
Year Produced 2014 
Impact Greater awareness of our research on CRISPR. 
URL http://groups.mrcfgu.ox.ac.uk/liu-group/publications/JGG_Jan2014_cover.pdf
 
Title Journal cover- Methods 09/14 
Description Cover image for the journal Methods (Sep 2014). 
Type Of Art Image 
Year Produced 2014 
Impact Greater awareness of our research on CRISPR. 
URL http://groups.mrcfgu.ox.ac.uk/liu-group/publications/Methods_Sep2014_cover.pdf
 
Description Postdoc funding - GK
Amount R$ 60,000 (BRL)
Organisation Government of Brazil 
Sector Public
Country Brazil
Start 08/2015 
End 07/2016
 
Description Student Grant - CSC - YH
Amount £26,000 (GBP)
Organisation Chinese Scholarshop Council 
Sector Academic/University
Country China
Start 10/2014 
End 09/2016
 
Description Student Grant - QS- CSC
Amount £130,000 (GBP)
Organisation University of Leeds 
Department China Scholarship Council
Sector Academic/University
Country United Kingdom
Start 10/2012 
End 09/2015
 
Description Studentship Grant -Kimi
Amount £107,163 (GBP)
Funding ID 880422355193 
Organisation Government of Malaysia 
Sector Public
Country Malaysia
Start 09/2014 
End 08/2017
 
Description Visiting scholarship - XY
Amount £8,400 (GBP)
Organisation Chinese Academy of Sciences 
Sector Public
Country China
Start 04/2014 
End 10/2014
 
Title CRISPR-1 
Description Developed a highly efficient targeted genome engineering technology in Drosophila using the CRISPR/Cas9 system 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Provided To Others? No  
Impact published a highly-cited paper on Cell Reports (97 citations in 16 months). PMID: 23827738. Published 3 more research papers on CRISPR. PMID: 24326186; 24576617; 25135198. Published a review article on CRISPR. PMID: 24480743. Created a resource website (Oxfcrispr.org) 
URL http://oxfcrispr.org
 
Title CRISPR-2 
Description We have applied the CRISPR/Cas9 system to Drosophila S2 cells to generate targeted genetic mutations in more than 85% of alleles. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2014 
Provided To Others? Yes  
Impact Published a research paper on CRISPR. PMID: 24326186. We deposited the plasmids to Addgene. Beneficial to the Drosophila community. 
URL http://www.addgene.org/Ji-Long_Liu/
 
Title CRISPR-3 
Description We have designed and built a genome-wide CRISPR library covering 13,501 genes, among which 8989 genes are targeted by three or more independent single guide RNAs (sgRNAs). Moreover, we describe strategies to monitor the population of guide RNAs by high throughput sequencing (HTS). This library will provide an invaluable resource for the community to screen loss of function mutations for cellular phenotypes, and as a source of guide RNA designs for future studies. 
Type Of Material Technology assay or reagent 
Year Produced 2015 
Provided To Others? Yes  
Impact Published a research paper on genome-wide CRISPR library. PMID: 26165496. We deposited the library to Addgene. Beneficial to the Drosophila community. 
URL http://www.sciencedirect.com/science/article/pii/S1673852715000594
 
Title CRISPR-4 
Description Long non-coding RNAs (lncRNAs) have emerged as regulators of gene expression across metazoa. Interestingly, some lncRNAs function independently of their transcripts - the transcription of the lncRNA locus itself affects target genes. However, current methods of loss-of-function analysis are insufficient to address the role of lncRNA transcription from the transcript which has impeded analysis of their function. Using the minimal CRISPR interference (CRISPRi) system, we show that coexpression of the catalytically inactive Cas9 (dCas9) and guide RNAs targeting the endogenous roX locus in the Drosophila cells results in a robust and specific knockdown of roX1 and roX2 RNAs, thus eliminating the need for recruiting chromatin modifying proteins for effective gene silencing. Additionally, we find that the human and Drosophila codon optimized dCas9 genes are functional and show similar transcription repressive activity. Finally, we demonstrate that the minimal CRISPRi system suppresses roX transcription efficiently in vivo resulting in loss-of-function phenotype, thus validating the method for the first time in a multicelluar organism. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2016 
Provided To Others? Yes  
Impact Our analysis expands the genetic toolkit available for interrogating lncRNA function in situ and is adaptable for targeting multiple genes across model organisms. This led a publication. PMID: 26850642 
URL http://nar.oxfordjournals.org/content/early/2016/02/04/nar.gkw063.long
 
