Research



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 by clicking on the images below.


Exon junction complex

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
EJC research

We currently investigate how the EJC is assembled in the first place, which factors are required for this, and how the EJC exerts its many functions on gene expression.

Key publications:

Boehm V, Britto-Borges T, Steckelberg AL, Singh KK, Gerbracht JV, Gueney E, Blazquez L, Altmüller J, Dieterich C, Gehring NH
Exon Junction Complexes Suppress Spurious Splice Sites to Safeguard Transcriptome Integrity
Mol Cell. 2018 Nov 1;72(3):482-495.e7. doi: 10.1016/j.molcel.2018.08.030.
PubMed

Productive splicing of human precursor messenger RNAs (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.


Gerbracht JV, Gehring NH
The exon junction complex: structural insights into a faithful companion of mammalian mRNPs
Biochem Soc Trans. 2018 Jan 19. pii: BST20170059. doi: 10.1042/BST20170059.
PubMed

During splicing, the exon junction complex (EJC) is deposited upstream of exon-exon boundaries. The EJC and its peripheral bound proteins play an essential role in mediating mRNA export, translation and turnover. However, the exact sequence of EJC assembly and the involved factors during splicing remain elusive. Recently published structures of the human C* spliceosome clarified the position of the EJC at this phase of splicing and have given insight into previously unidentified interactions between the EJC and spliceosomal proteins. Here, these new observations are presented and the significance for EJC assembly is discussed. Furthermore, the vast landscape of EJC interacting proteins and their manifold functions are described. Finally, the factors involved in EJC disassembly and recycling are recapitulated. This review aims to integrate structural, biochemical and physiological data to obtain a comprehensive picture of EJC components during the lifetime of the EJC.


Gehring NH, Wahle E, Fischer U
Deciphering the mRNP Code: RNA-Bound Determinants of Post-Transcriptional Gene Regulation
Trends Biochem Sci. 2017 Mar 3. pii: S0968-0004(17)30043-9. doi: 10.1016/j.tibs.2017.02.004.
PubMed

Eukaryotic cells determine the final protein output of their genetic program not only by controlling transcription but also by regulating the localization, translation and turnover rates of their mRNAs. Ultimately, the fate of any given mRNA is determined by the ensemble of all associated RNA-binding proteins (RBPs), non-coding RNAs and metabolites collectively known as the messenger ribonucleoprotein particle (mRNP). Although many mRNA-associated factors have been identified over the past years, little is known about the composition of individual mRNPs and the cooperation of their constituents. In this review we discuss recent progress that has been made on how this 'mRNP code' is established on individual transcripts and how it is interpreted during gene expression in eukaryotic cells.


Boehm V, Gehring NH
Exon Junction Complexes: Supervising the Gene Expression Assembly Line.
Trends Genet. 2016 Nov;32(11):724-735. doi: 10.1016/j.tig.2016.09.003. Review.
PubMed

The exon junction complex (EJC) is an RNA-binding protein complex that is assembled and deposited onto mRNAs during splicing. The EJC comprises four core components that bind to not only canonical sites upstream of exon-exon junctions, but also to noncanonical sites at other positions in exons. EJC-associated proteins are recruited by the EJC at different steps of gene expression to execute the multiple functions of the EJC. Recently, new insights have been obtained into how EJCs stimulate pre-mRNA splicing, and mRNA export, translation, and degradation. Furthermore, mutations in EJC core components were shown to result in severe disorders in humans, demonstrating the critical physiological role of the EJC. Hence, the EJC has been identified as an important player in post-transcriptional gene regulation in metazoans.


Steckelberg AL, Altmueller J, Dieterich C, Gehring NH
CWC22-dependent pre-mRNA splicing and eIF4A3 binding enables global deposition of exon junction complexes.
Nucleic Acids Res. 2015 May 19;43(9):4687-700. doi: 10.1093/nar/gkv320.
PubMed

In metazoan cells, spliced mRNAs are marked by the exon junction complex (EJC), a multi-protein complex that serves as a key regulator of post-transcriptional mRNA metabolism. Deposition of EJCs on mRNA is intimately linked to the splicing process. The spliceosomal protein CWC22 directly binds the core EJC-protein eIF4A3, guides it to the spliceosome and initiates EJC assembly. In addition, CWC22 is involved in the splicing process itself, but the molecular details of its dual function remain elusive. Here we analyze the mechanisms, by which CWC22 co-regulates pre-mRNA splicing and EJC assembly. We show that the core of CWC22 is sufficient to mediate both pre-mRNA splicing and EJC assembly. Nonetheless, both processes can be functionally uncoupled with an eIF4A3-binding deficient mutant of CWC22, which impedes EJC assembly. A C-terminal domain of CWC22 strongly enhances its spliceosomal interaction and likely regulates its function. High-throughput RNA-sequencing identifies global defects of pre-mRNA splicing and downregulation of diverse gene expression pathways in CWC22-depleted cells. We propose a model, in which CWC22 represents an integral component of the spliceosome and orchestrates pre-mRNA splicing and eIF4A3 binding to achieve global assembly of exon junction complexes.


