hGRAD: A versatile "one-fits-all" system to acutely deplete RNA binding proteins from condensates.

Nuclear RNA binding proteins (RBPs) are difficult to study because they often belong to large protein families and form extensive networks of auto- and crossregulation. They are highly abundant and many localize to condensates with a slow turnover, requiring long depletion times or knockouts that cannot distinguish between direct and indirect or compensatory effects. Here, we developed a system that is optimized for the rapid degradation of nuclear RBPs, called hGRAD. It comes as a "one-fits-all" plasmid, and integration into any cell line with endogenously GFP-tagged proteins allows for an inducible, rapid, and complete knockdown. We show that the nuclear RBPs SRSF3, SRSF5, SRRM2, and NONO are completely cleared from nuclear speckles and paraspeckles within 2 h. hGRAD works in various cell types, is more efficient than previous methods, and does not require the expression of exogenous ubiquitin ligases. Combining SRSF5 hGRAD degradation with Nascent-seq uncovered transient transcript changes, compensatory mechanisms, and an effect of SRSF5 on transcript stability.

Poison cassette exon splicing of SRSF6 regulates nuclear speckle dispersal and the response to hypoxia.

Hypoxia induces massive changes in alternative splicing (AS) to adapt cells to the lack of oxygen. Here, we identify the splicing factor SRSF6 as a key factor in the AS response to hypoxia. The SRSF6 level is strongly reduced in acute hypoxia, which serves a dual purpose: it allows for exon skipping and triggers the dispersal of nuclear speckles. Our data suggest that cells use dispersal of nuclear speckles to reprogram their gene expression during hypoxic adaptation and that SRSF6 plays an important role in cohesion of nuclear speckles. Down-regulation of SRSF6 is achieved through inclusion of a poison cassette exon (PCE) promoted by SRSF4. Removing the PCE 3' splice site using CRISPR/Cas9 abolishes SRSF6 reduction in hypoxia. Aberrantly high SRSF6 levels in hypoxia attenuate hypoxia-mediated AS and impair dispersal of nuclear speckles. As a consequence, proliferation and genomic instability are increased, while the stress response is suppressed. The SRSF4-PCE-SRSF6 hypoxia axis is active in different cancer types, and high SRSF6 expression in hypoxic tumors correlates with a poor prognosis. We propose that the ultra-conserved PCE of SRSF6 acts as a tumor suppressor and that its inclusion in hypoxia is crucial to reduce SRSF6 levels. This may prevent tumor cells from entering the metastatic route of hypoxia adaptation.

Npl3 functions in mRNP assembly by recruitment of mRNP components to the transcription site and their transfer onto the mRNA.

RNA-binding proteins (RBPs) control every RNA metabolic process by multiple protein-RNA and protein-protein interactions. Their roles have largely been analyzed by crude mutations, which abrogate multiple functions at once and likely impact the structural integrity of the large ribonucleoprotein particles (RNPs) these proteins function in. Using UV-induced RNA-protein crosslinking of entire cells, protein complex purification and mass spectrometric analysis, we identified >100 in vivo RNA crosslinks in 16 nuclear mRNP components in Saccharomyces cerevisiae. For functional analysis, we chose Npl3, which displayed crosslinks in its two RNA recognition motifs (RRMs) and in the connecting flexible linker region. Both RRM domains and the linker uniquely contribute to RNA recognition as revealed by NMR and structural analyses. Interestingly, mutations in these regions cause different phenotypes, indicating distinct functions of the different RNA-binding domains. Notably, an npl3-Linker mutation strongly impairs recruitment of several mRNP components to chromatin and incorporation of other mRNP components into nuclear mRNPs, establishing a so far unknown function of Npl3 in nuclear mRNP assembly. Taken together, our integrative analysis uncovers a specific function of the RNA-binding activity of the nuclear mRNP component Npl3. This approach can be readily applied to RBPs in any RNA metabolic process.

Nuclear export of plant pararetrovirus mRNAs involves the TREX complex, two viral proteins and the highly structured 5' leader region.

