Exon Junction Complexes Suppress Spurious Splice Sites to Safeguard Transcriptome Integrity.

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.

Comparative transcriptomics elucidates the cellular responses of an aeroterrestrial zygnematophyte to UV radiation.

The zygnematophytes are the closest relatives of land plants and comprise several lineages that adapted to a life on land. Species of the genus Serritaenia form colorful, mucilaginous capsules, which surround the cells and block harmful solar radiation, one of the major terrestrial stressors. In eukaryotic algae, this 'sunscreen mucilage' represents a unique photoprotective strategy, whose induction and chemical background are unknown. We generated a de novo transcriptome of Serritaenia testaceovaginata and studied its gene regulation under moderate UV radiation (UVR) that triggers sunscreen mucilage under experimental conditions. UVR induced the repair of DNA and the photosynthetic apparatus as well as the synthesis of aromatic specialized metabolites. Specifically, we observed pronounced expressional changes in the production of aromatic amino acids, phenylpropanoid biosynthesis genes, potential cross-membrane transporters of phenolics, and extracellular, oxidative enzymes. Interestingly, the most up-regulated enzyme was a secreted class III peroxidase, whose embryophyte homologs are involved in apoplastic lignin formation. Overall, our findings reveal a conserved, plant-like UVR perception system (UVR8 and downstream factors) in zygnematophyte algae and point to a polyphenolic origin of the sunscreen pigment of Serritaenia, whose synthesis might be extracellular and oxidative, resembling that of plant lignins.

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

The exon junction complex (EJC) is an essential constituent and regulator of spliced messenger ribonucleoprotein particles (mRNPs) in metazoans. As a core component of the EJC, CASC3 was described to be pivotal for EJC-dependent nuclear and cytoplasmic processes. However, recent evidence suggests that CASC3 functions differently from other EJC core proteins. Here, we have established human CASC3 knockout cell lines to elucidate the cellular role of CASC3. In the knockout cells, overall EJC composition and EJC-dependent splicing are unchanged. A transcriptome-wide analysis reveals that hundreds of mRNA isoforms targeted by nonsense-mediated decay (NMD) are upregulated. Mechanistically, recruiting CASC3 to reporter mRNAs by direct tethering or via binding to the EJC stimulates mRNA decay and endonucleolytic cleavage at the termination codon. Building on existing EJC-NMD models, we propose that CASC3 equips the EJC with the persisting ability to communicate with the NMD machinery in the cytoplasm. Collectively, our results characterize CASC3 as a peripheral EJC protein that tailors the transcriptome by promoting the degradation of EJC-dependent NMD substrates.

The exon junction complex: structural insights into a faithful companion of mammalian mRNPs.

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.

Comparative transcriptomics reveals the molecular toolkit used by an algivorous protist for cell wall perforation.

Microbial eukaryotes display a stunning diversity of feeding strategies, ranging from generalist predators to highly specialized parasites. The unicellular "protoplast feeders" represent a fascinating mechanistic intermediate, as they penetrate other eukaryotic cells (algae and fungi) like some parasites but then devour their cell contents by phagocytosis.1 Besides prey recognition and attachment, this complex behavior involves the local, pre-phagocytotic dissolution of the prey cell wall, which results in well-defined perforations of species-specific size and structure.2 Yet the molecular processes that enable protoplast feeders to overcome cell walls of diverse biochemical composition remain unknown. We used the flagellate Orciraptor agilis (Viridiraptoridae, Rhizaria) as a model protoplast feeder and applied differential gene expression analysis to examine its penetration of green algal cell walls. Besides distinct expression changes that reflect major cellular processes (e.g., locomotion and cell division), we found lytic carbohydrate-active enzymes that are highly expressed and upregulated during the attack on the alga. A putative endocellulase (family GH5_5) with a secretion signal is most prominent, and a potential key factor for cell wall dissolution. Other candidate enzymes (e.g., lytic polysaccharide monooxygenases) belong to families that are largely uncharacterized, emphasizing the potential of non-fungal microeukaryotes for enzyme exploration. Unexpectedly, we discovered various chitin-related factors that point to an unknown chitin metabolism in Orciraptor agilis, potentially also involved in the feeding process. Our findings provide first molecular insights into an important microbial feeding behavior and new directions for cell biology research on non-model eukaryotes.