Steckelberg AL, Gehring NH
Studying the composition of mRNPs in vitro using splicing-competent cell extracts.
Methods. 2014 Feb;65(3):342-9. doi: 10.1016/j.ymeth.2013.08.033.
PubMed

The correct processing and faithful decoding of mRNAs during gene expression depends on the interaction with RNA-binding proteins (RBPs). The association of RBPs with pre-mRNAs starts during transcription by RNA polymerase II and undergoes constant remodeling during pre-mRNA processing and later steps of genes expression. Recently developed high throughput methods enabled to define RBP binding sites in vivo and to identify a large number of novel RBPs in eukaryotic cells. However, the detailed characterization of RBP-RNA interactions as well as the analysis of functional RNPs is greatly facilitated by well-defined in vitro systems. Here, we describe a versatile method to study the assembly and splicing-dependent remodeling of mRNPs in vitro. This method employs splicing-competent whole cell extracts (WCE) generated from transfected human embryonic kidney (HEK) 293 cells. FLAG-tagged proteins present in the WCE are incorporated into mRNPs in vitro and afterwards used to immunoprecipitate substrate RNAs. We outline the principles of purifying in vitro assembled mRNPs and provide detailed protocols for the preparation and use of whole cell extracts. Alternative purification strategies and RNA substrates are discussed.



Nonsense-mediated mRNA decay

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
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.

Key publications:

Ottens F, Boehm V, Sibley CR, Ule J, Gehring NH
Transcript-specific characteristics determine the contribution of endo- and exonucleolytic decay pathways during the degradation of nonsense-mediated decay substrates.
RNA 2017 May 1. pii: rna.059659.116 doi: 10.1261/rna.059659.116
PubMed

Nonsense-mediated mRNA decay (NMD) controls gene expression by eliminating mRNAs with premature or aberrant translation termination. Degradation of NMD substrates is initiated by the central NMD factor UPF1, which recruits the endonuclease SMG6 and the deadenylation-promoting SMG5/7 complex. The extent to which SMG5/7 and SMG6 contribute to the degradation of individual substrates and their regulation by UPF1 remains elusive. Here we map transcriptome-wide sites of SMG6-mediated endocleavage via 3' fragment capture and degradome sequencing. This reveals that endogenous transcripts can have NMD-eliciting features at various positions, including upstream open reading frames (uORFs), premature termination codons (PTCs), and long 3' UTRs. We find that NMD substrates with PTCs undergo constitutive SMG6-dependent endocleavage, rather than SMG7-dependent exonucleolytic decay. In contrast, the turnover of NMD substrates containing uORFs and long 3' UTRs involves both SMG6- and SMG7-dependent endo- and exonucleolytic decay, respectively. This suggests that the extent to which SMG6 and SMG7 degrade NMD substrates is determined by the mRNA architecture.


Boehm V, Gerbracht JV, Marx MC, Gehring NH
Interrogating the degradation pathways of unstable mRNAs with XRN1-resistant sequences.
Nat Commun. 2016 Dec 5;7:13691. doi: 10.1038/ncomms13691.
PubMed

The turnover of messenger RNAs (mRNAs) is a key regulatory step of gene expression in eukaryotic cells. Due to the complexity of the mammalian degradation machinery, the contribution of decay factors to the directionality of mRNA decay is poorly understood. Here we characterize a molecular tool to interrogate mRNA turnover via the detection of XRN1-resistant decay fragments (xrFrag). Using nonsense-mediated mRNA decay (NMD) as a model pathway, we establish xrFrag analysis as a robust indicator of accelerated 5'-3' mRNA decay. In tethering assays, monitoring xrFrag accumulation allows to distinguish decapping and endocleavage activities from deadenylation. Moreover, xrFrag analysis of mRNA degradation induced by miRNAs, AU-rich elements (AREs) as well as the 3' UTRs of cytokine mRNAs reveals the contribution of 5'-3' decay and endonucleolytic cleavage. Our work uncovers formerly unrecognized modes of mRNA turnover and establishes xrFrag as a powerful tool for RNA decay analyses.


Fatscher T, Boehm V, Gehring NH
Mechanism, factors, and physiological role of nonsense-mediated mRNA decay.
Cell Mol Life Sci. 2015 Dec;72(23):4523-44. doi: 10.1007/s00018-015-2017-9. Review.
PubMed

Nonsense-mediated mRNA decay (NMD) is a translation-dependent, multistep process that degrades irregular or faulty messenger RNAs (mRNAs). NMD mainly targets mRNAs with a truncated open reading frame (ORF) due to premature termination codons (PTCs). In addition, NMD also regulates the expression of different types of endogenous mRNA substrates. A multitude of factors are involved in the tight regulation of the NMD mechanism. In this review, we focus on the molecular mechanism of mammalian NMD. Based on the published data, we discuss the involvement of translation termination in NMD initiation. Furthermore, we provide a detailed overview of the core NMD machinery, as well as several peripheral NMD factors, and discuss their function. Finally, we present an overview of diseases associated with NMD factor mutations and summarize the current state of treatment for genetic disorders caused by nonsense mutations.


