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Gehring Lab | RNA Biology

Gehring Lab | RNA Biology

RESEARCH

Mechanistic understanding of eukaryotic gene expression

Gene expression is the fundamental process that uses the genomic information in order to synthesize gene products. We focus on understanding the general mechanisms and processes that regulate gene expression and thereby ensure faithful protein production. Specifically, we are interested how ribonucleoprotein (RNP) complexes influence various stages of the messenger RNA (mRNA) lifecycle. More detailed information about ongoing research projects in the lab can be found below.

Mechanism of
nonsense-mediated mRNA decay (NMD)

NMD

Northern blot analysis of NMD targets

Nonsense-mediated mRNA decay (NMD)


We study the molecular mechanism how defective gene products (mRNAs) are detected co-translationally and removed by the NMD pathway

To maintain the correct translation of proteins in the cell, quality control mechanisms constantly monitor each step of gene expression. One of these mechanisms is the nonsense-mediated mRNA decay pathway, or short NMD. If due to mutations or other reasons, such as mistakes during pre-mRNA processing, a transcript contains a premature translation termination codon (PTC), this mRNA is detected by NMD and consequently degraded. Thereby, NMD generally protects the cell by preventing the translation of truncated proteins.

NMD pathway

Nevertheless, if the PTC-containing mRNAs in principle encode for partially functional proteins, as it is the case in certain diseases, the clinical phenotype could be alleviated by blocking NMD specifically. To better understand, how NMD is activated, how PTC-containing mRNAs are distinguished from normal ones, and how the transcripts are degraded in the end, we use molecular-biological and biochemical experimental approaches in cultured human cells.

Exon junction complex (EJC)
assembly and function

EJC

Structure of the core exon junction complex

Exon junction complex (EJC)


The EJC is a multi-protein complex that is deposited by the splicing process on mRNAs and influences the fate of the transcript. We study how and which processes are regulated by the EJC.

From the moment when a mRNA is first transcribed by the RNA polymerase, RNA-binding proteins associate with this transcript and influence its fate. One protein complex in particular, called the exon junction complex (EJC), is deposited on the mRNA during splicing. The EJC remains bound on the mRNA until it is displaced in the cytoplasm by ribosomes. Until then, the EJC regulates alternative splicing, enhances nuclear mRNA export, stimulates translation and ultimately, is involved in quality control (see nonsense-mediated mRNA decay).

EJC research

Other mRNA/mRNP research projects

Other

Immunofluorescence analysis of protein localization

Other mRNA/mRNP research projects


Other research areas involve studying mRNA export and ribosomal RNA biogenesis

Before an mRNA is translated in the cytoplasm, it has to be processed in the nucleus and transported to the cytoplasm. The export of mature mRNAs is tightly linked to nuclear pre-mRNA processing to ensure that only correctly processed transcripts are translated. The recruitment of the general mRNA export receptor NXF1/NXT1 to spliced mRNA involves different adaptor proteins. Some of these adaptor proteins (e.g. ALYREF) interact with the exon junction complex, which therefore provides a link between pre-mRNA splicing and export.

HIGHLIGHTS

News and impressions from the lab

NMD2FA

Now published in Nature Communications!

Check out the final version of our "two-factor authentication" NMD story - with new evidence supporting the role of SMG5-SMG7 in NMD (click picture for direct link)

Franz

New B.Sc. student - Welcome Franz!

Franz will illuminate the so far obscure early steps of NMD initiation, focussing on the interplay between multiple NMD factors

ScNapBar

Collaborative work on Nanopore - Single-cell RNA-seq

Interested in full-length RNA sequencing combined with single-cell approaches + NMD use-case? (click picture for direct link)

Ecd

Great collaboration with the Uhlirova lab on splicing regulation in Drosophila

Read more about how Ecd promotes U5 snRNP maturation and Prp8 stability in this work by Steffen Erkelenz and colleagues (click picture for direct link)

NewStudents

We welcome our new students in the lab!

