Congregating in the beautiful Czech city of Prague, more than 1,000 researchers from different corners of the world explored RNA in all its aspects last week at the RNA Society meeting. Lexogen is honored to be a regular supporter of this meeting, and already for the second time we organized a morning seminar on the first full day of the conference. Three excellent academic speakers presented their research to over 200 RNA scientists, who were very motivated to wake up early in the morning to learn about the wide range of applications Lexogen’s QuantSeq technology can support. The main topic of the workshop was transcriptome-wide and targeted gene expression profiling with the presented studies benefiting from QuantSeq protocol to evaluate poly(A) site usage, gene expression and profiling of co-immunoprecipitated RNAs.
The RNA Society meeting was also a chance to team up with some of the organizers of the international RNA Salons, which Lexogen is happy to support, too. These events enable young scientist to connect with other RNA researchers and to present their findings at a regional level.
We are looking forward to RNA 2018 in Berkeley in sunny California and to many more RNA Salon meetings in 2017 – expect Lexogen’s tools to advance your research!
Morning Seminar agenda:
Adapting QuantSeq 3’ RNA-Seq for Digital Gene Expression profiling of mRNAs and targeted RNAs
Lukas Paul, Senior Manager of Scientific Affairs, Lexogen, Vienna, Austria
Use of QuantSeq to study the regulation of 3’ end processing by TDP-43
Martina Hallegger1,2, Gregor Rot3, Nejc Haberman1,2, Ina Huppertz4, Christian von Mering3, Jernej Ule1,2
1 Department of Molecular Neuroscience, UCL Institute of Neurology, Queen Square, London, WC1N 3BG, UK;
2 Current address: The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK;
3 Institute of Molecular Life Sciences and Swiss Institute of Bioinformatics, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland;
4 European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany
Most motor neuron disease (MND) patients display neuronal, cytoplasmic TDP-43 protein aggregates, which give the RNA-binding protein (RBP) TDP-43 a central role in the development of this disease. We studied the function of this protein with the use of individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP), Lexogen QuantSeq 3’ mRNA-Seq and Lexogen SENSE mRNA-Seq.To integrate these data sets we developed expressRNA, a research platform for combining analysis of alternative splicing and alternative polyadenylation with the protein RNA binding sites identified by iCLIP and sequence analysis. We are visualising the regulatory principles using RNA maps, which link the binding position of RBPs to their regulatory functions. Similar to TDP-43’s splicing regulation, we find that TDP 43 binds UG rich motifs close to the repressed polyA sites, and further away from the enhanced polyA sites. Aggregation of TDP-43 is promoted by its low-complexity (LC) domain, which is also the region containing most of the disease-causing mutations. While most studies suggest that the LC domain mainly mediates protein-protein interactions, its function is poorly understood. We therefore examined the role of the TDP-43 LC domain in protein-RNA interactions and the functions of these interactions in regulating pre-mRNA processing. For this purpose, we created cell lines where the endogenous RBP is replaced by mutants lacking specific portions of the LC domain. We show that deletions in the LC domain affect the function of TDP-43 in splicing and 3’ end processing.
Molecular basis for cytoplasmic RNA surveillance by uridylation-triggered decay in Drosophila
Madalena M Reimão-Pinto1, Raphael A Manzenreither1, Thomas R Burkard1, Pawel Sledz2, Martin Jinek2, Karl Mechtler1 & Stefan L Ameres1
1 Institute of Molecular Biotechnology, IMBA, Vienna Biocenter Campus (VBC), Vienna, Austria;
2 Department of Biochemistry, University of Zurich, Zurich, Switzerland
The posttranscriptional addition of nucleotides to the 3´ end of RNA regulates the maturation, function, and stability of RNA species in all domains of life. Here, we show that in flies, 3´ terminal RNA uridylation triggers the processive, 3´-to-5´ exoribonucleolytic decay via the RNase II/Renzyme CG16940, a homolog of the human Perlman syndrome exoribonuclease Dis3l2. Together with the TUTase Tailor, dmDis3l2 forms the cytoplasmic, terminal RNA uridylation-mediated processing (TRUMP) complex that functionally cooperates in the degradation of structured RNA. RNA immunoprecipitation and high-throughput sequencing reveals a variety of TRUMP complex substrates, including abundant non-coding RNA, such as 5S rRNA, tRNA, snRNA, snoRNA, and the essential RNase MRP. Based on genetic and biochemical evidence, we propose a key function of the TRUMP complex in the cytoplasmic quality control of RNA polymerase III transcripts. Together with high-throughput biochemical characterization of dmDis3l2 and bacterial RNase R, our results imply a conserved molecular function of RNase II/R enzymes as “readers” of destabilizing posttranscriptional marks—uridylation in eukaryotes and adenylation in prokaryotes—that play important roles in RNA surveillance.
Thiol-linked alkylation for the metabolic sequencing of RNA
Veronika A. Herzog1, Brian Reichholf1, Philipp Rescheneder2, Tobias Neumann3, Pooja Bhat1, Thomas Burkard1, and Stefan L. Ameres1
1 Institute of Molecular Biotechnology (IMBA), Vienna Biocenter Campus (VBC), 1030 Vienna, Austria;
2 Center for Integrative Bioinformatics Vienna, Max F Perutz Laboratories, Medical University of Vienna, University of Vienna, Vienna Biocenter Campus (VBC), 1030 Vienna, Austria;
3 Research Institute of Molecular Pathology (IMP), Vienna Biocenter Campus (VBC), 1030 Vienna, Austria;
During the poster session on Friday night visit our poster (#305).
Identification of internal priming events in Lexogen’s QuantSeq library preparation leads to accurate gene quantification and detection of 3’ ends
Patrick Schagerl1, Michael Ante1, Lukas Paul1 and Andreas Tuerk1
1 Lexogen GmbH, Campus Vienna Biocenter 5, Vienna, Austria;
Accurate gene quantification and identification of 3’UTRs is important in many areas of biological research. Lexogen’s QuantSeq library preparation provides an easy and fast protocol for generating highly strand-specific NGS libraries close to the 3’ end of polyadenylated RNAs. The use of oligo(dT) primers in QuantSeq, however, also generates reads at long internal poly(A) stretches. Such reads can negatively affect the accuracy of end site detection and gene quantification. We, therefore, developed an internal priming filter (IPF) to remove reads associated with internal priming events. For this purpose, we investigate properties of the genome sequence and annotation around the read ends. In a window immediately downstream of the read end we count the number of A’s, upstream we search for 3’ motifs. We find the optimal cut-off for the number of A’s by fitting a logistic regression model to sets of high-confidence internal priming and end sites having the same size ratio as internal priming and end sites in one chromosome. This ratio is determined by fitting a Gaussian mixture to the bimodal distribution of A-count frequencies. On the MAQC dataset the IPF retained 85.32% of the original reads in 3’UTRs, whereas only 5% and 3.68% were retained in 5’UTRs and introns, respectively. An increased concentration of reads at the gene ends can also be observed in coverage plots. Base content around priming sites after IPF showed the typical end site signature with an increased frequency of CA at the read end site, A upstream and GU/U downstream. Correlation between log fold changes of read counts and qPCR values increased after IPF from R2=0.7234 to R2=0.8175 suggesting superior accuracy in detecting differentially expressed genes. The described method is part of the QuantSeq pipeline including gene quantification and differential expression analysis which can be downloaded from the Lexogen website.