Boehm V, Haberman N, Ottens F, Ule J, Gehring NH
3' UTR length and messenger ribonucleoprotein composition determine endocleavage efficiencies at termination codons.
Cell Rep. 2014 Oct 23;9(2):555-68. doi: 10.1016/j.celrep.2014.09.012.
PubMed

Nonsense-mediated mRNA decay (NMD) degrades different classes of mRNAs, including transcripts with premature termination codons (PTCs). The NMD factor SMG6 initiates degradation of substrate mRNAs by endonucleolytic cleavage. Here, we aim to delineate the cascade of NMD-activating events that culminate in endocleavage. We report that long 3' UTRs elicit SMG6-mediated endonucleolytic degradation. The presence of an exon-junction complex (EJC) within the 3' UTR strongly stimulates endocleavage in a distance-independent manner. The interaction of SMG6 with EJCs is not required for endocleavage. Whereas the core NMD component UPF2 supports endonucleolytic decay of long 3' UTR mRNAs, it is mostly dispensable during EJC-stimulated endocleavage. Using high-throughput sequencing, we map endocleavage positions of different PTC-containing reporter mRNAs and an endogenous NMD substrate to regions directly at and downstream of the termination codon. These results reveal how messenger ribonucleoprotein (mRNP) parameters differentially influence SMG6-executed endonucleolysis and uncover central characteristics of this phenomenon associated with translation termination.


Fatscher T, Boehm V, Weiche B, Gehring NH
The interaction of cytoplasmic poly(A)-binding protein with eukaryotic initiation factor 4G suppresses nonsense-mediated mRNA decay.
RNA. 2014 Oct;20(10):1579-92. doi: 10.1261/rna.044933.114.
PubMed

Nonsense-mediated mRNA decay (NMD) eliminates different classes of mRNA substrates including transcripts with long 3' UTRs. Current models of NMD suggest that the long physical distance between the poly(A) tail and the termination codon reduces the interaction between cytoplasmic poly(A)-binding protein (PABPC1) and the eukaryotic release factor 3a (eRF3a) during translation termination. In the absence of PABPC1 binding, eRF3a recruits the NMD factor UPF1 to the terminating ribosome, triggering mRNA degradation. Here, we have used the MS2 tethering system to investigate the suppression of NMD by PABPC1. We show that tethering of PABPC1 between the termination codon and a long 3' UTR specifically inhibits NMD-mediated mRNA degradation. Contrary to the current model, tethered PABPC1 mutants unable to interact with eRF3a still efficiently suppress NMD. We find that the interaction of PABPC1 with eukaryotic initiation factor 4G (eIF4G), which mediates the circularization of mRNAs, is essential for NMD inhibition by tethered PABPC1. Furthermore, recruiting either eRF3a or eIF4G in proximity to an upstream termination codon antagonizes NMD. While tethering of an eRF3a mutant unable to interact with PABPC1 fails to suppress NMD, tethered eIF4G inhibits NMD in a PABPC1-independent manner, indicating a sequential arrangement of NMD antagonizing factors. In conclusion, our results establish a previously unrecognized link between translation termination, mRNA circularization, and NMD suppression, thereby suggesting a revised model for the activation of NMD at termination codons upstream of long 3' UTR.



Other mRNA/mRNP research projects

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.

Key publications:

Gromadzka AM, Steckelberg AL, Singh KK, Hofmann K, Gehring NH
A short conserved motif in ALYREF directs cap- and EJC-dependent assembly of export complexes on spliced mRNAs.
Nucleic Acids Res. 2016 Mar 18;44(5):2348-61. doi: 10.1093/nar/gkw009.
PubMed

The export of messenger RNAs (mRNAs) is the final of several nuclear posttranscriptional steps of gene expression. The formation of export-competent mRNPs involves the recruitment of export factors that are assumed to facilitate transport of the mature mRNAs. Using in vitro splicing assays, we show that a core set of export factors, including ALYREF, UAP56 and DDX39, readily associate with the spliced RNAs in an EJC (exon junction complex)- and cap-dependent manner. In order to elucidate how ALYREF and other export adaptors mediate mRNA export, we conducted a computational analysis and discovered four short, conserved, linear motifs present in RNA-binding proteins. We show that mutation in one of the new motifs (WxHD) in an unstructured region of ALYREF reduced RNA binding and abolished the interaction with eIF4A3 and CBP80. Additionally, the mutation impaired proper localization to nuclear speckles and export of a spliced reporter mRNA. Our results reveal important details of the orchestrated recruitment of export factors during the formation of export competent mRNPs.




People




Group Leader

Niels

Niels H. Gehring
Email
Researchgate
Acting Director
Tel:+49 221 470 3873


Secretary

Jessy

Jessica Wiegel
Email
Tel: +49 221 470 5728



Technician

Juliane

Juliane Hancke
Email
Tel:+49 221 470 4515



Postdoc

Volker

Volker Böhm
Email
Researchgate
Tel: +49 221 470 5260


PhD Student

Jenny

Jennifer Gerbracht
Email
Researchgate
GSfBS representative
Tel: +49 221 470 5260


PhD Student

Lena

Lena Schlautmann
Email
Tel: +49 221 470 7848



M.Sc. Student

Sabrina

Sabrina Kückelmann
Tel: +49 221 470 7848




Visiting Scientist

Kusum

Kusum Singh
Email
Indian Institute of Technology Guwahati




Dominik Aschemeier

Simona Ciriello

Tobias Fatscher

Agnieszka Gromadzka

Marie Charlotte Marx

Franziska Ottens

Kusum Singh

Anna-Lena Steckelberg

Heidi Thelen

Benjamin Weiche


Publications




  1. Boehm V, Britto-Borges T, Steckelberg AL, Singh KK, Gerbracht JV, Gueney E, Blazquez L, Altmüller J, Dieterich C, Gehring NH
    Exon Junction Complexes Suppress Spurious Splice Sites to Safeguard Transcriptome Integrity
    Mol Cell. 2018 Nov 1;72(3):482-495.e7. doi: 10.1016/j.molcel.2018.08.030.
    PubMed

