CTIF is involved in mRNA export in human cells

In eukaryotic cells, translation of messenger RNA (mRNA) can be initiated either on transcripts associated with the cap-binding complex (CBC; consisting of CBP80 and CBP20) or on transcripts with the eukaryotic translation initiation factor (eIF) 4E bound to the cap. Together with eIF4G and eIF4A, eIF4E forms the eIF4F-complex, which mediates translation initiation during the bulk of cellular protein synthesis. Functionally substituting for eIF4G, the CBP80/20-dependent translation initiation factor (CTIF) has been reported to be part of the CBC-dependent translation initiation complex 1,2. CTIF consists of a N-terminal CBP80-binding domain and a conserved C-terminal MIF4G domain 1. This MIF4G domain has been shown to mediate the interaction between CTIF and different factors such as eIF3g and the stem-loop binding protein (SLBP) 2,3. Here we provide evidence that CTIF, besides its function in translation initiation, is also involved in mRNA translocation from the nucleus to the cytoplasm, possibly through a direct interaction with the nuclear export factor NFX1/TAP. Taken together our results suggest that CTIF can function as a platform that interacts with proteins involved in different steps of the mRNA metabolism.

Probing interactions of NMD factors in a distance-dependent manner by BioID

The understanding of molecular mechanisms requires the elucidation of protein-protein interaction in vivo. For large multi-factor complexes like those assembling on mRNA, co-immunoprecipitation assays often identify many peripheral interactors that complicate the interpretation of such results and that might conceal other insightful mechanistic connections. Here we address the protein-protein interaction network for key factors in the nonsense-mediated mRNA decay (NMD) pathway in a distant-dependent manner using BioID1,2. In this novel approach, the mutant E. coli biotin-protein ligase BirAR118G is fused to the bait protein and biotinylates proximal proteins promiscuously. Hence, interactors positioned close to the bait in vivo are enriched by streptavidin purification and identified by mass spectrometry or western blotting. We present a validation of the BioID assay and preliminary results for close interactors of UPF1 and other key players in NMD.

SMG6 mediated degradation of nonsense mRNA requires phosphorylation-independent interaction with the helicase domain of UPF1

Eukaryotic mRNAs with premature translation-termination codons (PTCs) are recognized and eliminated by nonsense-mediated mRNA decay (NMD). NMD targeted mRNAs can be degraded by different routes that all involve phosphorylated UPF1 (P-UPF1) as a starting point. The endonuclease SMG6, which cleaves mRNA near the PTC, is one of three known NMD factors thought to be recruited to nonsense mRNAs by interaction with P-UPF1, leading to eventual mRNA degradation. By MS2-mediated tethering of SMG6 and mutants thereof to a reporter RNA combined with knockdowns of various NMD factors, we demonstrate that besides its endonucleolytic activity, SMG6 also requires UPF1 and SMG1 for inducing RNA decay. Our experiments revealed a phosphorylation-independent interaction between SMG6 and UPF1 that is important for SMG6-mediated mRNA decay and using yeast two hybrid assays, we mapped this interaction to the unique stalk region of the UPF1 helicase domain. This region of UPF1 is essential for SMG6-mediated reporter RNA decay and also for NMD. Our results postulate that besides recruiting SMG6 to its RNA substrates, UPF1 is also required to activate its endonuclease activity.

Nonsense-mediated mRNA decay (NMD) restricts replication of mammalian RNA viruses

A genome-wide siRNA screen against host factors that affect the infection of Semliki Forest virus (SFV), a positive-strand (+)RNA virus, revealed that components of the nonsense-mediated mRNA decay (NMD) pathway restrict early, post-entry steps of the infection cycle. In HeLa cells and primary human fibroblasts, knockdown of UPF1, SMG5 and SMG7 leads to increased levels of viral proteins and RNA and to higher titers of released virus. The inhibitory effect of NMD was stronger when the efficiency of virus replication was impaired by mutations or deletions in the replicase proteins. Accordingly, impairing NMD resulted in a more than 20-fold increased production of these attenuated viruses. Our data suggest that intrinsic features of genomic and sub-genomic viral mRNAs, most likely the extended 3'-UTR length, make them susceptible to NMD. The fact that SFV replication is entirely cytoplasmic strongly suggests that degradation of the viral RNA occurs through the exon junction complex (EJC)-independent mode of NMD. Collectively, our findings uncover a new biological function for NMD as an intrinsic barrier to the translation of early viral proteins and the amplification of (+)RNA viruses in animal cells. Thus, in addition to its role in mRNA surveillance and post-transcriptional gene regulation, NMD also contributes to protect cells from RNA viruses.

