Revealing the elusive molecular biology of the vault RNA

Recently several novel and previously reported non-protein-coding RNAs (ncRNAs) have been identified to be upregulated upon Epstein-Barr virus (EBV) infection in human B-lymphocytes. A group of these significantly upregulated ncRNAs are called vault RNAs (vtRNAs). ,b Only about 5% of the total cellular vtRNAs are connected to the vault particle, the largest known ribonucleoprotein particle (RNP) in eukaryotic cells. However the function of this ncRNA family and moreover of the vault particle remains still rather unclear. Our previous findings suggest a link between EBV infection and vtRNA expression. Consequently we are interested which part of the viral genome is responsible for the upregulation and moreover which function the vtRNAs might possess during virus propagation. To address this question we have separately overexpressed specific EBV-encoded, latently expressed proteins in BL2-cells to determine the influence on the vault RNA levels. Thereby we identified one EBV-encoded protein, called Latent Membrane Protein 1 (LMP1), which significantly contributes to the vtRNA upregulation. We used LMP1 mutants to characterize the region of the protein and the responsible pathway for triggering the elevated vtRNA expression. Our results suggest that the NFkB- pathway might be involved in this process. To investigate a possible functional connection between the vtRNA and EBV infection, we have overexpressed vtRNA1-1 in BL41, a cell line usually not expressing this vault RNA. We show that overexpression of vtRNA1-1 leads to a better viral establishment and markedly protects cells from undergoing apoptosis. Knock-down of the major vault protein, the main component of the vault particle, had no effect on EBV infection and apoptosis resistance. Thus these results support the view that the observed phenotype is caused by the vtRNA rather than the vault particle.

Characterization of the potential role for RNA-binding protein FUS/TLS in DNA damage response: A quantitative proteomic approach

FUS/TLS (fused in sarcoma/translocated in liposarcoma) is a ubiquitously expressed RNA-binding protein of the hnRNP family, that has been discovered as fused to transcription factors, through chromosomal translocations, in several human sarcomas and found in protein aggregates in neurons of patients with an inherited form of Amyotrophic Lateral Sclerosis (ALS) [1]. To date, FUS/TLS has been implicated in a variety of cellular processes such as gene expression control, transcriptional regulation, pre-mRNA splicing and miRNA processing [2]. In addition, some evidences link FUS/TLS to genome stability control and DNA damage response. In fact, mice lacking FUS/TLS are hypersensitive to ionizing radiation (IR) and show high levels of chromosome instability and in response to double-strand breaks, FUS/TLS gets phosphorylated by the protein kinase ATM [3,4,5]. Furthermore, the inducible depletion of FUS/TLS in a neuroblastoma cell line (SH-SY5Y FUS/TLS TET-off iKD) subjected to genotoxic stress (IR) resulted in an increased phosphorylation of γH2AX respect to control cells, suggesting an higher activation of the DNA damage response. The study aims to investigate the specific role of FUS/TLS in DNA damage response through the characterization of the proteomic profile of SH-SY5Y FUS/TLS iKD cells subjected to DNA damage stress, by mass spectrometry-based quantitative proteomics (e.g. SILAC). Preliminary results of mass spectrometric identification of FUS/TLS interacting proteins in HEK293 cells, expressing a recombinant flag-tagged FUS/TLS protein, highlighted the interactions with several proteins involved in DNA damage response, such as DNA-PK, XRCC-5/-6, and ERCC-6, raising the possibilities that FUS/TLS is involved in this pathway, even thou its exact role still need to be addressed.

Unravelling the multiple roles of FUS in RNA processing and on ALS pathogenesis

Two genes with related functions in RNA biogenesis were recently reported in patients with familial ALS: the FUS/TLS gene at the ALS6 locus and the TARDBP/TDP-43 gene at the ALS10 locus [1, 2]. FUS has been implicated to function in several steps of gene expression, including transcription regulation [3], RNA splicing [4, 5], mRNA transport in neurons [6] and, interestingly, in microRNA (miRNA) processing [7]. The goal of this project is to identify the molecular mechanisms leading to the development of FUS mutations-associated ALS. Specifically, we want to test the hypothesis that these FUS mutations misregulate miRNA levels that in turn affect the expression of genes critical for motor neuron survival. In addition we want to test whether misregulation of the miRNA profile is a common feature in ALS. We have performed immunoprecipitations from total extracts of 293T cells expressing FLAG-tagged FUS to characterize its interactome by mass spectrometry. This proteomic study not only revealed a strong interaction of FUS with splicing factors, but shows that FUS might be involved in many, quite different pathways. To map which parts of the FUS protein contribute to the interaction with splicing factors, we have performed a set of experiments with a series of missense and deletion mutants. With this approach, we will not only gain information on the binding partners of FUS along with a map of the required domains for the interactions, but it will also help to unravel whether certain ALS-associated FUS mutations lead to a loss or gain of function due to gain or loss of interactors. Additionally, we have performed quantitative interactomics using SILAC to identify interactome differences of ALS-associated FUS mutants. To this end we have performed immunoprecipitations of total extract from 293T cells, stably transduced with constructs expressing wild-type FUS-FLAG as well as three different ALS-associated mutants (G156E, R244C, P525L). First results indicate striking differences in the interactome with certain RNA binding proteins. We are now validating these candidates in order to reveal the importance of these differential interactions in the context of ALS.

