Methods for Analysis of Circular RNAs: No Tautology

We are excited to announce funding by EMBO for implementing a workshop on circular RNAs.

Organizers: Vladimir Benes (main), Irene Bozzoni, Marie-Laure Baudet and
Christoph Dieterich
Category: EMBO Practical Course
Title: EMBO Practical Course: Methods for analysis of circular RNAs: No tautology
Dates: 17 November 2019 – 22 November 2019
Location: DE–Heidelberg

https://www.embl.de/training/events/2019/CIR19-01/index.html

 

Advanced Training with Oxford Nanopore Technologies

Join us for an advanced training experience using the Oxford Nanopore Technologies (ONT) platform. We will start with an introduction into ONT technology and devices, with the goal of covering end to end workflows for the preparation and analysis of human and yeast samples using whole genome and barcoded cDNA sequencing approaches. This course will cover the wet lab preparation of libraries from genomic DNA and total RNA, with a focus on the critical steps and potential pitfalls and understanding what constitutes a ‘good’ sample for purpose of best results using the technology. The training includes an overview of the MinKNOW GUI for GridION and MinION devices. We then cover methods available for basecalling and analysis of samples for structural variants and differential gene expression, using both Oxford Nanopore and open source tools.

More information and application at https://www.embl.de/training/events/2019/NAN19-01/index.html

Guardians of the transcriptome

Our new article on “Exon junction complexes suppress spurious splice sites to safeguard transcriptome integrity” is in press and will appear in Molecular Cell beginning of November. Congratulations to the team of authors: 

Volker Boehm1, Thiago Britto-Borges2,3, Anna-Lena Steckelberg1,4, Kusum K. Singh1,5, Jennifer V. Gerbracht1, Elif Gueney1, Lorea Blazquez6,7, Janine Altmüller8,9,10, Christoph Dieterich2,3, Niels H. Gehring1,11


Productive splicing of human pre-mRNAs requires the correct selection of authentic splice sites (SS) from the large pool of potential SS. Although SS consensus sequence and splicing regulatory proteins are known to influence SS usage, the mechanisms ensuring the effective suppression of cryptic SS are insufficiently explored. Here, we find that many aberrant exonic SS are efficiently silenced by the exon junction complex (EJC), a multi-protein complex that is deposited on spliced mRNA near the exon-exon junction. Upon depletion of EJC proteins, cryptic SS are de-repressed, leading to the mis-splicing of a broad set of mRNAs. Mechanistically, the EJC-mediated recruitment of the splicing regulator RNPS1 inhibits cryptic 5′SS usage, while the deposition of the EJC core directly masks reconstituted 3′SS, thereby precluding transcript disintegration. Thus, the EJC protects the transcriptome of mammalian cells from inadvertent loss of exonic sequences and safeguards the expression of intact, full length mRNAs.

New publication on circular RNAs in the heart

Identification of circular RNAs with host gene-independent expression in human model systems for cardiac differentiation and disease.

Siede D(1), Rapti K(2), Gorska AA(2), Katus HA(2), Altmüller J(3), Boeckel JN(2), Meder B(2), Maack C(4), Völkers M(2), Müller OJ(2), Backs J(5), Dieterich C(6).

AIMS: Cardiovascular disease, one of the most common causes of death in western populations, is characterized by changes in RNA splicing and expression. Circular RNAs (circRNA) originate from back-splicing events, which link a downstream 5′ splice site to an upstream 3′ splice site. Several back-splicing junctions (BSJ) have been described in heart biopsies from human, rat and mouse hearts (Werfel et al., 2016; Jakobi et al., 2016 ). Here, we use human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) to identify circRNA and host gene dynamics in cardiac development and disease. In parallel, we explore candidate interactions of selected homologs in mouse and rat via RIP-seq experiments.

METHODS AND RESULTS: Deep RNA sequencing of cardiomyocyte development and β-adrenergic stimulation uncovered 4518 circRNAs. The set of circular RNA host genes is enriched for chromatin modifiers and GTPase activity regulators. RNA-seq and qRT-PCR data showed that circular RNA expression is highly dynamic in the hiPSC-CM model with 320 circRNAs showing significant expression changes. Intriguingly, 82 circRNAs are independently regulated to their host genes. We validated the same circRNA dynamics for circRNAs from ATXN10, CHD7, DNAJC6 and SLC8A1 in biopsy material from human dilated cardiomyopathy (DCM) and control patients. Finally, we could show that rodent homologs of circMYOD, circSLC8A1, circATXN7 and circPHF21A interact with either the ribosome or Argonaute2 protein complexes.

CONCLUSION: CircRNAs are dynamically expressed in a hiPSC-CM model of cardiac development and stress response. Some circRNAs show similar, host-gene independent expression dynamics in patient samples and may interact with the ribosome and RISC complex. In summary, the hiPSC-CM model uncovered a new signature of potentially disease relevant circRNAs which may serve as novel therapeutic targets.

https://www.ncbi.nlm.nih.gov/pubmed/28676412

 

Flexbar 3.0 – SIMD and multicore parallelization

Motivation:

High throughput sequencing machines can process many samples in a single run. For Illumina systems, sequencing reads are barcoded with an additional DNA tag that is contained in the respective sequencing adapters. The recognition of barcode and adapter sequences is hence commonly needed for the analysis of next generation sequencing data. Flexbar performs demultiplexing based on barcodes and adapter trimming for such data. read more…

A microRNA-129-5p/Rbfox crosstalk coordinates homeostatic downscaling of excitatory synapses – New paper

Abstract: Synaptic downscaling is a homeostatic mechanism that allows neurons to reduce firing rates during chronically elevated network activity. Although synaptic downscaling is important in neural circuit development and epilepsy, the underlying mechanisms are poorly described. We performed small RNA profiling in picrotoxin (PTX)-treated hippocampal neurons, a model of synaptic downscaling. Thereby, we identified eight microRNAs (miRNAs) that were increased in response to PTX, including miR-129-5p, whose inhibition blocked synaptic downscaling in vitro and reduced epileptic seizure severity in vivo Using transcriptome, proteome, and bioinformatic analysis, we identified the calcium pump Atp2b4 and doublecortin (Dcx) as miR-129-5p targets. Restoring Atp2b4 and Dcx expression was sufficient to prevent synaptic downscaling in PTX-treated neurons. Furthermore, we characterized a functional crosstalk between miR-129-5p and the RNA-binding protein (RBP) Rbfox1. In the absence of PTX, Rbfox1 promoted the expression of Atp2b4 and Dcx. Upon PTX treatment, Rbfox1 expression was downregulated by miR-129-5p, thereby allowing the repression of Atp2b4 and Dcx. We therefore identified a novel activity-dependent miRNA/RBP crosstalk during synaptic scaling, with potential implications for neural network homeostasis and epileptogenesis.

Find me on Pubmed: https://www.ncbi.nlm.nih.gov/pubmed/28487411