Genetic Engineering of Induced Pluripotent Stem Cells and Reprogramming to Motor Neurons to Elucidate Selective Motor Neuron Death in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease, fatal within 1 to 5 years after onset of symptoms. About 3 out of 100’000 persons are diagnosed with ALS and there is still no cure available [1, 2]. 95% of all cases occur sporadically and the aetiology remains largely unknown [XXXX]. However, up to now 16 genes were identified to play a role in the development of familial ALS. One of these genes is FUS that encodes for the protein fused in sarcoma/translocated in liposarcoma (FUS/TLS). Mutations in this gene are responsible for some cases of sporadic as well as of inherited ALS [3]. FUS belongs to the family of heterogeneous nuclear ribonucleoproteins and is predicted to be involved in several cellular functions like transcription regulation [4], RNA splicing [5, 6], mRNA transport in neurons [7] and microRNA processing [8]. Aberrant accumulation of mutated FUS has been found in the cytoplasm of motor neurons from ALS patients [9]. The mislocalization of FUS is based on a mutation in the nuclear localization signal of FUS [10]. However, it is still unclear if the cytoplasmic localization of FUS leads to a toxic gain of cytoplasmic function and/or a loss of nuclear function that might be crucial in the course of ALS. The goal of this project is to characterize the impact of ALS-associated FUS mutations on in vitro differentiated motor neurons. To this end, we edit the genome of induced pluripotent stem cells (iPSC) using transcription activator-like effector nucleases (TALENs) [11,12] to create three isogenic cell lines, each carrying an ALS-associated FUS mutation (G156E, R244C and P525L). These iPSC’s will then be differentiated to motor neurons according to a recently establishe protocol (Ref Wichterle) and serve to study alterations in the transcriptome, proteome and metabolome upon the expression of ALS-associated FUS. With this approach, we hope to unravel the molecular mechanism leading to FUS-associated ALS and to provide new insight into the emerging connection between misregulation of RNA metabolism and neurodegeneration, a connection that is currently implied in a variety of additional neurological diseases, including spinocerebellar ataxia 2 (SCA-2), spinal muscular atrophy (SMA), fragile X syndrome, and myotonic dystrophy.

CTIF is involved in mRNA export in human cells

In eukaryotic cells translation initiation of messenger RNA (mRNA) transcripts can be initiated either by the cap-binding complex (CBC) consisting of CBP80 and CBP20, or by the eukaryotic translation initiation factor (eIF) 4E. Together with eIF4G and eIF4A, eIF4E forms the eIF4F-complex, which mediates initiation of the bulk of cellular translation. Analogous to eIF4G, the CBP80/20-dependent translation initiation factor (CTIF) has been reported to be part of the CBC-dependent translation initiation complex. CTIF consists of a N-terminal CBP80-binding domain and a conserved C-terminal MIF4G domain. This MIF4G domain has been shown to mediate the interaction between CTIF and different factors such as eIF3g and the stem-loop binding protein (SLBP). Here we show data indicating that CTIF, besides its function in translation initiation, is involved in mRNA translocation from the nucleus to the cytoplasm, possibly through a direct interaction with the nuclear export factor NFX1/TAP. Taken together our results suggest that CTIF can function as a platform that interacts with proteins involved in different steps of mRNA metabolism.