Title Drosophila genome-wide CRISPR library 
Description Analysis of sgRNA sequences in the raw library and at different time points after transfection into Drosophila S2R+ cells. 
Type Of Material Database/Collection of data 
Year Produced 2015 
Provided To Others? Yes  
Impact Beneficial to the Drosophila community. 
URL http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE67339
 
Description Autism 
Organisation University of Oxford
Department Department of Physiology, Anatomy and Genetics
Country United Kingdom 
Sector Academic/University 
PI Contribution All fly genetics was done in my group
Collaborator Contribution All bioinformatic analysis was done by my collaborator.
Impact Published a paper in PLOS Genetics
Start Year 2013
 
Description Drosophila non-coding RNA 
Organisation Medical Research Council (MRC)
Department MRC Functional Genomics Unit
Country United Kingdom 
Sector Public 
PI Contribution I co-supervise a PhD student who conducts experiments in my lab. I lead on the study of long intergenic noncoding RNAs in the fruit fly, a work package supported by an ERC advanced grant awarded to Prof Chris Ponting.
Collaborator Contribution regular discussion.
Impact The data derived from this collaboration trigged a following larger-scale collaboration on study of non-coding RNAs. Three researchers funded my Prof Ponting's ERC grant are conducting research in my laboratory. Seven papers published: PMID: 22403033; PMID: 23827738; PMID: 24326186; PMID: 25135198; PMID: 24480743; PMID: 24576617; PMID: 26850642
Start Year 2008
 
Description GARS 
Organisation John Radcliffe Hospital
Department Department of Clinical Neurology
Country United Kingdom 
Sector Hospitals 
PI Contribution Using fruit fly as a model to study GARS protein, which is related to motor neuron disease
Collaborator Contribution link to clinic medicine
Impact A manuscript derived from this collaboration has been submitted to a peer-reviewed journal
Start Year 2010
 
Description IMPDH Cytoophidia 
Organisation National Taiwan University
Country Taiwan, Province of China 
Sector Academic/University 
PI Contribution My group provided experimental designs
Collaborator Contribution My partner performed the experiments.
Impact This collaboration led to a publication.
Start Year 2014
 
Description Model Parkinson's disease in Drosophila 
Organisation University of Oxford
Department Nuffield Department of Clinical Neurosciences
Country United Kingdom 
Sector Academic/University 
PI Contribution The fly work is conduced in my lab. I and my student will train the collaborators on various techniques on fly genetics. We will aslo conduct some experiments.
Collaborator Contribution modeling human disease in the fruit fly.
Impact A manuscript has been submitted to a peer-reviewed journal.
Start Year 2010
 
Description SMN in ES cells 
Organisation National Taiwan University
Department Institute of Biotechnology
Country Taiwan, Province of China 
Sector Academic/University 
PI Contribution My group has identified that SMN plays important role in Drosophila stem cells
Collaborator Contribution work on SMN in germ cells and embryonic stem cells in mice
Impact a manuscript on this collaboration has been submitted to a peer-reviewed journal.
Start Year 2010
 
Description inactive CTPS 
Organisation Chang Gung University
Country Taiwan, Province of China 
Sector Academic/University 
PI Contribution The essential metabolic enzyme CTP synthase (CTPsyn) can be compartmentalised to form an evolutionarily-conserved intracellular structure termed the cytoophidium. We show that cytoophidium formation occurs during nutrient deprivation in cultured cells, as well as in quiescent and starved neuroblasts of the Drosophila larval central nervous system. We also show that cytoophidia formation is reversible during neurogenesis, indicating that filament formation regulates pyrimidine synthesis in a normal developmental context. Together, our study provides experimental evidence, and a mathematical model, for the hypothesis that inactive CTPsyn is incorporated into cytoophidia.
Collaborator Contribution Our partners helped us analyse our global metabolic profiling, suggesting that cytoophidium formation facilitates metabolic stabilisation. In addition, our partners showed that overexpression of CTPsyn only results in moderate increase of CTP pool in human stable cell lines.
Impact This collaboration led to a publication.
Start Year 2014
 