In eukaryotes, the major nuclear export pathway for mature mRNAs uses the dimeric receptor TAP/p15, which is recruited to mRNAs via the multisubunit TREX complex, comprising the THO core and different export adaptors. Viruses that replicate in the nucleus adopt different strategies to hijack cellular export factors and achieve cytoplasmic translation of their mRNAs. No export receptors are known in plants, but Arabidopsis TREX resembles the mammalian complex, with a conserved hexameric THO core associated with ALY and UIEF proteins, as well as UAP56 and MOS11. The latter protein is an orthologue of mammalian CIP29. The nuclear export mechanism for viral mRNAs has not been described in plants. To understand this process, we investigated the export of mRNAs of the pararetrovirus CaMV in Arabidopsis and demonstrated that it is inhibited in plants deficient in ALY, MOS11 and/or TEX1. Deficiency for these factors renders plants partially resistant to CaMV infection. Two CaMV proteins, the coat protein P4 and reverse transcriptase P5, are important for nuclear export. P4 and P5 interact and co-localise in the nucleus with the cellular export factor MOS11. The highly structured 5' leader region of 35S RNAs was identified as an export enhancing element that interacts with ALY1, ALY3 and MOS11 in vitro.

Identification and Regulation of Tomato Serine/Arginine-Rich Proteins Under High Temperatures.

Alternative splicing is an important mechanism for the regulation of gene expression in eukaryotes during development, cell differentiation or stress response. Alterations in the splicing profiles of genes under high temperatures that cause heat stress (HS) can impact the maintenance of cellular homeostasis and thermotolerance. Consequently, information on factors involved in HS-sensitive alternative splicing is required to formulate the principles of HS response. Serine/arginine-rich (SR) proteins have a central role in alternative splicing. We aimed for the identification and characterization of SR-coding genes in tomato (Solanum lycopersicum), a plant extensively used in HS studies. We identified 17 canonical SR and two SR-like genes. Several SR-coding genes show differential expression and altered splicing profiles in different organs as well as in response to HS. The transcriptional induction of five SR and one SR-like genes is partially dependent on the master regulator of HS response, HS transcription factor HsfA1a. Cis-elements in the promoters of these SR genes were predicted, which can be putatively recognized by HS-induced transcription factors. Further, transiently expressed SRs show reduced or steady-state protein levels in response to HS. Thus, the levels of SRs under HS are regulated by changes in transcription, alternative splicing and protein stability. We propose that the accumulation or reduction of SRs under HS can impact temperature-sensitive alternative splicing.

SRSF3 and SRSF7 modulate 3'UTR length through suppression or activation of proximal polyadenylation sites and regulation of CFIm levels.

BACKGROUND: Alternative polyadenylation (APA) refers to the regulated selection of polyadenylation sites (PASs) in transcripts, which determines the length of their 3' untranslated regions (3'UTRs). We have recently shown that SRSF3 and SRSF7, two closely related SR proteins, connect APA with mRNA export. The mechanism underlying APA regulation by SRSF3 and SRSF7 remained unknown. RESULTS: Here we combine iCLIP and 3'-end sequencing and find that SRSF3 and SRSF7 bind upstream of proximal PASs (pPASs), but they exert opposite effects on 3'UTR length. SRSF7 enhances pPAS usage in a concentration-dependent but splicing-independent manner by recruiting the cleavage factor FIP1, generating short 3'UTRs. Protein domains unique to SRSF7, which are absent from SRSF3, contribute to FIP1 recruitment. In contrast, SRSF3 promotes distal PAS (dPAS) usage and hence long 3'UTRs directly by counteracting SRSF7, but also indirectly by maintaining high levels of cleavage factor Im (CFIm) via alternative splicing. Upon SRSF3 depletion, CFIm levels decrease and 3'UTRs are shortened. The indirect SRSF3 targets are particularly sensitive to low CFIm levels, because here CFIm serves a dual function; it enhances dPAS and inhibits pPAS usage by binding immediately downstream and assembling unproductive cleavage complexes, which together promotes long 3'UTRs. CONCLUSIONS: We demonstrate that SRSF3 and SRSF7 are direct modulators of pPAS usage and show how small differences in the domain architecture of SR proteins can confer opposite effects on pPAS regulation.

