The art of hijacking: how Nsp1 impacts host gene expression during coronaviral infections.

Non-structural protein 1 (Nsp1) is one of the first proteins produced during coronaviral infections. It plays a pivotal role in hijacking and rendering the host gene expression under the service of the virus. With a focus on SARS-CoV-2, this review presents how Nsp1 selectively inhibits host protein synthesis and induces mRNA degradation of host but not viral mRNAs and blocks nuclear mRNA export. The clinical implications of this protein are highlighted by showcasing the pathogenic role of Nsp1 through the repression of interferon expression pathways and the features of viral variants with mutations in the Nsp1 coding sequence. The ability of SARS-CoV-2 Nsp1 to hinder host immune responses at an early step, the absence of homology to any human proteins, and the availability of structural information render this viral protein an ideal drug target with therapeutic potential

Transcriptome-wide identification of NMD-targeted human mRNAs reveals extensive redundancy between SMG6- and SMG7-mediated degradation pathways

Besides degrading aberrant mRNAs that harbor a premature translation termination codon (PTC), nonsense-mediated mRNA decay (NMD) also targets many seemingly "normal" mRNAs that encode for full-length proteins. To identify a bona fide set of such endogenous NMD targets in human cells, we applied a meta-analysis approach in which we combined transcriptome profiling of knockdowns and rescues of the three NMD factors UPF1, SMG6 and SMG7. We provide evidence that this combinatorial approach identifies NMD-targeted transcripts more reliably than previous attempts that focused on inactivation of single NMD factors. Our data revealed that SMG6 and SMG7 act on essentially the same transcripts, indicating extensive redundancy between the endo- and exonucleolytic decay routes. Besides mRNAs, we also identified as NMD targets many long non-coding RNAs as well as miRNA and snoRNA host genes. The NMD target feature with the most predictive value is an intron in the 3' UTR, followed by the presence of upstream open reading frames (uORFs) and long 3' UTRs. Furthermore, the 3' UTRs of NMD-targeted transcripts tend to have an increased GC content and to be phylogenetically less conserved when compared to 3' UTRs of NMD insensitive transcripts

Coronavirus takeover of host cell translation and intracellular antiviral response: a molecular perspective.

Coronaviruses are a group of related RNA viruses that cause respiratory diseases in humans and animals. Understanding the mechanisms of translation regulation during coronaviral infections is critical for developing antiviral therapies and preventing viral spread. Translation of the viral single-stranded RNA genome in the host cell cytoplasm is an essential step in the life cycle of coronaviruses, which affects the cellular mRNA translation landscape in many ways. Here we discuss various viral strategies of translation control, including how members of the Betacoronavirus genus shut down host cell translation and suppress host innate immune functions, as well as the role of the viral non-structural protein 1 (Nsp1) in the process. We also outline the fate of viral RNA, considering stress response mechanisms triggered in infected cells, and describe how unique viral RNA features contribute to programmed ribosomal -1 frameshifting, RNA editing, and translation shutdown evasion

The broader sense of nonsense

Nonsense-mediated mRNA decay (NMD) serves as a quality control mechanism by degrading mRNAs with premature termination codons (PTCs), and it also regulates the abundance of physiological RNAs encoding functional proteins. Recent work showed that NMD can be triggered with a certain probability by each translation termination event. Readthrough of the PTC can render NMD-sensitive mRNA molecules immune to NMD, which can explain why a certain fraction of an otherwise NMD-sensitive mRNA population persists. Accurate prediction of NMD-sensitive transcripts based on sequence information is currently not possible; experimental testing is necessary. NMD modulates the severity of many genetic diseases and affects antigen presentation in cancer cells and the progression of developmental diseases. NMD targets many viral RNAs, but viruses circumvent this by expressing NMD-inhibiting proteins and harboring NMD-suppressing sequence elements in their RNAs