Characterization of the potential role for RNA-binding protein FUS in DNA damage response: A quantitative proteomic approach

FUS/TLS (fused in sarcoma/translocated in liposarcoma) is a ubiquitously expressed protein of the hnRNP family, that has been discovered as fused to transcription factors in several human sarcomas and found in protein aggregates in neurons of patients with an inherited form of Amyotrophic Lateral Sclerosis [Vance C. et al., 2009]. FUS is a 53 kDa nuclear protein that contains structural domains, such as a RNA Recognition Motif (RRM) and a zinc finger motif, that give to FUS the ability to bind to both RNA and DNA sequences. It has been implicated in a variety of cellular processes, such as pre-mRNA splicing, miRNA processing, gene expression control and transcriptional regulation [Fiesel FC. and Kahle PJ., 2011]. Moreover, some evidences link FUS to genome stability control and DNA damage response: mice lacking FUS are hypersensitive to ionizing radiation (IR) and show high levels of chromosome instability and, in response to double-strand breaks, FUS is phosphorylated by the protein kinase ATM [Kuroda M. et al., 2000; Hicks GG. et al., 2000; Gardiner M. et al., 2008]. Furthermore, preliminary results of mass spectrometric identification of FUS interacting proteins in HEK293 cells, expressing a recombinant flag-tagged FUS protein, highlighted the interactions with proteins involved in DNA damage response, such as DNA-PK, XRCC-5/-6, and ERCC-6, raising the possibilities that FUS is involved in this pathway, even though its role still needs to be clarified. This study aims to investigate the biological roles of FUS in human cells and in particular the putative role in DNA damage response through the characterization of the proteomic profile of the neuroblastoma cell line SH-SY5Y upon FUS inducible depletion, by a quantitative proteomic approach. The SH-SY5Y cell line that will be used in this study expresses, in presence of tetracycline, a shRNA that targets FUS mRNA, leading to FUS protein depletion (SH-SY5Y FUS iKD cells). To quantify changes in proteins expression levels a SILAC strategy (Stable Isotope Labeling by Amino acids in Cell culture) will be conducted on SH-SY5Y FUS iKD cells and a control SH-SY5Y cell line (that expresses a mock shRNA) and the relative changes in proteins levels will be evaluated after five and seven days upon FUS depletion, by nanoliquid chromatography coupled to tandem mass spectrometry (nLC-MS/MS) and bioinformatics analysis. Preliminary experiments demonstrated that the SH-SY5Y FUS iKD cells, when subjected to genotoxic stress (high dose of IR), upon inducible depletion of FUS, showed a increased phosphorylation of gH2AX with respect to control cells, suggesting an higher activation of the DNA damage response.

The Host Nonsense-Mediated mRNA Decay Pathway Restricts Mammalian RNA Virus Replication

In addition to classically defined immune mechanisms, cell-intrinsic processes can restrict virus infection and have shaped virus evolution. The details of this virus-host interaction are still emerging. Following a genome-wide siRNA screen for host factors affecting replication of Semliki Forest virus (SFV), a positive-strand RNA (+RNA) virus, we found that depletion of nonsense-mediated mRNA decay (NMD) pathway components Upf1, Smg5, and Smg7 led to increased levels of viral proteins and RNA and higher titers of released virus. The inhibitory effect of NMD was stronger when virus replication efficiency was impaired by mutations or deletions in the replicase proteins. Consequently, depletion of NMD components resulted in a more than 20-fold increase in production of these attenuated viruses. These findings indicate that a cellular mRNA quality control mechanism serves as an intrinsic barrier to the translation of early viral proteins and the amplification of +RNA viruses in animal cells.