[Nuclear export of viral RNAs in metazoan]. / Export nucléaire des ARN viraux chez les métazoaires.

The nuclear export of mRNAs is a complex process, involving the participaton of numerous proteins, the recruitement of which starts during the early steps of mRNAs biosynthesis and maturation. This strategy allows the cell to export only mature and non-defective transcripts to the cytoplasm where they are directed to the translational machinery. The vast majority of mRNAs is exported by the dimeric transport receptor TAP-p15, which is mainly recruited by the large multiprotein complex TREX-1. Other mRNAs that do not display all typical features of a mature transcript use variants of the TAP-p15 export pathway or recruit the alternative export receptor CRM1. Most DNA viruses, retroviruses, and influenza viruses, the mRNAs of which are synthesized in the nucleus, also use TAP-p15 and/or CRM1 to export their mRNAs. The highjacking of the cellular export machinery by viral mRNAs usually involves the presence of constitutive structural elements that directly load cellular export factors and/or viral adaptor proteins. Associated with the host export machinery, viral mRNAs escape host surveillance, are efficiently exported in the cytoplasm in order to be translated, and thus make possible the progress toward the later events of the virus life cycles.

miRNAs involved in transcriptome remodeling during pollen development and heat stress response in Solanum lycopersicum.

Cellular transitions during development and stress response depend on coordinated transcriptomic and proteomic alterations. Pollen is particular because its development is a complex process that includes meiotic and mitotic divisions which causes a high heat sensitivity of these cells. Development and stress response are accompanied by a reprogramming of the transcriptome, e.g. by post-transcriptional regulation via miRNAs. We identified known and potentially novel miRNAs in the transcriptome of developing and heat-stressed pollen of Solanum lycopersicum (tomato). The prediction of target mRNAs yielded an equal number of predicted target-sites in CDS and 3'UTR regions of target mRNAs. The result enabled the postulation of a possible link between miRNAs and a fine-tuning of transcription factor abundance during pollen development. miRNAs seem to play a role in the pollen heat stress response as well. We identified several heat stress transcription factors and heat shock proteins as putative targets of miRNAs in response to heat stress, thereby placing these miRNAs as important elements of thermotolerance. Moreover, for members of the AP2, SBP and ARF family members we could predict a miRNA-mediated regulation during development via the miR172, mir156 and mir160-family strengthening the current concept of a cross-connection between development and stress response in plants.

The coupling of transcriptome and proteome adaptation during development and heat stress response of tomato pollen.

BACKGROUND: Pollen development is central for plant reproduction and is assisted by changes of the transcriptome and proteome. At the same time, pollen development and viability is largely sensitive to stress, particularly to elevated temperatures. The transcriptomic and proteomic changes during pollen development and of different stages in response to elevated temperature was targeted to define the underlying molecular principles. RESULTS: The analysis of the transcriptome and proteome of Solanum lycopersicum pollen at tetrad, post-meiotic and mature stage before and after heat stress yielded a decline of the transcriptome but an increase of the proteome size throughout pollen development. Comparison of the transcriptome and proteome led to the discovery of two modes defined as direct and delayed translation. Here, genes of distinct functional processes are under the control of direct and delayed translation. The response of pollen to elevated temperature occurs rather at proteome, but not as drastic at the transcriptome level. Heat shock proteins, proteasome subunits, ribosomal proteins and eukaryotic initiation factors are most affected. On the example of heat shock proteins we demonstrate a decoupling of transcript and protein levels as well as a distinct regulation between the developmental stages. CONCLUSIONS: The transcriptome and proteome of developing pollen undergo drastic changes in composition and quantity. Changes at the proteome level are a result of two modes assigned as direct and delayed translation. The response of pollen to elevated temperature is mainly regulated at the proteome level, whereby proteins related to synthesis and degradation of proteins are most responsive and might play a central role in the heat stress response of pollen.

Formation of large viroplasms and virulence of Cauliflower mosaic virus in turnip plants depend on the N-terminal EKI sequence of viral protein TAV.