Nanopore sequencing reveals endogenous NMD-targeted isoforms in human cells

Background: Nonsense-mediated mRNA decay (NMD) is a eukaryotic, translation-dependent degradation pathway that targets mRNAs with premature termination codons and also regulates the expression of some mRNAs that encode full-length proteins. Although many genes express NMD-sensitive transcripts, identifying them based on short-read sequencing data remains a challenge. Results: To identify and analyze endogenous targets of NMD, we apply cDNA Nanopore sequencing and short-read sequencing to human cells with varying expression levels of NMD factors. Our approach detects full-length NMD substrates that are highly unstable and increase in levels or even only appear when NMD is inhibited. Among the many new NMD-targeted isoforms that our analysis identifies, most derive from alternative exon usage. The isoform-aware analysis reveals many genes with significant changes in splicing but no significant changes in overall expression levels upon NMD knockdown. NMD-sensitive mRNAs have more exons in the 3΄UTR and, for those mRNAs with a termination codon in the last exon, the length of the 3΄UTR per se does not correlate with NMD sensitivity. Analysis of splicing signals reveals isoforms where NMD has been co-opted in the regulation of gene expression, though the main function of NMD seems to be ridding the transcriptome of isoforms resulting from spurious splicing events. Conclusions: Long-read sequencing enables the identification of many novel NMD-sensitive mRNAs and reveals both known and unexpected features concerning their biogenesis and their biological role. Our data provide a highly valuable resource of human NMD transcript targets for future genomic and transcriptomic applications. Keywords: Long-read sequencing; NMD; Nanopore sequencing; Nonsense-mediated mRNA decay; cDNA sequencing; mRNA degradation; mRNA isoforms; transcriptomics

A subtle alternative splicing event gives rise to a widely expressed human RNase k isoform.

Subtle alternative splicing leads to the formation of RNA variants lacking or including a small number of nucleotides. To date, the impact of subtle alternative splicing phenomena on protein biosynthesis has been studied in frame-preserving incidents. On the contrary, mRNA isoforms derived from frame-shifting events were poorly studied and generally characterized as non-coding. This work provides evidence for a frame-shifting subtle alternative splicing event which results in the production of a novel protein isoform. We applied a combined molecular approach for the cloning and expression analysis of a human RNase κ transcript (RNase κ-02) which lacks four consecutive bases compared to the previously isolated RNase κ isoform. RNase κ-02 mRNA is expressed in all human cell lines tested end encodes the synthesis of a 134-amino-acid protein by utilizing an alternative initiation codon. The expression of RNase κ-02 in the cytoplasm of human cells was verified by Western blot and immunofluorescence analysis using a specific polyclonal antibody developed on the basis of the amino-acid sequence difference between the two protein isoforms. The results presented here show that subtle changes during mRNA splicing can lead to the expression of significantly altered protein isoforms

Human NMD ensues independently of stable ribosome stalling

Nonsense-mediated mRNA decay (NMD) was thought to ensue when ribosomes fail to terminate translation properly. However, the authors observe similar ribosome occupancy at stop codons of NMD sensitive and insensitive mRNAs, showing that human NMD is not activated by stable ribosome stalling as previously suggested

RNase κ-01/RNase κ-02 mRNA ratio in human cell lines.

<p>(A) A histogram of RNase κ mRNA isoforms ratio. Total RNA isolated from 12 human cell lines was reverse transcribed and amplified for 10 cycles by regular PCR. Equal amount of the PCR products from each reaction were incubated in the presence or in the absence of FatI and the samples were re-amplified by Real Time PCR. The comparative ΔC_T analysis performed as described in Materials and Methods resulted in the relative quantification of RNase κ mRNA isoforms. Error bars denote the standard error of the mean of triplicate reactions performed three times for each cell line. (B) The Real Time PCR products from the digested samples were overnight incubated with FatI and analyzed by electrophoresis in a 2% agarose gel. In a parallel experiment, 200 ng of RNAse κ-01 cDNA amplified by the same primers were digested under the same conditions as a control reaction.</p

Immunofluorescence microscopy analysis of RNase κ-02.

<p>HEK-293 cells (A) and RNAi depleted RNase κ-02 HEK-293 cells (B) were treated with K02N specific polyclonal antibody and protein – primary antibody complexes were visualized with rabbit matched Alexa-488 secondary antibody. DNA was stained with propidium iodide (B,E). Merged image of the two stains (C,F).</p

Schematic representation of the hybrid capture protocol for the selection of RNase κ-02 alternative transcript.

<p>The method consists of the following steps: (i) Reverse transcription of poly(A)⁺ mRNA and construction of a single stranded cDNA library. (ii) Hybridization of cDNA molecules with a biotinylated single stranded RNase κ specific DNA probe immobilized on streptavidin magnetic beads. (iii) Complete digestion of the hybrids with an appropriate restriction enzyme. (iv) PCR amplification of the selected target cDNA.</p