    Productive splicing of human precursor messenger RNAs (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.
  2. Gerbracht JV, Gehring NH
    The exon junction complex: structural insights into a faithful companion of mammalian mRNPs.
    Biochem Soc Trans. 2018 Jan 19. pii: BST20170059. doi: 10.1042/BST20170059.
    PubMed

    During splicing, the exon junction complex (EJC) is deposited upstream of exon-exon boundaries. The EJC and its peripheral bound proteins play an essential role in mediating mRNA export, translation and turnover. However, the exact sequence of EJC assembly and the involved factors during splicing remain elusive. Recently published structures of the human C* spliceosome clarified the position of the EJC at this phase of splicing and have given insight into previously unidentified interactions between the EJC and spliceosomal proteins. Here, these new observations are presented and the significance for EJC assembly is discussed. Furthermore, the vast landscape of EJC interacting proteins and their manifold functions are described. Finally, the factors involved in EJC disassembly and recycling are recapitulated. This review aims to integrate structural, biochemical and physiological data to obtain a comprehensive picture of EJC components during the lifetime of the EJC.

  1. Gerbracht JV, Boehm V, Gehring NH
    Plasmid transfection influences the readout of nonsense-mediated mRNA decay reporter assays in human cells.
    Sci Rep. 2017 Sep 6;7(1):10616. doi: 10.1038/s41598-017-10847-4.
    PubMed

    Messenger RNA (mRNA) turnover is a crucial and highly regulated step of gene expression in mammalian cells. This includes mRNA surveillance pathways such as nonsense-mediated mRNA decay (NMD), which assesses the fidelity of transcripts and eliminates mRNAs containing a premature translation termination codon (PTC). When studying mRNA degradation pathways, reporter mRNAs are commonly expressed in cultivated cells. Traditionally, the molecular mechanism of NMD has been characterized using pairs of reporter constructs that express the same mRNA with ("PTC-containing mRNA") or without ("wild-type mRNA") a PTC. Cell lines stably expressing an NMD reporter have been reported to yield very robust and highly reproducible results, but establishing the cell lines can be very time-consuming. Therefore, transient transfection of such reporter constructs is frequently used and allows analysis of many samples within a short period of time. However, the behavior of transiently and stably transfected NMD constructs has not been systematically compared so far. Here, we report that not all commonly used human cell lines degrade NMD targets following transient transfection. Furthermore, the degradation efficiency of NMD substrates can depend on the manner of transfection within the same cell line. This has substantial implications for the interpretation of NMD assays based on transient transfections.
  2. Ottens F, Boehm V, Sibley CR, Ule J, Gehring NH
    Transcript-specific characteristics determine the contribution of endo- and exonucleolytic decay pathways during the degradation of nonsense-mediated decay substrates.
    RNA 2017 May 1. pii: rna.059659.116 doi: 10.1261/rna.059659.116
    PubMed

    Nonsense-mediated mRNA decay (NMD) controls gene expression by eliminating mRNAs with premature or aberrant translation termination. Degradation of NMD substrates is initiated by the central NMD factor UPF1, which recruits the endonuclease SMG6 and the deadenylation-promoting SMG5/7 complex. The extent to which SMG5/7 and SMG6 contribute to the degradation of individual substrates and their regulation by UPF1 remains elusive. Here we map transcriptome-wide sites of SMG6-mediated endocleavage via 3' fragment capture and degradome sequencing. This reveals that endogenous transcripts can have NMD-eliciting features at various positions, including upstream open reading frames (uORFs), premature termination codons (PTCs), and long 3' UTRs. We find that NMD substrates with PTCs undergo constitutive SMG6-dependent endocleavage, rather than SMG7-dependent exonucleolytic decay. In contrast, the turnover of NMD substrates containing uORFs and long 3' UTRs involves both SMG6- and SMG7-dependent endo- and exonucleolytic decay, respectively. This suggests that the extent to which SMG6 and SMG7 degrade NMD substrates is determined by the mRNA architecture.
  3. Gehring NH, Wahle E, Fischer U
    Deciphering the mRNP Code: RNA-Bound Determinants of Post-Transcriptional Gene Regulation
    Trends Biochem Sci. 2017 Mar 3. pii: S0968-0004(17)30043-9. doi: 10.1016/j.tibs.2017.02.004.
    PubMed

    Eukaryotic cells determine the final protein output of their genetic program not only by controlling transcription but also by regulating the localization, translation and turnover rates of their mRNAs. Ultimately, the fate of any given mRNA is determined by the ensemble of all associated RNA-binding proteins (RBPs), non-coding RNAs and metabolites collectively known as the messenger ribonucleoprotein particle (mRNP). Although many mRNA-associated factors have been identified over the past years, little is known about the composition of individual mRNPs and the cooperation of their constituents. In this review we discuss recent progress that has been made on how this 'mRNP code' is established on individual transcripts and how it is interpreted during gene expression in eukaryotic cells.

