Pamela Nicholson

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

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.

Cutting the nonsense: the degradation of PTC-containing mRNAs

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.

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

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.

A novel phosphorylation-independent interaction between SMG6 and UPF1 is essential for human NMD

Eukaryotic mRNAs with premature translation-termination codons (PTCs) are recognized and eliminated by nonsense-mediated mRNA decay (NMD). NMD substrates can be degraded by different routes that all require phosphorylated UPF1 (P-UPF1) as a starting point. The endonuclease SMG6, which cleaves mRNA near the PTC, is one of the three known NMD factors thought to be recruited to nonsense mRNAs via an interaction with P-UPF1, leading to eventual mRNA degradation. By artificial tethering of SMG6 and mutants thereof to a reporter mRNA combined with knockdowns of various NMD factors, we demonstrate that besides its endonucleolytic activity, SMG6 also requires UPF1 and SMG1 to reduce reporter mRNA levels. Using in vivo and in vitro approaches, we further document that SMG6 and the unique stalk region of the UPF1 helicase domain, along with a contribution from the SQ domain, form a novel interaction and we also show that this region of the UPF1 helicase domain is critical for SMG6 function and NMD. Our results show that this interaction is required for NMD and for the capability of tethered SMG6 to degrade its bound RNA, suggesting that it contributes to the intricate regulation of UPF1 and SMG6 enzymatic activities.

Detection of Tilapia Lake Virus in Egyptian fish farms experiencing high mortalities in 2015

Institute of Veterinary Bacteriology, University of Bern, Bern, Switzerland WorldFish, Abbassa Research Center, Sharkia, Egypt National Institute of Oceanography and Fisheries, Cairo, Egypt International Livestock Research Institute, Nairobi, Kenya WorldFish, Bayan Lepas, Penang, Malaysia Center for Infection and Immunity, Mailman School of Public Health, Columbia University, New York, NY, USA Institute of Geography, University of Bern, Bern, Switzerland International Livestock Research Institute, Addis Ababa, Ethiopia

Analysis of Nonsense‐Mediated mRNA Decay in Mammalian Cells

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.

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

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.

Brachyspira hyodysenteriae detection in the large intestine of slaughtered pigs

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.

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

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.

Corrigendum: Clostridium chauvoei, an Evolutionary Dead-End Pathogen

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