Cauliflower mosaic virus (CaMV) TAV protein (TransActivator/Viroplasmin) plays a pivotal role during the infection cycle since it activates translation reinitiation of viral polycistronic RNAs and suppresses RNA silencing. It is also the major component of cytoplasmic electron-dense inclusion bodies (EDIBs) called viroplasms that are particularly evident in cells infected by the virulent CaMV Cabb B-JI isolate. These EDIBs are considered as virion factories, vehicles for CaMV intracellular movement and reservoirs for CaMV transmission by aphids. In this study, focused on different TAV mutants in vivo, we demonstrate that three physically separated domains collectively participate to the formation of large EDIBs: the N-terminal EKI motif, a sequence of the MAV domain involved in translation reinitiation and a C-terminal region encompassing the zinc finger. Surprisingly, EKI mutant TAVm3, corresponding to a substitution of the EKI motif at amino acids 11-13 by three alanines (AAA), which completely abolished the formation of large viroplasms, was not lethal for CaMV but highly reduced its virulence without affecting the rate of systemic infection. Expression of TAVm3 in a viral context led to formation of small irregularly shaped inclusion bodies, mild symptoms and low levels of viral DNA and particles accumulation, despite the production of significant amounts of mature capsid proteins. Unexpectedly, for CaMV-TAVm3 the formation of viral P2-containing electron-light inclusion body (ELIB), which is essential for CaMV aphid transmission, was also altered, thus suggesting an indirect role of the EKI tripeptide in CaMV plant-to-plant propagation. This important functional contribution of the EKI motif in CaMV biology can explain the strict conservation of this motif in the TAV sequences of all CaMV isolates.

Alternative splicing in tomato pollen in response to heat stress.

Alternative splicing (AS) is a key control mechanism influencing signal response cascades in different developmental stages and under stress conditions. In this study, we examined heat stress (HS)-induced AS in the heat sensitive pollen tissue of two tomato cultivars. To obtain the entire spectrum of HS-related AS, samples taken directly after HS and after recovery were combined and analysed by RNA-seq. For nearly 9,200 genes per cultivar, we observed at least one AS event under HS. In comparison to control, for one cultivar we observed 76% more genes with intron retention (IR) or exon skipping (ES) under HS. Furthermore, 2,343 genes had at least one transcript with IR or ES accumulated under HS in both cultivars. These genes are involved in biological processes like protein folding, gene expression and heat response. Transcriptome assembly of these genes revealed that most of the alternative spliced transcripts possess truncated coding sequences resulting in partial or total loss of functional domains. Moreover, 141 HS specific and 22 HS repressed transcripts were identified. Further on, we propose AS as layer of stress response regulating constitutively expressed genes under HS by isoform abundance.

Cauliflower mosaic virus Transcriptome Reveals a Complex Alternative Splicing Pattern.

The plant pararetrovirus Cauliflower mosaic virus (CaMV) uses alternative splicing to generate several isoforms from its polycistronic pregenomic 35S RNA. This pro-cess has been shown to be essential for infectivity. Previous works have identified four splice donor sites and a single splice acceptor site in the 35S RNA 5' region and suggested that the main role of CaMV splicing is to downregulate expression of open reading frames (ORFs) I and II. In this study, we show that alternative splicing is a conserved process among CaMV isolates. In Cabb B-JI and Cabb-S isolates, splicing frequently leads to different fusion between ORFs, particularly between ORF I and II. The corresponding P1P2 fusion proteins expressed in E. coli interact with viral proteins P2 and P3 in vitro. However, they are detected neither during infection nor upon transient expression in planta, which suggests rapid degradation after synthesis and no important biological role in the CaMV infectious cycle. To gain a better understanding of the functional relevance of 35S RNA alternative splicing in CaMV infectivity, we inactivated the previously described splice sites. All the splicing mutants were as pathogenic as the corresponding wild-type isolate. Through RT-PCR-based analysis we demonstrate that CaMV 35S RNA exhibits a complex splicing pattern, as we identify new splice donor and acceptor sites whose selection leads to more than thirteen 35S RNA isoforms in infected turnip plants. Inactivating splice donor or acceptor sites is not lethal for the virus, since disrupted sites are systematically rescued by the activation of cryptic and/or seldom used splice sites. Taken together, our data depict a conserved, complex and flexible process, involving multiple sites, that ensures splicing of 35S RNA.