Investigation of premature termination codon recognition in nonsense-mediated mRNA decay

Nonsense-mediated mRNA decay (NMD) is best known for its role in quality control of mRNAs, where it recognizes premature translation termination codons (PTCs) and rapidly degrades the corresponding mRNA. The basic mechanism of NMD appears to be conserved among eukaryotes: aberrant translation termination triggers NMD. According to the current working model, correct termination requires the interaction of the ribosome with the poly(A)-binding protein (PABPC1) mediated through the eukaryotic release factors 1 (eRF1) and 3 (eRF3). The model predicts that in the absence of this interaction, the NMD core factor UPF1 binds to eRF3 instead and initiates the events ultimately leading to mRNA degradation. However, the exact mechanism of how the decision between proper and aberrant (i.e. NMD-inducing) translation termination occurs is not yet well understood. We address this question using a tethering approach in which proteins of interest are bound to a reporter transcript into the vicinity of a PTC. Subsequently, the ability of the tethered proteins to inhibit NMD and thus stabilize the reporter transcript is assessed. Our results revealed that the C-terminal domain interacting with eRF3 seems not to be necessary for tethered PABPC1 to suppress NMD. In contrast, the N-terminal part of PABPC1, consisting of 4 RNA recognition motifs (RRMs) and interacting with eukaryotic initiation factor 4G (eIF4G), retains the ability to inhibit NMD. We find that eIF4G is able to inhibit NMD in a similar manner as PABPC1 when tethered to the reporter mRNA. This stabilization by eIF4G depends on two key interactions. One of these interactions is to PABPC1, the other is to eukaryotic initiation factor 3 (eIF3). These results confirm the importance of PABPC1 in inhibiting NMD but additionally reveal a role of translation initiation factors in the distinction between bona fide termination codons and PTCs.

Probing interactions of NMD factors in a distance-dependent manner by BioID

The understanding of molecular mechanisms requires the elucidation of protein-protein interaction in vivo. For large multi-factor complexes like those assembling on mRNA, co-immunoprecipitation assays often identify many peripheral interactors that complicate the interpretation of such results and that might conceal other insightful mechanistic connections. Here we address the protein-protein interaction network for key factors in the nonsense-mediated mRNA decay (NMD) pathway in a distant-dependent manner using BioID1,2. In this novel approach, the mutant E. coli biotin-protein ligase BirAR118G is fused to the bait protein and biotinylates proximal proteins promiscuously. Hence, interactors positioned close to the bait in vivo are enriched by streptavidin purification and identified by mass spectrometry or western blotting. We present a validation of the BioID assay and preliminary results for close interactors of UPF1 and other key players in NMD.

SMG6 mediated degradation of nonsense mRNA requires phosphorylation-independent interaction with the helicase domain of UPF1

Eukaryotic mRNAs with premature translation-termination codons (PTCs) are recognized and eliminated by nonsense-mediated mRNA decay (NMD). NMD targeted mRNAs can be degraded by different routes that all involve phosphorylated UPF1 (P-UPF1) as a starting point. The endonuclease SMG6, which cleaves mRNA near the PTC, is one of three known NMD factors thought to be recruited to nonsense mRNAs by interaction with P-UPF1, leading to eventual mRNA degradation. By MS2-mediated tethering of SMG6 and mutants thereof to a reporter RNA combined with knockdowns of various NMD factors, we demonstrate that besides its endonucleolytic activity, SMG6 also requires UPF1 and SMG1 for inducing RNA decay. Our experiments revealed a phosphorylation-independent interaction between SMG6 and UPF1 that is important for SMG6-mediated mRNA decay and using yeast two hybrid assays, we mapped this interaction to the unique stalk region of the UPF1 helicase domain. This region of UPF1 is essential for SMG6-mediated reporter RNA decay and also for NMD. Our results postulate that besides recruiting SMG6 to its RNA substrates, UPF1 is also required to activate its endonuclease activity.