  1. Boehm V, Gerbracht JV, Marx MC, Gehring NH
    Interrogating the degradation pathways of unstable mRNAs with XRN1-resistant sequences.
    Nat Commun. 2016 Dec 5;7:13691. doi: 10.1038/ncomms13691.
    PubMed

    The turnover of messenger RNAs (mRNAs) is a key regulatory step of gene expression in eukaryotic cells. Due to the complexity of the mammalian degradation machinery, the contribution of decay factors to the directionality of mRNA decay is poorly understood. Here we characterize a molecular tool to interrogate mRNA turnover via the detection of XRN1-resistant decay fragments (xrFrag). Using nonsense-mediated mRNA decay (NMD) as a model pathway, we establish xrFrag analysis as a robust indicator of accelerated 5'-3' mRNA decay. In tethering assays, monitoring xrFrag accumulation allows to distinguish decapping and endocleavage activities from deadenylation. Moreover, xrFrag analysis of mRNA degradation induced by miRNAs, AU-rich elements (AREs) as well as the 3' UTRs of cytokine mRNAs reveals the contribution of 5'-3' decay and endonucleolytic cleavage. Our work uncovers formerly unrecognized modes of mRNA turnover and establishes xrFrag as a powerful tool for RNA decay analyses.
  2. Fatscher T, Gehring NH
    Harnessing short poly(A)-binding protein-interacting peptides for the suppression of nonsense-mediated mRNA decay.
    Sci Rep. 2016 Nov 22;6:37311. doi: 10.1038/srep37311.
    PubMed

    Nonsense-mediated mRNA decay (NMD) is a cellular process that eliminates messenger RNA (mRNA) substrates with premature translation termination codons (PTCs). In addition, NMD regulates the expression of a number of physiological mRNAs, for example transcripts containing long 3' UTRs. Current models implicate the interaction between cytoplasmic poly(A)-binding protein (PABPC1) and translation termination in NMD. Accordingly, PABPC1 present within close proximity of a termination codon antagonizes NMD. Here, we use reporter mRNAs with different NMD-inducing 3' UTRs to establish a general NMD-inhibiting property of PABPC1. NMD-inhibition is not limited to PABPC1, but can also be achieved by peptides consisting of the PABP-interacting motif 2 (PAM2) of different proteins when recruited to an NMD-inhibiting position of NMD reporter transcripts. The short PAM2 peptides efficiently suppress NMD activated by a long 3' UTR, an exon-junction complex (EJC) and individual EJC components, and stabilize a PTC-containing β-globin mRNA. In conclusion, our results establish short PABPC1-recruiting peptides as potent but position-dependent inhibitors of mammalian NMD.
  3. Boehm V, Gehring NH
    Exon Junction Complexes: Supervising the Gene Expression Assembly Line.
    Trends Genet. 2016 Nov;32(11):724-735. doi: 10.1016/j.tig.2016.09.003. Review.
    PubMed

    The exon junction complex (EJC) is an RNA-binding protein complex that is assembled and deposited onto mRNAs during splicing. The EJC comprises four core components that bind to not only canonical sites upstream of exon-exon junctions, but also to noncanonical sites at other positions in exons. EJC-associated proteins are recruited by the EJC at different steps of gene expression to execute the multiple functions of the EJC. Recently, new insights have been obtained into how EJCs stimulate pre-mRNA splicing, and mRNA export, translation, and degradation. Furthermore, mutations in EJC core components were shown to result in severe disorders in humans, demonstrating the critical physiological role of the EJC. Hence, the EJC has been identified as an important player in post-transcriptional gene regulation in metazoans.
  4. Ottens F, Gehring NH
    Physiological and pathophysiological role of nonsense-mediated mRNA decay.
    Pflugers Arch. 2016 Jun;468(6):1013-28. doi: 10.1007/s00424-016-1826-5. Review.
    PubMed

    Nonsense-mediated messenger RNA (mRNA) decay (NMD) is a quality control mechanism that degrades irregular or faulty mRNAs. NMD mainly degrades mRNAs, which contain a premature termination codon (PTC) and therefore encode a truncated protein. Furthermore, NMD alters the expression of different types of cellular mRNAs, the so-called endogenous NMD substrates. In this review, we focus on the impact of NMD on cellular and molecular physiology. We specify key classes of NMD substrates and provide a detailed overview of the physiological function of gene regulation by NMD. We also describe different mechanisms of NMD substrate degradation and how the regulation of the NMD machinery affects cellular physiology. Finally, we outline the physiological functions of central NMD factors.
  5. Gromadzka AM, Steckelberg AL, Singh KK, Hofmann K, Gehring NH
    A short conserved motif in ALYREF directs cap- and EJC-dependent assembly of export complexes on spliced mRNAs.
    Nucleic Acids Res. 2016 Mar 18;44(5):2348-61. doi: 10.1093/nar/gkw009.
    PubMed

    The export of messenger RNAs (mRNAs) is the final of several nuclear posttranscriptional steps of gene expression. The formation of export-competent mRNPs involves the recruitment of export factors that are assumed to facilitate transport of the mature mRNAs. Using in vitro splicing assays, we show that a core set of export factors, including ALYREF, UAP56 and DDX39, readily associate with the spliced RNAs in an EJC (exon junction complex)- and cap-dependent manner. In order to elucidate how ALYREF and other export adaptors mediate mRNA export, we conducted a computational analysis and discovered four short, conserved, linear motifs present in RNA-binding proteins. We show that mutation in one of the new motifs (WxHD) in an unstructured region of ALYREF reduced RNA binding and abolished the interaction with eIF4A3 and CBP80. Additionally, the mutation impaired proper localization to nuclear speckles and export of a spliced reporter mRNA. Our results reveal important details of the orchestrated recruitment of export factors during the formation of export competent mRNPs.