The composition of the global and feature specific cyanobacterial core-genomes.

Cyanobacteria are photosynthetic prokaryotes important for many ecosystems with a high potential for biotechnological usage e.g., in the production of bioactive molecules. Either asks for a deep understanding of the functionality of cyanobacteria and their interaction with the environment. This in part can be inferred from the analysis of their genomes or proteomes. Today, many cyanobacterial genomes have been sequenced and annotated. This information can be used to identify biological pathways present in all cyanobacteria as proteins involved in such processes are encoded by a so called core-genome. However, beside identification of fundamental processes, genes specific for certain cyanobacterial features can be identified by a holistic genome analysis as well. We identified 559 genes that define the core-genome of 58 analyzed cyanobacteria, as well as three genes likely to be signature genes for thermophilic and 57 genes likely to be signature genes for heterocyst-forming cyanobacteria. To get insights into cyanobacterial systems for the interaction with the environment we also inspected the diversity of the outer membrane proteome with focus on ß-barrel proteins. We observed that most of the transporting outer membrane ß-barrel proteins are not globally conserved in the cyanobacterial phylum. In turn, the occurrence of ß-barrel proteins shows high strain specificity. The core set of outer membrane proteins globally conserved in cyanobacteria comprises three proteins only, namely the outer membrane ß-barrel assembly protein Omp85, the lipid A transfer protein LptD, and an OprB-type porin. Thus, we conclude that cyanobacteria have developed individual strategies for the interaction with the environment, while other intracellular processes like the regulation of the protein homeostasis are globally conserved.

Identification and Expression Analysis of Ribosome Biogenesis Factor Co-orthologs in Solanum lycopersicum.

Ribosome biogenesis involves a large inventory of proteinaceous and RNA cofactors. More than 250 ribosome biogenesis factors (RBFs) have been described in yeast. These factors are involved in multiple aspects like rRNA processing, folding, and modification as well as in ribosomal protein (RP) assembly. Considering the importance of RBFs for particular developmental processes, we examined the complexity of RBF and RP (co-)orthologs by bioinformatic assignment in 14 different plant species and expression profiling in the model crop Solanum lycopersicum. Assigning (co-)orthologs to each RBF revealed that at least 25% of all predicted RBFs are encoded by more than one gene. At first we realized that the occurrence of multiple RBF co-orthologs is not globally correlated to the existence of multiple RP co-orthologs. The transcript abundance of genes coding for predicted RBFs and RPs in leaves and anthers of S. lycopersicum was determined by next generation sequencing (NGS). In combination with existing expression profiles, we can conclude that co-orthologs of RBFs by large account for a preferential function in different tissue or at distinct developmental stages. This notion is supported by the differential expression of selected RBFs during male gametophyte development. In addition, co-regulated clusters of RBF and RP coding genes have been observed. The relevance of these results is discussed.

Le virus de la mosaïque du chou-fleur (CaMV), encore et toujours.

As a pararetrovirus and because of the non-canonical translation of its polycistronic pregenomic 35S RNA, Cauliflower mosaic virus (CaMV) is an original model system that has been extensively studied. Recent advances have improved our understanding of CaMV aphid transmission, cell-to-cell movement, protein expression and virus counter-defense strategy against host plant defense. Since P6/TAV is involved in many aspects of viral pathogenesis as well as in some replication steps, it is considered as the key player of CaMV infectious cycle. This paper reviews our current knowledge on CaMV multiplication and pathogenesis, with special emphasis on steps in which P6/TAV has a major role.

TOR and S6K1 promote translation reinitiation of uORF-containing mRNAs via phosphorylation of eIF3h.