Barbara will work in her B.Sc. thesis on different ways to inhibit NMD in human cells.
Dominik will focus in his M.Sc. thesis on different NMD aspects in collaboration with the Kolanus lab (LIMES Institute Bonn)

TiG

Anything but Ordinary - Emerging Splicing Mechanisms in Eukaryotic Gene Regulation

Review together with Jean-Yves Roignant on unconventional splicing (click picture for direct link)

2FA

New preprint with mechanistic insights into NMD activation

Nonsense-mediated mRNA decay relies on “two-factor authentication” by SMG5-SMG7; now on bioRxiv (click picture for direct link)

CASC3

CASC3 promotes transcriptome-wide activation of nonsense-mediated decay by the exon junction complex

Now published in Nucleic Acids Research (click picture for direct link)!

Day-EJC

A Day in the Life of the Exon Junction Complex

Review from Lena and Niels on a regular "day" in the life of the EJC (click picture for direct link)

CASC3

CASC3 promotes transcriptome-wide activation of nonsense-mediated decay by the exon junction complex

First preprint from the Gehring lab, many thanks to all co-authors!

Oktoberfest2019
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Oktoberfest 2019

Oktoberfest 2019

O'Zapft is! Celebrating Bavarian beer culture

HD2019

Protein Synthesis and Translational Control

Presented our latest story in the mRNA turnover session. Delighted about the return of the EMBO photo booth.

RNA2019
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RNA2019 Conference

RNA 2019 conference in beautiful Kraków

Exciting talks in a great venue and city, plus another reunion of Gehringians!

NProt

Detailed xrRNA protocol is online

Find out how to use XRN1-resistant RNAs to visualize and track RNA degradation in this collaborative Nature Protocol with the Chao Lab in Basel

Karneval2019
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Karneval2019

Karneval2019

Kölle Alaaf 2019!

PEOPLE

Meet the Gehring Lab Team

Niels

Niels H. Gehring

Group Leader

Jessy

Jessica Wiegel

Secretary

Juliane

Juliane Hancke

Technician

Volker

Volker Böhm

Postdoc

Lena

Lena Schlautmann

PhD Student

Sabrina

Sabrina Kückelmann

PhD Student

Damaris

Damaris Wallmeroth

PhD Student

Vanessa

Vanessa Schwietzke

Technician

Dominik

Dominik Aschemeier

M. Sc. student

Barbara

Barbara Maubach

B. Sc. student

Franz

Franz Reichel

B. Sc. student

Unexcused days of absence: 1


Simona Ciriello

Tobias Fatscher

Jennifer Gerbracht

Agnieszka Gromadzka

Julia Keloglou

Marie Charlotte Marx

Franziska Ottens

Kusum Singh

Anna-Lena Steckelberg

Joanna Swierz

Heidi Thelen

Benjamin Weiche

PUBLICATIONS

Published research from the Gehring Lab

  1. Boehm V, Kueckelmann S, Gerbracht JV, Kallabis S, Britto-Borges T, Altmüller J, Krüger M, Dieterich C, Gehring NH
    SMG5-SMG7 authorize nonsense-mediated mRNA decay by enabling SMG6 endonucleolytic activity
    Nat Commun. 2021 Jun 25;12(1):3965. doi: 10.1038/s41467-021-24046-3.
    PubMed
    Abstract
    Eukaryotic gene expression is constantly controlled by the translation-coupled nonsense-mediated mRNA decay (NMD) pathway. Aberrant translation termination leads to NMD activation, resulting in phosphorylation of the central NMD factor UPF1 and robust clearance of NMD targets via two seemingly independent and redundant mRNA degradation branches. Here, we uncover that the loss of the first SMG5-SMG7-dependent pathway also inactivates the second SMG6-dependent branch, indicating an unexpected functional connection between the final NMD steps. Transcriptome-wide analyses of SMG5-SMG7-depleted cells confirm exhaustive NMD inhibition resulting in massive transcriptomic alterations. Intriguingly, we find that the functionally underestimated SMG5 can substitute the role of SMG7 and individually activate NMD. Furthermore, the presence of either SMG5 or SMG7 is sufficient to support SMG6-mediated endonucleolysis of NMD targets. Our data support an improved model for NMD execution that features two-factor authentication involving UPF1 phosphorylation and SMG5-SMG7 recruitment to access SMG6 activity.
    2FA
    ×