  1. Ajamian L, Abel K, Rao S, Vyboh K, García-de-Gracia F, Soto-Rifo R, Kulozik AE, Gehring NH, Mouland AJ
    HIV-1 Recruits UPF1 but Excludes UPF2 to Promote Nucleocytoplasmic Export of the Genomic RNA.
    Biomolecules. 2015 Oct 20;5(4):2808-39. doi: 10.3390/biom5042808.
    PubMed

    Unspliced, genomic HIV-1 RNA (vRNA) is a component of several ribonucleoprotein complexes (RNP) during the viral replication cycle. In earlier work, we demonstrated that the host upframeshift protein 1 (UPF1), a key factor in nonsense-mediated mRNA decay (NMD), colocalized and associated to the viral structural protein Gag during viral egress. In this work, we demonstrate a new function for UPF1 in the regulation of vRNA nuclear export. OPEN ACCESS Biomolecules 2015, 5 2809 We establish that the nucleocytoplasmic shuttling of UPF1 is required for this function and demonstrate that UPF1 exists in two essential viral RNPs during the late phase of HIV-1 replication: the first, in a nuclear export RNP that contains Rev, CRM1, DDX3 and the nucleoporin p62, and the second, which excludes these nuclear export markers but contains Gag in the cytoplasm. Interestingly, we observed that both UPF2 and the long isoform of UPF3a, UPF3aL, but not the shorter isoforms UPF3aS and UPF3b, are excluded from the UPF1-Rev-CRM1-DDX3 complex as they are negative regulators of vRNA nuclear export. In silico protein-protein docking analyses suggest that Rev binds UPF1 in a region that overlaps the UPF2 binding site, thus explaining the exclusion of this negative regulatory factor by HIV-1 that is necessary for vRNA trafficking. This work uncovers a novel and unique regulatory circuit involving several UPF proteins that ultimately regulate vRNA nuclear export and trafficking.
  2. Fatscher T, Boehm V, Gehring NH
    Mechanism, factors, and physiological role of nonsense-mediated mRNA decay.
    Cell Mol Life Sci. 2015 Dec;72(23):4523-44. doi: 10.1007/s00018-015-2017-9. Review.
    PubMed

    Nonsense-mediated mRNA decay (NMD) is a translation-dependent, multistep process that degrades irregular or faulty messenger RNAs (mRNAs). NMD mainly targets mRNAs with a truncated open reading frame (ORF) due to premature termination codons (PTCs). In addition, NMD also regulates the expression of different types of endogenous mRNA substrates. A multitude of factors are involved in the tight regulation of the NMD mechanism. In this review, we focus on the molecular mechanism of mammalian NMD. Based on the published data, we discuss the involvement of translation termination in NMD initiation. Furthermore, we provide a detailed overview of the core NMD machinery, as well as several peripheral NMD factors, and discuss their function. Finally, we present an overview of diseases associated with NMD factor mutations and summarize the current state of treatment for genetic disorders caused by nonsense mutations.
  3. Linder B, Fischer U, Gehring NH
    mRNA metabolism and neuronal disease.
    FEBS Lett. 2015 Jun 22;589(14):1598-606. doi: 10.1016/j.febslet.2015.04.052. Review.
    PubMed

    To serve as templates for translation eukaryotic mRNAs undergo an elaborate processing and maturation pathway. In eukaryotes this process comprises the synthesis of mRNA precursors, their processing and transport to the site of translation and eventually their decay. During the entire life cycle, mRNAs interact with distinct sets of trans-acting factors that determine their fate at any given phase of gene expression. Recent studies have shown that mutations in components acting in trans on mRNAs are frequent causes of a large variety of different human disorders. The etiology of most of these diseases is, however, only poorly understood, mostly because the consequences for mRNA-metabolism are unclear. Here we discuss three prominent genetic diseases that fall into this category, namely spinal muscular atrophy (SMA), retinitis pigmentosa (RP) and X-linked syndromic mental retardation (XLMR). Whereas SMA and RP can be directly linked to mRNA processing, XLMR results from mutations in the mRNA surveillance system. We discuss how defects in mRNA maturation and turnover might lead to the tissue specific defects seen in these diseases.
  4. Steckelberg AL, Altmueller J, Dieterich C, Gehring NH
    CWC22-dependent pre-mRNA splicing and eIF4A3 binding enables global deposition of exon junction complexes.
    Nucleic Acids Res. 2015 May 19;43(9):4687-700. doi: 10.1093/nar/gkv320.
    PubMed