The synthesis and application of a diazirine-modified uridine analogue for investigating RNA-protein interactions

The roles played by many ncRNAs remain largely unknown. Similarly, relatively little is known about the RNA binding proteins involved in processing ncRNA. Identification of new RNA/RNA binding protein (RBP) interactions may pave the way to gain a better understanding of the complex events occurring within cells during gene expression and ncRNA biogenesis. The development of chemical tools for the isolation of RBPs is of paramount importance. In this context, we report on the synthesis of the uridine phosphoramidite U Dz that bears a diazirine moiety on the nucleobase. RNA probes containing U Dz units were irradiated in the presence of single-stranded DNA binding protein (SSB), which is also known to bind ssRNAs, and shown to efficiently (15% yield) and selectively cross-link to the protein. The corresponding diazirine-modified uridine triphosphate U DzTP was synthesized and its capacity to act as a substrate for the T7 RNA polymerase was tested in transcription assays. U DzTP was accepted with a maximum yield of 38% for a 26mer RNA containing a single incorporation and 28% yield for triple consecutive incorporations. Thus, this uridine analogue represents a convenient biochemical tool for the identification of RNA binding proteins and unraveling the role and function played by ncRNAs.

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.

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.

Expression of the vault RNA protects cells from undergoing apoptosis

Non-protein-coding RNAs are a functionally versatile class of transcripts found in all domains of life exerting their biological role at the RNA level. Recently, we demonstrated that the vault-associated RNAs (vtRNAs) were significantly up-regulated in human B cells upon Epstein-Barr virus (EBV) infection [1,2]. vtRNAs are an integral part of the vault complex, a huge and evolutionarily conserved cytoplasmic ribonucleoprotein complex. The major vault protein (MVP) is the main structural component of the complex while vtRNA accounts for only 5% of its mass. Very little is known about the function(s) of the vtRNAs or the vault complex. In particular the role and significance of the previously observed vtRNA up-regulation upon EBV infection remained unclear. We individually expressed EBV-encoded genes in B cells and found the latent membrane protein 1 (LMP1) as trigger for vtRNA up-regulation. To unravel a putative functional interconnection between vtRNA expression and EBV infection, we ectopically expressed vtRNA1-1 in human B cells and observed an improved viral establishment. Furthermore, expression of vtRNA1-1 but not of the other vtRNA paralogs protected cells from undergoing apoptosis. Knock-down of MVP had no effect on these phenotypes thus revealing the vtRNA and not the vault complex to contribute to the enhanced EBV establishment and apoptosis resistance. Mutational analysis highlighted the central domain of the vtRNA to be involved in the anti-apoptotic effect. Ongoing research aims at characterizing the target of vtRNA1-1 in the apoptotic pathway. In summary, our data reveal a crucial cellular function for the so far elusive RNA biology of the vtRNAs.

Identification of gunshots to the head by detection of RNA in backspatter primarily expressed in brain tissue

Traces of backspatter recovered from the inside of the barrel of a gun that was used to deliver suicidal or homicidal contact shots may be a source of valuable forensic evidence and first systematic investigations of the persistence of victim DNA from inside firearms have been presented. The aim of the present study was to include victim RNA in such analyses to determine the origin of tissues in addition and parallel to standard DNA profiling for forensic identification purposes. In a first step, suitable mRNA (C1orf61) and micro-RNAs (miR-124a and miR-124*) that are primarily expressed in brain tissue were selected from potential candidates and confirmed using quantitative PCR (qPCR). Secondly, a co-extraction procedure for RNA and DNA was established and brain differentiability of the selected RNAs was demonstrated via qPCR using samples from experimental shots at ballistic models. In a third step, this procedure was successfully applied to analyse samples from real casework comprising eight cases of suicidal contact shots. In this pilot study, we are first to report the possibility of co-extracting mRNA, miRNA and DNA from ballistic trace samples collected from the inside of firearms and we demonstrate that RNA and DNA based analyses can be performed in parallel to produce informative and highly complementary evidence.

RNA interference screening identifies a novel role for PCTK1/CDK16 in medulloblastoma with c-Myc amplification.