Description inactive CTPS 
Organisation Chinese Academy of Sciences
Country China 
Sector Public 
PI Contribution The essential metabolic enzyme CTP synthase (CTPsyn) can be compartmentalised to form an evolutionarily-conserved intracellular structure termed the cytoophidium. We show that cytoophidium formation occurs during nutrient deprivation in cultured cells, as well as in quiescent and starved neuroblasts of the Drosophila larval central nervous system. We also show that cytoophidia formation is reversible during neurogenesis, indicating that filament formation regulates pyrimidine synthesis in a normal developmental context. Together, our study provides experimental evidence, and a mathematical model, for the hypothesis that inactive CTPsyn is incorporated into cytoophidia.
Collaborator Contribution Our partners helped us analyse our global metabolic profiling, suggesting that cytoophidium formation facilitates metabolic stabilisation. In addition, our partners showed that overexpression of CTPsyn only results in moderate increase of CTP pool in human stable cell lines.
Impact This collaboration led to a publication.
Start Year 2014
 
Description inactive CTPS 
Organisation National Taiwan University
Country Taiwan, Province of China 
Sector Academic/University 
PI Contribution The essential metabolic enzyme CTP synthase (CTPsyn) can be compartmentalised to form an evolutionarily-conserved intracellular structure termed the cytoophidium. We show that cytoophidium formation occurs during nutrient deprivation in cultured cells, as well as in quiescent and starved neuroblasts of the Drosophila larval central nervous system. We also show that cytoophidia formation is reversible during neurogenesis, indicating that filament formation regulates pyrimidine synthesis in a normal developmental context. Together, our study provides experimental evidence, and a mathematical model, for the hypothesis that inactive CTPsyn is incorporated into cytoophidia.
Collaborator Contribution Our partners helped us analyse our global metabolic profiling, suggesting that cytoophidium formation facilitates metabolic stabilisation. In addition, our partners showed that overexpression of CTPsyn only results in moderate increase of CTP pool in human stable cell lines.
Impact This collaboration led to a publication.
Start Year 2014
 
Description nuclear Cytoophidia 
Organisation National Taiwan University
Country Taiwan, Province of China 
Sector Academic/University 
PI Contribution We discovered that CTPsyn forms cytoophidia in the nucleus of human cells.
Collaborator Contribution Our partner made live imaging to support our results.
Impact The collaboration led to a publication.
Start Year 2012
 
Description ASCBTV 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Cell biology and physics - interviewed by ASCBTV - What Ji-Long Liu was looking forward to at the 2013 ASCB Annual Meeting, 14 December 2013.

not known
Year(s) Of Engagement Activity 2013
 
Description Castaways at the Ashmolean 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Castaways at the Ashmolean - Ji-Long Liu's choice of castaway object was reported on Oxfordshire Limited Edition, 9 January 2014, and also featured in Sylvia Vetta's new book Oxford Castaways 2, April 2014.

greater international awareness of research undertaken in MRC and FGU.
Year(s) Of Engagement Activity 2014
 
Description Cheltenham Science Festival 2013 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Cheltenham Science Festival 2013 - 3 members of staff (Stephen Meader, Charlotte Tibbit and Mayte Siswick) from the Webber and Liu groups represented the MRC at the Festival, 7 June 2013.

public awareness of scientific research
Year(s) Of Engagement Activity 2013
 
Description HW 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Ji-Long Liu hosted a 9-week long work experience for a 17 year old pupil from D'overbroeck's College, July-September 2014.

increase the pupil experience on research
Year(s) Of Engagement Activity 2014
 
Description MRC Centenary mini-Science Festival 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Dr Charlotte Tibbit, Research Assistant in the Liu Group, helps the MRC Centenary mini-Science Festival in Oxford, June 2013

public awareness of research undertaken by MRC
Year(s) Of Engagement Activity 2013
 
Description Media - Newspaper 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Being reported in an international newspaper to reach a wider audiences.
Year(s) Of Engagement Activity 2015
 
Description QLZB 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Gave an online live talk which was well received
Year(s) Of Engagement Activity 2016
 
Description SanLian Weekly 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Live and work in Oxford - Ji-Long Liu gave an interview to Sanlian LifeWeek, the Chinese equivalent of TIME magazine, 9 June 2014.

greater international awareness of research and life in Oxford.
Year(s) Of Engagement Activity 2014
 
Description School Visit - HS2 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact 200 pupils attended for a school visit, which sparked questions and discussion afterwards.
Year(s) Of Engagement Activity 2015
 
Description School Visit - JJ1 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact 250 pupils attended for a school visit, which sparked questions and discussion afterwards. This has been reported in local media.
Year(s) Of Engagement Activity 2015
 
Description School Visit - JJ2 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact 300 pupils attended for a school visit, which sparked questions and discussion afterwards. This has been listed as one of the top 10 events in 2015 in the school.
Year(s) Of Engagement Activity 2015
 
Description ZZFZ 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Wrote a pupular article on cytoophidia
Year(s) Of Engagement Activity 2016