SMG5-SMG7 authorize nonsense-mediated mRNA decay by enabling SMG6 endonucleolytic activity.

Eukaryotic gene expression is constantly controlled by the translation-coupled nonsense-mediated mRNA decay (NMD) pathway. Aberrant translation termination leads to NMD activation, resulting in phosphorylation of the central NMD factor UPF1 and robust clearance of NMD targets via two seemingly independent and redundant mRNA degradation branches. Here, we uncover that the loss of the first SMG5-SMG7-dependent pathway also inactivates the second SMG6-dependent branch, indicating an unexpected functional connection between the final NMD steps. Transcriptome-wide analyses of SMG5-SMG7-depleted cells confirm exhaustive NMD inhibition resulting in massive transcriptomic alterations. Intriguingly, we find that the functionally underestimated SMG5 can substitute the role of SMG7 and individually activate NMD. Furthermore, the presence of either SMG5 or SMG7 is sufficient to support SMG6-mediated endonucleolysis of NMD targets. Our data support an improved model for NMD execution that features two-factor authentication involving UPF1 phosphorylation and SMG5-SMG7 recruitment to access SMG6 activity.

The E3 ubiquitin ligase UBR5 interacts with the H/ACA ribonucleoprotein complex and regulates ribosomal RNA biogenesis in embryonic stem cells.

UBR5 is an E3 ubiquitin ligase involved in distinct processes such as transcriptional regulation and development. UBR5 is highly upregulated in embryonic stem cells (ESCs), whereas its expression decreases with differentiation, suggesting a role for UBR5 in ESC function. However, little is known about how UBR5 regulates ESC identity. Here, we define the protein interactome of UBR5 in ESCs and find interactions with distinct components of the H/ACA ribonucleoprotein complex, which is required for proper maturation of ribosomal RNA (rRNA). Notably, loss of UBR5 induces an abnormal accumulation of rRNA processing intermediates, resulting in diminished ribosomal levels. Consequently, lack of UBR5 triggers an increase in p53 levels and a concomitant decrease in cellular proliferation rates. Thus, our results indicate a link between UBR5 and rRNA maturation.

Detection and quantification of RNA decay intermediates using XRN1-resistant reporter transcripts.

RNA degradation ensures appropriate levels of mRNA transcripts within cells and eliminates aberrant RNAs. Detailed studies of RNA degradation dynamics have been heretofore infeasible because of the inherent instability of degradation intermediates due to the high processivity of the enzymes involved. To visualize decay intermediates and to characterize the spatiotemporal dynamics of mRNA decay, we have developed a set of methods that apply XRN1-resistant RNA sequences (xrRNAs) to protect mRNA transcripts from 5'-3' exonucleolytic digestion. To our knowledge, this approach is the only method that can detect the directionality of mRNA degradation and that allows tracking of degradation products in unperturbed cells. Here, we provide detailed procedures for xrRNA reporter design, transfection and cell line generation. We explain how to extract xrRNA reporter mRNAs from mammalian cells, as well as their detection and quantification using northern blotting and quantitative PCR. The procedure further focuses on how to detect and quantify intact reporter mRNAs and XRN1-resistant degradation intermediates using single-molecule fluorescence microscopy. It provides detailed instructions for sample preparation and image acquisition using fixed, as well as living, cells. The procedure puts special emphasis on detailed descriptions of high-throughput image analysis pipelines, which are provided along with the article and were designed to perform spot co-localization, detection efficiency normalization and the quality control steps necessary for interpretation of results. The aim of the analysis software published here is to enable nonexpert readers to detect and quantify RNA decay intermediates within 4-6 d after reporter mRNA expression.

Plasmid transfection influences the readout of nonsense-mediated mRNA decay reporter assays in human cells.

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.

Interrogating the degradation pathways of unstable mRNAs with XRN1-resistant sequences.

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.