Trying to make sense in nonsense-mediated mRNA decay

Despite over 30 years of research, the molecular mechanisms of nonsense-mediated mRNA decay (NMD) are still not well understood. NMD appears to exist in most eukaryotes and is intensively studied in S. cerevisiae, C. elegans, D. melanogaster and in mammalian cells. Current evidence suggests that the core of NMD – involving UPF1, UPF2 and UPF3 – is evolutionarily conserved, but that different species may have evolved slightly different ways to identify target mRNAs for NMD and to degrade them. Our lab has shown that the exon junction complex (EJC) is not absolutely required for NMD in human cells (Bühler et al., NSMB 2006) and that it is neither restricted to CBP80-bound mRNAs as classical models claim (Rufener & Mühlemann, NSMB 2013). Together with the finding that long 3’ UTRs often are an NMD-inducing feature (Eberle et al, PLoS Biol 2008; Yepiskoposyan et al., RNA 2011), our data is consistent with much of the data from other species and hence has led to a “unified” working model for NMD (Stalder & Mühlemann, Trends Cell Biol 2008; Schweingruber et al., Biochim Biophys Acta 2013). Our recent iCLIP experiments with endogenous UPF1 indicate that UPF1 binds mRNAs indiscriminately with respect to being an NMD target or not before they engage with ribosomes (Zünd et al., NSMB 2013). After onset of translation, UPF1 is cleared from the coding region but remains bound to the 3’ UTR of mRNAs. Why this 3’ UTR-associated in some cases induces NMD and in others not is currently being investigated and not yet understood. Following assembly of a phospho-UPF1-containing NMD complex, decay adaptors (SMG5, SMG7, PNRC2) and/or the endonuclease SMG6 are recruited. While the latter cleaves the mRNA in the vicinity of the termination codon, the former proteins induce deadenylation, decapping and exonucleolytic degradation of the mRNA. In my talk, I will give an overview about the latest developments in NMD – with a focus on our own work – and try to integrate the bits and pieces into a somewhat coherent working model.

An mRNA-derived ncRNA targets and regulates the ribosome

Small non-protein-coding RNA (ncRNA) molecules are key players in controlling gene expression at multiple steps in all domains of life. While the list of validated ncRNAs that regulate crucial cellular processes grows steadily (such as micro RNAs and small-interfering RNAs), not a single ncRNA has been identified that directly interacts and regulates the ribosome during protein biosynthesis (with the notable exceptions of 7SL RNA and tmRNA). This is unexpected, given the central position the ribosome plays during gene expression. To investigate whether such a class of regulatory ncRNAs does exist we performed genomic screens for small ribosome-associated RNAs in various model organisms of all three domains [1,2]. Here we show that an mRNA-derived 18 nucleotide long ncRNA is capable of down-regulating translation in Saccharomyces cerevisiae by directly targeting the ribosome [3]. This 18-mer ncRNA binds to polysomes upon salt stress and is crucial for efficient growth under hyperosmotic conditions. Although the 18-mer RNA originates from the TRM10 locus, which encodes a tRNA methyltransferase, genetic analyses revealed the 18-mer RNA nucleotide sequence, rather than the mRNA-encoded enzyme, as the translation regulator under these stress conditions. Our data reveal the ribosome as a target for small regulatory ncRNAs and unveil the existence of a novel mechanism of translation regulation. Analogous genomic screens in organisms spanning all three domains of life demonstrate the existence of thousands of ncRNA candidates putatively regulating the ribosome. We therefore anticipate that ribosome-bound ncRNAs are capable of fine tuning translation and might represent a so far largely unexplored class of regulatory ncRNAs.

An mRNA-derived ncRNA targets and regulates the ribosome

Small non-protein-coding RNAs (ncRNAs) are key players in controlling gene expression. The advantage of ncRNA regulators is their almost immediate availability since they act on the RNA level. The list of validated ncRNAs regulating translation, such as micro RNAs, is growing steadily, however, they almost exclusively target the mRNA rather than the ribosome. This is unexpected given the central position the ribosome plays. Here we show that an mRNA-derived 18 nucleotide long ncRNA is capable of down-regulating translation in Saccharomyces cerevisiae by targeting the ribosome. This 18-mer ncRNA binds to polysomes upon salt stress and is crucial for efficient growth. Although the 18-mer RNA originates from the TRM10 locus, which encodes a tRNA methyltransferase, genetic analyses revealed the 18-mer RNA nucleotide sequence as the translation regulator. Our data reveal the ribosome as a target for a small regulatory ncRNA and demonstrate the existence of a yet unknown mechanism of translation regulation.