detection-tilapia-lake-virus-using-conventional-rt-pcr-sybr-green-rt

Detection of Tilapia Lake Virus Using Conventional RT-PCR and SYBR Green RT-qPCR

The aim of this method is to facilitate the fast, sensitive and specific detection of Tilapia Lake Virus (TiLV) in tilapia tissues. This protocol can be ...

University of Bern

Nonsense-mediated decay is well known by the lucid definition of being a RNA surveillance mechanism that ensures the speedy degradation of mRNAs containing premature translation termination codons. However, as we review here, NMD is far from being a simple quality control mechanism; it also regulates the stability of many wild-type transcripts. We summarise the abundance of research that has characterised each of the NMD factors and present a unified model for the recognition of NMD substrates. The contentious issue of how and where NMD occurs is also discussed, particularly with regard to P-bodies and SMG6-driven endonucleolytic degradation. In recent years, the discovery of additional functions played by several of the NMD factors has further complicated the picture. Therefore, we also review the reported roles of UPF1, SMG1 and SMG6 in other cellular processes.

Nonsense-mediated mRNA decay in human cells: mechanistic insights, functions beyond quality control and the double-life of NMD factors.

In eukaryotes, mRNAs harbouring PTCs (premature translation-termination codons) are recognized and eliminated by NMD (nonsense-mediated mRNA decay). In addition to its quality-control function, NMD constitutes a translation-dependent post-transcriptional pathway to regulate the expression levels of physiological mRNAs. In contrast with PTC recognition, little is known about the mechanisms that trigger the rapid degradation of mammalian nonsense mRNA. Studies have shown that mammalian NMD targets can be degraded via both an SMG6 (where SMG is suppressor of morphological defects on genitalia)-dependent endonucleolytic pathway and a deadenylation and decapping-dependent exonucleolytic pathway, with the possible involvement of SMG5 and SMG7. In contrast, Drosophila melanogaster NMD is confined to the former and Saccharomyces cerevisiae NMD to the latter decay pathway. Consistent with this conclusion, mammals possess both SMG6 and SMG7, whereas D. melanogaster lacks an SMG7 homologue and yeast have no SMG6 equivalent. In the present paper, we review what is known about the degradation of PTC-containing mRNAs so far, paying particular attention to the properties of the NMD-specific factors SMG5-SMG7 and to what is known about the mechanism of degrading mRNAs after they have been committed to the NMD pathway.

Cutting the nonsense: the degradation of PTC-containing mRNAs.

Aberrant mRNAs with premature translation termination codons (PTCs) are recognized and eliminated by the nonsense-mediated mRNA decay (NMD) pathway in eukaryotes. We employed a novel live-cell imaging approach to investigate the kinetics of mRNA synthesis and release at the transcription site of PTC-containing (PTC+) and PTC-free (PTC-) immunoglobulin-μ reporter genes. Fluorescence recovery after photobleaching (FRAP) and photoconversion analyses revealed that PTC+ transcripts are specifically retained at the transcription site. Remarkably, the retained PTC+ transcripts are mainly unspliced, and this RNA retention is dependent upon two important NMD factors, UPF1 and SMG6, since their depletion led to the release of the PTC+ transcripts. Finally, ChIP analysis showed a physical association of UPF1 and SMG6 with both the PTC+ and the PTC- reporter genes in vivo. Collectively, our data support a mechanism for regulation of PTC+ transcripts at the transcription site.

Cotranscriptional effect of a premature termination codon revealed by live-cell imaging.