    Two-factor authentication

  2. Wang Q, Boenigk S, Boehm V, Gehring NH, Altmueller J, Dieterich C
    Single cell transcriptome sequencing on the Nanopore platform with ScNapBar
    RNA. 2021 Apr 27;rna.078154.120. doi: 10.1261/rna.078154.120. Online ahead of print.
    PubMed
    Abstract
    The current ecosystem of single cell RNA-seq platforms is rapidly expanding, but robust solutions for single cell and single molecule full- length RNA sequencing are virtually absent. A high-throughput solution that covers all aspects is necessary to study the complex life of mRNA on the single cell level. The Nanopore platform offers long read sequencing and can be integrated with the popular single cell sequencing method on the 10x Chromium platform. However, the high error-rate of Nanopore reads poses a challenge in downstream processing (e.g. for cell barcode assignment). We propose a solution to this particular problem by using a hybrid sequencing approach on Nanopore and Illumina platforms. Our software ScNapBar enables cell barcode assignment with high accuracy, especially if sequencing satura- tion is low. ScNapBar uses unique molecular identifier (UMI) or Naıve Bayes probabilistic approaches in the barcode assignment, depending on the available Illumina sequencing depth. We have benchmarked the two approaches on simulated and real Nanopore datasets. We further applied ScNapBar to pools of cells with an active or a silenced non-sense mediated RNA decay pathway. Our Nanopore read assignment distinguishes the respective cell populations and reveals characteristic nonsense-mediated mRNA decay events depending on cell status.
  3. Erkelenz S, Stanković D, Mundorf J, Bresser T, Claudius AK, Boehm V, Gehring NH, Uhlirova M
    Ecd promotes U5 snRNP maturation and Prp8 stability
    Nucleic Acids Res. 2021 Feb 22;49(3):1688-1707. doi: 10.1093/nar/gkaa1274.
    PubMed
    Abstract
    Pre-mRNA splicing catalyzed by the spliceosome represents a critical step in the regulation of gene expression contributing to transcriptome and proteome diversity. The spliceosome consists of five small nuclear ribonucleoprotein particles (snRNPs), the biogenesis of which remains only partially understood. Here we define the evolutionarily conserved protein Ecdysoneless (Ecd) as a critical regulator of U5 snRNP assembly and Prp8 stability. Combining Drosophila genetics with proteomic approaches, we demonstrate the Ecd requirement for the maintenance of adult healthspan and lifespan and identify the Sm ring protein SmD3 as a novel interaction partner of Ecd. We show that the predominant task of Ecd is to deliver Prp8 to the emerging U5 snRNPs in the cytoplasm. Ecd deficiency, on the other hand, leads to reduced Prp8 protein levels and compromised U5 snRNP biogenesis, causing loss of splicing fidelity and transcriptome integrity. Based on our findings, we propose that Ecd chaperones Prp8 to the forming U5 snRNP allowing completion of the cytoplasmic part of the U5 snRNP biogenesis pathway necessary to meet the cellular demand for functional spliceosomes.