Mammalian target-of-rapamycin (mTOR) triggers S6 kinase (S6K) activation to phosphorylate targets linked to translation in response to energy, nutrients, and hormones. Pathways of TOR activation in plants remain unknown. Here, we uncover the role of the phytohormone auxin in TOR signalling activation and reinitiation after upstream open reading frame (uORF) translation, which in plants is dependent on translation initiation factor eIF3h. We show that auxin triggers TOR activation followed by S6K1 phosphorylation at T449 and efficient loading of uORF-mRNAs onto polysomes in a manner sensitive to the TOR inhibitor Torin-1. Torin-1 mediates recruitment of inactive S6K1 to polysomes, while auxin triggers S6K1 dissociation and recruitment of activated TOR instead. A putative target of TOR/S6K1-eIF3h-is phosphorylated and detected in polysomes in response to auxin. In TOR-deficient plants, polysomes were prebound by inactive S6K1, and loading of uORF-mRNAs and eIF3h was impaired. Transient expression of eIF3h-S178D in plant protoplasts specifically upregulates uORF-mRNA translation. We propose that TOR functions in polysomes to maintain the active S6K1 (and thus eIF3h) phosphorylation status that is critical for translation reinitiation.

Over-sulfated chondroitin sulfate derivatives induce osteogenic differentiation of hMSC independent of BMP-2 and TGF-ß1 signalling.

Natural glycosaminoglycans (GAGs) and chemically modified GAG derivatives are known to support osteogenic differentiation of mesenchymal stromal cells (MSC). This effect has mainly been described to be mediated by increasing the effectiveness of bone anabolic growth factors such as bone morphogenetic proteins (BMPs) due to the binding and presentation of the growth factor or by modulating its signal transduction pathway. In the present study, the influence of chondroitin sulfate (CS) and two chemically over-sulfated CS derivatives on osteogenic differentiation of human mesenchymal stromal cells (hMSC) and on BMP-2 and transforming growth factor ß1 (TGF-ß1) signalling was investigated. Over-sulfated CS derivatives induced an increase of tissue non-specific alkaline phosphatase (TNAP) activity and calcium deposition, whereas collagen synthesis was slightly decreased. The BMP-2-induced Smad1/5 activation was inhibited in the presence of over-sulfated CS derivatives leading to a loss of BMP-2-induced TNAP activity and calcium deposition. In contrast, the TGF-ß1-induced activation of Smad2/3 and collagen synthesis were not affected by the over-sulfated CS derivatives. BMP-2 and TGF-ß1 did not activate the extracellular signal-regulated kinase 1/2 or mitogen-activated protein kinase p38 in hMSC. These data suggest that over-sulfated CS derivatives themselves are able to induce osteogenic differentiation, probably independent of BMP-2 and TGF-ß1 signalling, and offer therefore an interesting approach for the improvement of bone healing.

Two preferentially expressed proteins protect vascular endothelial cells from an attack by peptide-specific CTL.

Vascular endothelial cells (EC) are an exposed tissue with intimate contact with circulating Ag-specific CTL. Experimental in vitro and clinical data suggested that endothelial cells present a different repertoire of MHC class I-restricted peptides compared with syngeneic leukocytes or epithelial cells. This endothelial-specific peptide repertoire might protect EC from CTL-mediated cell death. The HLA-A*02-restricted peptide profile of human EC and syngeneic B lymphoblastoid cells was biochemically analyzed and compared. For EC selective peptides, source protein expression, peptide binding affinity, and peptide-HLA-A*02 turnover were measured. The significance of abundant peptide presentation for target cell recognition by immunodominant CTL was tested by small interfering RNA treatment of EC to knock down the source proteins. High amounts of two peptides, PTRF(56-64) and CD59(106-114), were consistently detected in EC. This predominance of two endothelial peptides was explained by cell type-specific source protein expression that compensated for poor HLA-A*02 binding affinity and short half-live of peptide/HLA-A*02 complexes. Knocking down the source proteins containing the abundant endothelial peptide motifs led to a nearly 100-fold increase of surface expression of SMCY(311-319), an immunodominant minor histocompatibility Ag, as detected by cytotoxicity assays using SMCY(311-319)-specific CTL. We conclude that EC express and present preferentially two distinct HLA-A*02-restricted peptides at extraordinary high levels. These abundant self-peptides may protect EC from CTL-mediated lysis by competing for HLA-A*02 binding sites with immunodominant scarcely expressed antigenic peptides.