    In metazoan cells, spliced mRNAs are marked by the exon junction complex (EJC), a multi-protein complex that serves as a key regulator of post-transcriptional mRNA metabolism. Deposition of EJCs on mRNA is intimately linked to the splicing process. The spliceosomal protein CWC22 directly binds the core EJC-protein eIF4A3, guides it to the spliceosome and initiates EJC assembly. In addition, CWC22 is involved in the splicing process itself, but the molecular details of its dual function remain elusive. Here we analyze the mechanisms, by which CWC22 co-regulates pre-mRNA splicing and EJC assembly. We show that the core of CWC22 is sufficient to mediate both pre-mRNA splicing and EJC assembly. Nonetheless, both processes can be functionally uncoupled with an eIF4A3-binding deficient mutant of CWC22, which impedes EJC assembly. A C-terminal domain of CWC22 strongly enhances its spliceosomal interaction and likely regulates its function. High-throughput RNA-sequencing identifies global defects of pre-mRNA splicing and downregulation of diverse gene expression pathways in CWC22-depleted cells. We propose a model, in which CWC22 represents an integral component of the spliceosome and orchestrates pre-mRNA splicing and eIF4A3 binding to achieve global assembly of exon junction complexes.
  5. Stockklausner C, Klotter AC, Dickemann N, Kuhlee IN, Duffert CM, Kerber C, Gehring NH, Kulozik AE
    The thrombopoietin receptor P106L mutation functionally separates receptor signaling activity from thrombopoietin homeostasis.
    Blood. 2015 Feb 12;125(7):1159-69. doi: 10.1182/blood-2014-07-587170.
    PubMed

    The interaction between thrombopoietin (THPO) and its receptor c-Mpl regulates downstream cytokine signaling and platelet homeostasis. Hereditary mutations of c-Mpl can either result in loss-of-function and thrombocytopenia or in gain-of-function and thrombocythemia (HT), and are important models to analyze the mechanism of c-Mpl activity. We have analyzed the effect of the c-Mpl P106L gain-of-function and the nearby loss-of-function R102P and F104S mutations, which cause HT or thrombocytopenia, respectively, on posttranslational processing, intracellular trafficking, cell surface expression, and cell proliferation. In contrast to R102P and F104S, the P106L mutant confers cytokine-independent growth and stimulates downstream signaling after THPO treatment in Ba/F3 cells. Despite their opposite function, R102P and P106L, both lead to abnormal subcellular receptor distribution, lack of membrane localization, impaired glycosylation, and elevated THPO serum levels in effected patients. These findings indicate that the activation of downstream signaling by c-Mpl P106L does not require correct processing, trafficking, and cell surface expression of c-Mpl, whereas the negative feedback loop controlling THPO serum levels requires cell surface expression of the receptor. Thus, we propose that the P106L mutation functionally separates the activity of c-Mpl in downstream signaling from that in maintaining platelet homeostasis.

  1. Boehm V, Haberman N, Ottens F, Ule J, Gehring NH
    3' UTR length and messenger ribonucleoprotein composition determine endocleavage efficiencies at termination codons.
    Cell Rep. 2014 Oct 23;9(2):555-68. doi: 10.1016/j.celrep.2014.09.012.
    PubMed

    Nonsense-mediated mRNA decay (NMD) degrades different classes of mRNAs, including transcripts with premature termination codons (PTCs). The NMD factor SMG6 initiates degradation of substrate mRNAs by endonucleolytic cleavage. Here, we aim to delineate the cascade of NMD-activating events that culminate in endocleavage. We report that long 3' UTRs elicit SMG6-mediated endonucleolytic degradation. The presence of an exon-junction complex (EJC) within the 3' UTR strongly stimulates endocleavage in a distance-independent manner. The interaction of SMG6 with EJCs is not required for endocleavage. Whereas the core NMD component UPF2 supports endonucleolytic decay of long 3' UTR mRNAs, it is mostly dispensable during EJC-stimulated endocleavage. Using high-throughput sequencing, we map endocleavage positions of different PTC-containing reporter mRNAs and an endogenous NMD substrate to regions directly at and downstream of the termination codon. These results reveal how messenger ribonucleoprotein (mRNP) parameters differentially influence SMG6-executed endonucleolysis and uncover central characteristics of this phenomenon associated with translation termination.
  2. Fatscher T, Boehm V, Weiche B, Gehring NH
    The interaction of cytoplasmic poly(A)-binding protein with eukaryotic initiation factor 4G suppresses nonsense-mediated mRNA decay.
    RNA. 2014 Oct;20(10):1579-92. doi: 10.1261/rna.044933.114.
    PubMed