Medulloblastoma (MB) is the most common malignant brain tumor in children and is associated with a poor outcome. cMYC amplification characterizes a subgroup of MB with very poor prognosis. However, there exist so far no targeted therapies for the subgroup of MB with cMYC amplification. Here we used kinome-wide RNA interference screening to identify novel kinases that may be targeted to inhibit the proliferation of c-Myc-overexpressing MB. The RNAi screen identified a set of 5 genes that could be targeted to selectively impair the proliferation of c-Myc-overexpressing MB cell lines: AKAP12 (A-kinase anchor protein), CSNK1α1 (casein kinase 1, alpha 1), EPHA7 (EPH receptor A7) and PCTK1 (PCTAIRE protein kinase 1). When using RNAi and a pharmacological inhibitor selective for PCTK1, we could show that this kinase plays a crucial role in the proliferation of MB cell lines and the activation of the mammalian target of rapamycin (mTOR) pathway. In addition, pharmacological PCTK1 inhibition reduced the expression levels of c-Myc. Finally, targeting PCTK1 selectively impaired the tumor growth of c-Myc-overexpressing MB cells in vivo. Together our data uncover a novel and crucial role for PCTK1 in the proliferation and survival of MB characterized by cMYC amplification.

Characterization of the role of the RNA-binding protein FUS in the DNA damage response

FUS/TLS (fused in sarcoma/translocated in liposarcoma), a ubiquitously expressed RNA-binding protein, has been linked to a variety of cellular processes, including RNA metabolism, microRNA biogenesis and DNA repair. However, the precise cellular function of FUS remains unclear. Recently, mutations in the FUS gene have been found in ∼5% of familial Amyotrophic Lateral Sclerosis, a neurodegenerative disorder characterized by the dysfunction and death of motor neurons. Since MEFs and B-lymphocytes derived from FUS knockdown mice display major sensitivity to ionizing radiation and chromosomal aberrations [1,2], we are investigating the effects of DNA damage both in the presence or in the absence of FUS. To this purpose, we have generated a SH-SY5Y human neuroblastoma cell line expressing a doxycycline-induced shRNA targeting FUS, which specifically depletes the protein. We have found that FUS depletion induces an activation of the DNA damage response (DDR). However, treatment with genotoxic agents did not induce any strong changes in ATM (Ataxia Telangiectasia Mutated)-mediated DDR signaling. Interestingly, genotoxic treatment results in changes in the subcellular localization of FUS in normal cells. We are currently exploring on one hand the mechanism by which FUS depletion leads to DNA damage, and on the other the functional significance of FUS relocalization after genotoxic stress.

To sense or not to sense viral RNA--essentials of coronavirus innate immune evasion.

An essential function of innate immunity is to distinguish self from non-self and receptors have evolved to specifically recognize viral components and initiate the expression of antiviral proteins to restrict viral replication. Coronaviruses are RNA viruses that replicate in the host cytoplasm and evade innate immune sensing in most cell types, either passively by hiding their viral signatures and limiting exposure to sensors or actively, by encoding viral antagonists to counteract the effects of interferons. Since many cytoplasmic viruses exploit similar mechanisms of innate immune evasion, mechanistic insight into the direct interplay between viral RNA, viral RNA-processing enzymes, cellular sensors and antiviral proteins will be highly relevant to develop novel antiviral targets and to restrict important animal and human infections.

Targeting membrane-bound viral RNA synthesis reveals potent inhibition of diverse coronaviruses including the middle East respiratory syndrome virus.

Coronaviruses raise serious concerns as emerging zoonotic viruses without specific antiviral drugs available. Here we screened a collection of 16671 diverse compounds for anti-human coronavirus 229E activity and identified an inhibitor, designated K22, that specifically targets membrane-bound coronaviral RNA synthesis. K22 exerts most potent antiviral activity after virus entry during an early step of the viral life cycle. Specifically, the formation of double membrane vesicles (DMVs), a hallmark of coronavirus replication, was greatly impaired upon K22 treatment accompanied by near-complete inhibition of viral RNA synthesis. K22-resistant viruses contained substitutions in non-structural protein 6 (nsp6), a membrane-spanning integral component of the viral replication complex implicated in DMV formation, corroborating that K22 targets membrane bound viral RNA synthesis. Besides K22 resistance, the nsp6 mutants induced a reduced number of DMVs, displayed decreased specific infectivity, while RNA synthesis was not affected. Importantly, K22 inhibits a broad range of coronaviruses, including Middle East respiratory syndrome coronavirus (MERS-CoV), and efficient inhibition was achieved in primary human epithelia cultures representing the entry port of human coronavirus infection. Collectively, this study proposes an evolutionary conserved step in the life cycle of positive-stranded RNA viruses, the recruitment of cellular membranes for viral replication, as vulnerable and, most importantly, druggable target for antiviral intervention. We expect this mode of action to serve as a paradigm for the development of potent antiviral drugs to combat many animal and human virus infections.