The nonsense-mediated mRNA decay (NMD) pathway acts to selectively identify and degrade mRNAs that contain a premature translation termination codon (PTC), and hence reduce the accumulation of potentially toxic truncated proteins. NMD is one of the best studied mRNA quality-control mechanisms in eukaryotes, and it has become clear during recent years that many physiological mRNAs are also NMD substrates, signifying a role for NMD beyond mRNA quality control as a translation-dependent post-transcriptional regulator of gene expression. Despite a great deal of scientific research for over twenty years, the process of NMD is far from being fully understood with regard to its physiological relevance to the cell, the molecular mechanisms that underpin this pathway, all of the factors that are involved, and the exact cellular locations of NMD. This unit details some of the fundamental RNA based approaches taken to examine aspects of NMD, such as creating PTC+ reporter genes, knocking down key NMD factors via RNAi, elucidating the important functions of NMD factors by complementation assays or Tethered Function Assays, and measuring RNA levels by reverse-transcription quantitative PCR.

Analysis of nonsense-mediated mRNA decay in mammalian cells.

Animal replication-dependent histone genes produce histone proteins for the packaging of newly replicated genomic DNA. The expression of these histone genes occurs during S phase and is linked to DNA replication via S-phase checkpoints. The histone RNA-binding protein HBP/SLBP (hairpin-binding protein/stem-loop binding protein), an essential regulator of histone gene expression, binds to the conserved hairpin structure located in the 3'UTR (untranslated region) of histone mRNA and participates in histone pre-mRNA processing, translation and histone mRNA degradation. Here, we report the accumulation of alternatively spliced HBP/SLBP transcripts lacking exons 2 and/or 3 in HeLa cells exposed to replication stress. We also detected a shorter HBP/SLBP protein isoform under these conditions that can be accounted for by alternative splicing of HBP/SLBP mRNA. HBP/SLBP mRNA alternative splicing returned to low levels again upon removal of replication stress and was abrogated by caffeine, suggesting the involvement of checkpoint kinases. Analysis of HBP/SLBP cellular localization using GFP (green fluorescent protein) fusion proteins revealed that HBP/SLBP protein and isoforms lacking the domains encoded by exon 2 and exons 2 and 3 were found in the nucleus and cytoplasm, whereas HBP/SLBP lacking the domain encoded by exon 3 was predominantly localised to the nucleus. This isoform lacks the conserved region important for protein-protein interaction with the CTIF [CBP80/20 (cap-binding protein 80/20)]-dependent initiation translation factor and the eIF4E (eukaryotic initiation factor 4E)-dependent translation factor SLIP1/MIF4GD (SLBP-interacting protein 1/MIF4G domain). Consistent with this, we have previously demonstrated that this region is required for the function of HBP/SLBP in cap-dependent translation. In conclusion, alternative splicing allows the synthesis of HBP/SLBP isoforms with different properties that may be important for regulating HBP/SLBP functions during replication stress. 

Replication stress-induced alternative mRNA splicing alters properties of the histone RNA-binding protein HBP/SLBP: a key factor in the control of histone gene expression.

Full genome sequences of 20 strains of , the etiological agent of blackleg of cattle and sheep, isolated from four different continents over a period of 64 years (1951-2015) were determined and analyzed. The study reveals that the genome of the species  is highly homogeneous compared to the closely related species , a widespread pathogen that affects human and many animal species. Analysis of the CRISPR locus is sufficient to differentiate most  strains and is the most heterogenous region in the genome, containing in total 187 different spacer elements that are distributed as 30 - 77 copies in the various strains. Some genetic differences are found in the 3 allelic variants of ,  and  genes that encode structural flagellin proteins, and certain strains do only contain one or two alleles. However, the major virulence genes including the highly toxic , the sialidase and the two hyaluronidases are fully conserved as are the metabolic and structural genes of . These data indicate that  is a strict ruminant-associated pathogen that has reached a dead end in its evolution.

, an Evolutionary Dead-End Pathogen.