    Nonsense-mediated mRNA decay (NMD) eliminates different classes of mRNA substrates including transcripts with long 3' UTRs. Current models of NMD suggest that the long physical distance between the poly(A) tail and the termination codon reduces the interaction between cytoplasmic poly(A)-binding protein (PABPC1) and the eukaryotic release factor 3a (eRF3a) during translation termination. In the absence of PABPC1 binding, eRF3a recruits the NMD factor UPF1 to the terminating ribosome, triggering mRNA degradation. Here, we have used the MS2 tethering system to investigate the suppression of NMD by PABPC1. We show that tethering of PABPC1 between the termination codon and a long 3' UTR specifically inhibits NMD-mediated mRNA degradation. Contrary to the current model, tethered PABPC1 mutants unable to interact with eRF3a still efficiently suppress NMD. We find that the interaction of PABPC1 with eukaryotic initiation factor 4G (eIF4G), which mediates the circularization of mRNAs, is essential for NMD inhibition by tethered PABPC1. Furthermore, recruiting either eRF3a or eIF4G in proximity to an upstream termination codon antagonizes NMD. While tethering of an eRF3a mutant unable to interact with PABPC1 fails to suppress NMD, tethered eIF4G inhibits NMD in a PABPC1-independent manner, indicating a sequential arrangement of NMD antagonizing factors. In conclusion, our results establish a previously unrecognized link between translation termination, mRNA circularization, and NMD suppression, thereby suggesting a revised model for the activation of NMD at termination codons upstream of long 3' UTR.
  3. Burgute BD, Peche VS, Steckelberg AL, Glöckner G, Gaßen B, Gehring NH, Noegel AA
    NKAP is a novel RS-related protein that interacts with RNA and RNA binding proteins.
    Nucleic Acids Res. 2014 Mar;42(5):3177-93. doi: 10.1093/nar/gkt1311.
    PubMed

    NKAP is a highly conserved protein with roles in transcriptional repression, T-cell development, maturation and acquisition of functional competency and maintenance and survival of adult hematopoietic stem cells. Here we report the novel role of NKAP in splicing. With NKAP-specific antibodies we found that NKAP localizes to nuclear speckles. NKAP has an RS motif at the N-terminus followed by a highly basic domain and a DUF 926 domain at the C-terminal region. Deletion analysis showed that the basic domain is important for speckle localization. In pull-down experiments, we identified RNA-binding proteins, RNA helicases and splicing factors as interaction partners of NKAP, among them FUS/TLS. The FUS/TLS-NKAP interaction takes place through the RS domain of NKAP and the RGG1 and RGG3 domains of FUS/TLS. We analyzed the ability of NKAP to interact with RNA using in vitro splicing assays and found that NKAP bound both spliced messenger RNA (mRNA) and unspliced pre-mRNA. Genome-wide analysis using crosslinking and immunoprecipitation-seq revealed NKAP association with U1, U4 and U5 small nuclear RNA, and we also demonstrated that knockdown of NKAP led to an increase in pre-mRNA percentage. Our results reveal NKAP as nuclear speckle protein with roles in RNA splicing and processing.
  4. Clerici M, Deniaud A, Boehm V, Gehring NH, Schaffitzel C, Cusack S
    Structural and functional analysis of the three MIF4G domains of nonsense-mediated decay factor UPF2.
    Nucleic Acids Res. 2014 Feb;42(4):2673-86. doi: 10.1093/nar/gkt1197.
    PubMed

    Nonsense-mediated decay (NMD) is a eukaryotic quality control pathway, involving conserved proteins UPF1, UPF2 and UPF3b, which detects and degrades mRNAs with premature stop codons. Human UPF2 comprises three tandem MIF4G domains and a C-terminal UPF1 binding region. MIF4G-3 binds UPF3b, but the specific functions of MIF4G-1 and MIF4G-2 are unknown. Crystal structures show that both MIF4G-1 and MIF4G-2 contain N-terminal capping helices essential for stabilization of the 10-helix MIF4G core and that MIF4G-2 interacts with MIF4G-3, forming a rigid assembly. The UPF2/UPF3b/SMG1 complex is thought to activate the kinase SMG1 to phosphorylate UPF1 in vivo. We identify MIF4G-3 as the binding site and in vitro substrate of SMG1 kinase and show that a ternary UPF2 MIF4G-3/UPF3b/SMG1 complex can form in vitro. Whereas in vivo complementation assays show that MIF4G-1 and MIF4G-2 are essential for NMD, tethering assays reveal that UPF2 truncated to only MIF4G-3 and the UPF1-binding region can still partially accomplish NMD. Thus UPF2 MIF4G-1 and MIF4G-2 appear to have a crucial scaffolding role, while MIF4G-3 is the key module required for triggering NMD.
  5. Steckelberg AL, Gehring NH
    Studying the composition of mRNPs in vitro using splicing-competent cell extracts.
    Methods. 2014 Feb;65(3):342-9. doi: 10.1016/j.ymeth.2013.08.033.
    PubMed

    The correct processing and faithful decoding of mRNAs during gene expression depends on the interaction with RNA-binding proteins (RBPs). The association of RBPs with pre-mRNAs starts during transcription by RNA polymerase II and undergoes constant remodeling during pre-mRNA processing and later steps of genes expression. Recently developed high throughput methods enabled to define RBP binding sites in vivo and to identify a large number of novel RBPs in eukaryotic cells. However, the detailed characterization of RBP-RNA interactions as well as the analysis of functional RNPs is greatly facilitated by well-defined in vitro systems. Here, we describe a versatile method to study the assembly and splicing-dependent remodeling of mRNPs in vitro. This method employs splicing-competent whole cell extracts (WCE) generated from transfected human embryonic kidney (HEK) 293 cells. FLAG-tagged proteins present in the WCE are incorporated into mRNPs in vitro and afterwards used to immunoprecipitate substrate RNAs. We outline the principles of purifying in vitro assembled mRNPs and provide detailed protocols for the preparation and use of whole cell extracts. Alternative purification strategies and RNA substrates are discussed.

For the full list of publications by the Gehring Lab, follow this link


Funding




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