  1. Gehring NH, Roignant JY
    Anything but Ordinary - Emerging Splicing Mechanisms in Eukaryotic Gene Regulation
    Trends Genet. 2020 Nov 14;S0168-9525(20)30297-3. doi: 10.1016/j.tig.2020.10.008.
    PubMed
    Abstract
    Splicing of precursor mRNAs (pre-mRNA) is an important step during eukaryotic gene expression. The identification of the actual splice sites and the proper removal of introns are essential for the production of the desired mRNA isoforms and their encoded proteins. While the basic mechanisms of splicing regulation are well understood, recent work has uncovered a growing number of noncanonical splicing mechanisms that play key roles in the regulation of gene expression. In this review, we summarize the current principles of splicing regulation, including the impact of cis and trans regulatory elements, as well as the influence of chromatin structure, transcription, and RNA modifications. We further discuss the recent development of emerging splicing mechanisms, such as recursive and back splicing, and their impact on gene expression.
  2. Boehm V, Kueckelmann S, Gerbracht JV, Britto-Borges T, Altmüller J, Dieterich C, Gehring NH.
    Nonsense-mediated mRNA decay relies on “two-factor authentication” by SMG5-SMG7
    bioRxiv 2020.07.07.191437; doi: https://doi.org/10.1101/2020.07.07.191437
    BioRxiv
    Abstract
    Eukaryotic gene expression is constantly regulated and controlled by the translation-coupled nonsense-mediated mRNA decay (NMD) pathway. Aberrant translation termination leads to NMD activation and robust clearance of NMD targets via two seemingly independent and redundant mRNA degradation branches. Here, we uncover that the loss of the first SMG5-SMG7-dependent pathway also inactivates the second SMG6-dependent branch, indicating an unexpected functional hierarchy of the final NMD steps. Transcriptome-wide analyses of SMG5-SMG7-depleted cells confirm complete NMD inhibition resulting in massive transcriptomic alterations. The NMD activity conferred by SMG5-SMG7 is determined to varying degrees by their interaction with the central NMD factor UPF1, heterodimer formation and the initiation of deadenylation. Surprisingly, we find that SMG5 functionally substitutes SMG7 and vice versa. Our data support an improved model for NMD execution that requires two-factor authentication involving UPF1 phosphorylation and SMG5-SMG7 recruitment to access SMG6 activity.
    2FA
    ×

    Two-factor authentication

  3. Gerbracht JV, Boehm V, Britto-Borges T, Kallabis S, Wiederstein JL, Ciriello S, Aschemeier DU, Krüger M, Frese CK, Altmüller J, Dieterich C, Gehring NH.
    CASC3 promotes transcriptome-wide activation of nonsense-mediated decay by the exon junction complex
    Nucleic Acids Res. 2020 Jul 4:gkaa564. doi: 10.1093/nar/gkaa564.
    PubMed
    Abstract
    The exon junction complex (EJC) is an essential constituent and regulator of spliced messenger ribonucleoprotein particles (mRNPs) in metazoans. As a core component of the EJC, CASC3 was described to be pivotal for EJC-dependent nuclear and cytoplasmic processes. However, recent evidence suggests that CASC3 functions differently from other EJC core proteins. Here, we have established human CASC3 knockout cell lines to elucidate the cellular role of CASC3. In the knockout cells, overall EJC composition and EJC-dependent splicing are unchanged. A transcriptome-wide analysis reveals that hundreds of mRNA isoforms targeted by nonsense-mediated decay (NMD) are upregulated. Mechanistically, recruiting CASC3 to reporter mRNAs by direct tethering or via binding to the EJC stimulates mRNA decay and endonucleolytic cleavage at the termination codon. Building on existing EJC-NMD models, we propose that CASC3 equips the EJC with the persisting ability to communicate with the NMD machinery in the cytoplasm. Collectively, our results characterize CASC3 as a peripheral EJC protein that tailors the transcriptome by promoting the degradation of EJC-dependent NMD substrates.
  4. Schlautmann LP, Gehring NH
    A Day in the Life of the Exon Junction Complex
    Biomolecules. 2020 Jun 5;10(6):E866. doi: 10.3390/biom10060866.
    PubMed
    Abstract
    The exon junction complex (EJC) is an abundant messenger ribonucleoprotein (mRNP) component that is assembled during splicing and binds to mRNAs upstream of exon-exon junctions. EJCs accompany the mRNA during its entire life in the nucleus and the cytoplasm and communicate the information about the splicing process and the position of introns. Specifically, the EJC's core components and its associated proteins regulate different steps of gene expression, including pre-mRNA splicing, mRNA export, translation, and nonsense-mediated mRNA decay (NMD). This review summarizes the most important functions and main protagonists in the life of the EJC. It also provides an overview of the latest findings on the assembly, composition and molecular activities of the EJC and presents them in the chronological order, in which they play a role in the EJC's life cycle.

FUNDING

OPPORTUNITIES

Opportunities for Master and Bachelor Theses, Lab Modules

Please send inquiries for Bachelor and Master theses or laboratory modules including a short CV to Niels Gehring