Detection of subclinical Brachyspira hyodysenteriae infection in pig herds using feces is challenging. However, the ability to detect the pathogen in intestinal samples of slaughtered pigs has not been investigated, to our knowledge. Therefore, we determined the detection of B. hyodysenteriae in the colon, cecum, and rectum from slaughtered pigs. We analyzed the correlation between detection rates and intestinal lesions, ingesta or fecal consistency, and time from sample collection until processing. A total of 400 ingesta-mucosal (colon, cecum) and 200 fecal (rectum) samples from 200 pigs originating from 20 different herds were bacteriologically examined using selective culture followed by Brachyspira spp. identification by PCR and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Ingesta or fecal consistency and intestinal lesions were scored. Brachyspira hyodysenteriae was detected in 23 samples from 16 intestines originating from 7 herds. Brachyspira spp. were detected in 96 samples. More intestinal (16) than fecal (7) samples tested positive for B. hyodysenteriae. For Brachyspira spp., this difference was significant (69 vs. 27; p < 0.01). In particular, colon samples tested positive ( n = 42, p = 0.06). Most (91%) of the intestines showed no lesions typical for clinical B. hyodysenteriae infection, and median ingesta or fecal consistency was "soft and formed," indicating subclinical infection, colonization, or absence of infection. Ingesta from slaughtered pigs, in particular from the colon and sites with lesions, is useful material for detection of B. hyodysenteriae.

Brachyspira hyodysenteriae detection in the large intestine of slaughtered pigs.

The term "nonsense-mediated mRNA decay" (NMD) originally described the degradation of mRNAs with premature translation-termination codons (PTCs), but its meaning has recently been extended to be a translation-dependent post-transcriptional regulator of gene expression affecting 3%-10% of all mRNAs. The degradation of NMD target mRNAs involves both exonucleolytic and endonucleolytic pathways in mammalian cells. While the latter is mediated by the endonuclease SMG6, the former pathway has been reported to require a complex of SMG5-SMG7 or SMG5-PNRC2 binding to UPF1. However, the existence, dominance, and mechanistic details of these exonucleolytic pathways are divisive. Therefore, we have investigated the possible exonucleolytic modes of mRNA decay in NMD by examining the roles of UPF1, SMG5, SMG7, and PNRC2 using a combination of functional assays and interaction mapping. Confirming previous work, we detected an interaction between SMG5 and SMG7 and also a functional need for this complex in NMD. In contrast, we found no evidence for the existence of a physical or functional interaction between SMG5 and PNRC2. Instead, we show that UPF1 interacts with PNRC2 and that it triggers 5'-3' exonucleolytic decay of reporter transcripts in tethering assays. PNRC2 interacts mainly with decapping factors and its knockdown does not affect the RNA levels of NMD reporters. We conclude that PNRC2 is probably an important mRNA decapping factor but that it does not appear to be required for NMD.

Dissecting the functions of SMG5, SMG7, and PNRC2 in nonsense-mediated mRNA decay of human cells.

Treatment of Swine Dysentery (SD) caused by Brachyspira hyodysenteriae (B. hyodysenteriae) is carried out using antimicrobials such as macrolides, lincosamides and pleuromutilins leading to the selection of resistant strains. Whole genome sequencing of a multidrug-resistant B. hyodysenteriae strain called BH718 belonging to sequence type (ST) 83 revealed the presence of the lincosamide resistance gene lnu(C) on the small 1724-bp transposon MTnSag1. The strain also contains an A to T substitution at position 2058 (A2058T) in the 23S rRNA gene which is known to be associated with macrolide and lincosamide resistance in B. hyodysenteriae. Testing of additional strains showed that those containing lnu(C) exhibited a higher minimal inhibitory concentration (MIC) of lincomycin (MIC ≥ 64 mg/L) compared to strains lacking lnu(C), even if they also harbor the A2058T mutation. Resistance to pleuromutilins could not be explained by the presence of already reported mutations in the 23S rRNA gene and in the ribosomal protein L3. This study shows that B. hyodysenteriae has the ability to acquire mobile genetic elements conferring resistance to antibiotics.

Transposon-associated lincosamide resistance lnu(C) gene identified in Brachyspira hyodysenteriae ST83.

 is a foodborne pathogen that causes abortion, septicemia, gastroenteritis and central nervous system (CNS) infections in ruminants and humans.  strains mainly belong to two distinct phylogenetic groups, named lineages I and II. In general, clinical cases in humans and animals, in particular CNS infections, are caused by lineage I strains, while most of the environmental and food strains belong to lineage II. Little is known about why lineage I is more virulent than lineage II, even though various molecular factors and mechanisms associated with pathogenesis are known. In this study, we have used a variety of whole genome sequence analyses and comparative genomic tools in order to find characteristics that distinguish lineage I from lineage II strains and CNS infection strains from non-CNS strains. We analyzed 225 strains and identified single nucleotide variants between lineages I and II, as well as differences in the gene content. Using a novel approach based on Reads Per Kilobase per Million Mapped (RPKM), we identified 167 genes predominantly absent in lineage II but present in lineage I. These genes are mostly encoding for membrane-associated proteins. Additionally, we found 77 genes that are largely absent in the non-CNS associated strains, while 39 genes are especially lacking in our defined "non-clinical" group. Based on the RPKM analysis and the metadata linked to the  strains, we identified 6 genes potentially associated with CNS cases, which include a transcriptional regulator, an ABC transporter and a non-coding RNA. Although there is not a clear separation between pathogenic and non-pathogenic strains based on phylogenetic lineages, the presence of the genes identified in our study reveals potential pathogenesis traits in ruminant  strains. Ultimately, the differences that we have found in our study will help steer future studies in understanding the virulence mechanisms of the most pathogenic  strains.

Genetic Separation of  Causing Central Nervous System Infections in Animals.

[This corrects the article on p. 1054 in vol. 8, PMID: 28649238.].

Corrigendum: , an Evolutionary Dead-End Pathogen.

Corrigendum to "Transposon-associated lincosamide resistance lnu(C) gene identified in Brachyspira hyodysenteriae ST83" [Vet. Microbiol. 214 (2018) 51-55].

Article	Year
Nonsense-mediated mRNA decay in human cells: mechanistic insights, functions beyond quality control and the double-life of NMD factors. Cellular and molecular life sciences : CMLS\| PubMed ID: 19859661	2010
Cutting the nonsense: the degradation of PTC-containing mRNAs. Biochemical Society transactions\| PubMed ID: 21118136	2010
Cotranscriptional effect of a premature termination codon revealed by live-cell imaging. RNA (New York, N.Y.)\| PubMed ID: 22028363	2011
Analysis of nonsense-mediated mRNA decay in mammalian cells. Current protocols in cell biology\| PubMed ID: 22733442	2012
Replication stress-induced alternative mRNA splicing alters properties of the histone RNA-binding protein HBP/SLBP: a key factor in the control of histone gene expression. Bioscience reports\| PubMed ID: 23941746	2013
, an Evolutionary Dead-End Pathogen. Frontiers in microbiology\| PubMed ID: 28649238	2017
Brachyspira hyodysenteriae detection in the large intestine of slaughtered pigs. Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc\| PubMed ID: 28906177	2018
Dissecting the functions of SMG5, SMG7, and PNRC2 in nonsense-mediated mRNA decay of human cells. RNA (New York, N.Y.)\| PubMed ID: 29348139	2018
Transposon-associated lincosamide resistance lnu(C) gene identified in Brachyspira hyodysenteriae ST83. Veterinary microbiology\| PubMed ID: 29408032	2018
Genetic Separation of Causing Central Nervous System Infections in Animals. Frontiers in cellular and infection microbiology\| PubMed ID: 29459888	2018
Corrigendum: , an Evolutionary Dead-End Pathogen. Frontiers in microbiology\| PubMed ID: 29531522	2018
Corrigendum to "Transposon-associated lincosamide resistance lnu(C) gene identified in Brachyspira hyodysenteriae ST83" [Vet. Microbiol. 214 (2018) 51-55]. Veterinary microbiology\| PubMed ID: 29754947	2018

Pamela Nicholson

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