Evidence that Xrn1 is in complex with Gcn1, and is required for full levels of eIF2α phosphorylation.

The protein kinase Gcn2 and its effector protein Gcn1 are part of the general amino acid control signalling (GAAC) pathway best known in yeast for its function in maintaining amino acid homeostasis. Under amino acid limitation, Gcn2 becomes activated, subsequently increasing the levels of phosphorylated eIF2α (eIF2α-P). This leads to the increased translation of transcriptional regulators, such as Gcn4 in yeast and ATF4 in mammals, and subsequent re-programming of the cell's gene transcription profile, thereby allowing cells to cope with starvation. Xrn1 is involved in RNA decay, quality control and processing. We found that Xrn1 co-precipitates Gcn1 and Gcn2, suggesting that these three proteins are in the same complex. Growth under starvation conditions was dependent on Xrn1 but not on Xrn1-ribosome association, and this correlated with reduced eIF2α-P levels. Constitutively active Gcn2 leads to a growth defect due to eIF2α-hyperphosphorylation, and we found that this phenotype was independent of Xrn1, suggesting that xrn1 deletion does not enhance eIF2α de-phosphorylation. Our study provides evidence that Xrn1 is required for efficient Gcn2 activation, directly or indirectly. Thus, we have uncovered a potential new link between RNA metabolism and the GAAC.

Functional analysis of RYR1 variants in patients with confirmed susceptibility to malignant hyperthermia.

BACKGROUND: A major bottleneck to the introduction of noninvasive presymptomatic diagnostic tests for the pharmacogenetic disorder malignant hyperthermia is the lack of functional data for associated variants. METHODS: We screened 50 genes having a potential role in skeletal muscle calcium homeostasis using the HaloPlex™ (Agilent Technologies, Santa Clara, CA, USA) target enrichment system and next-generation sequencing. Twenty-one patients with a history of a clinical malignant hyperthermia reaction together with a positive in vitro contracture test were included. Eight variants in RYR1 were subsequently introduced into the cDNA for the human ryanodine receptor gene and tested in cultured human embryonic kidney (HEK293) cells for their effect on calcium release from intracellular stores in response to the ryanodine receptor-1 agonist 4-chloro-m-cresol using fura-2 as calcium indicator. Each variant was subjected to in silico curation using the European Malignant Hyperthermia Group scoring matrix and ClinGen RYR1 variant curation expert panel guidelines. RESULTS: Potentially causative RYR1 variants were identified in 15 patients. Of these, two families carried two RYR1 variants, five variants had been previously reported as 'pathogenic', two variants had been previously reported as 'likely benign', and eight were of 'uncertain significance'. Of these eight variants, four showed hypersensitivity to 4-chloro-m-cresol. Three variants were reclassified as either 'pathogenic' or 'likely pathogenic'. Two were classified as 'benign', whilst three remained of 'uncertain significance'. CONCLUSIONS: Three (p.Tyr1711Cys, p.Val2280Ile, and p.Arg4737Gln) additional variants can be added to the list of RYR1 disease-associated variants managed by the European Malignant Hyperthermia Group. These can therefore be used diagnostically in the future. Three variants (p.Glu2348Gly, p.Asn2634Lys, and p.Arg3629Trp) that remained classified as of uncertain significance require further family studies or a different functional test to determine clinical relevance in malignant hyperthermia.

Functional characterization of 2 known ryanodine receptor mutations causing malignant hyperthermia.

BACKGROUND: Malignant hyperthermia (MH) is a potentially lethal pharmacogenetic disorder. More than 300 variants in the ryanodine receptor 1 (RYR1) have been associated with MH; however, only 31 have been identified as causative. To confirm a mutation in RYR1 as being causative for MH, segregation of the potential mutation in at least 2 unrelated families with MH susceptibility must be demonstrated and functional assays must show abnormal calcium release compared with wild-type RYR1. METHODS: We used "Hot-spot" DNA screening to identify mutations in RYR1 in 3 New Zealand families. B-lymphoblastoid cells were used to compare the amount of calcium released on stimulation with 4-chloro-m-cresol between wild-type RYR1 cells and cells carrying the new variants in RYR1. RESULTS: We identified a known RYR1 mutation (R2355W) in 2 families and another more recently identified (V2354M) mutation in another family. Both mutations segregated with MH susceptibility in the respective families. Cell lines carrying a mutation in RYR1 showed increased sensitivity to 4-chloro-m-cresol. CONCLUSIONS: We propose that R2355W is confirmed as being an MH-causative mutation and suggest that V2354M is a RYR1 mutation likely to cause MH.

GCN2 in Viral Defence and the Subversive Tactics Employed by Viruses.

The recent SARS-CoV-2 pandemic and associated COVID19 disease illustrates the important role of viral defence mechanisms in ensuring survival and recovery of the host or patient. Viruses absolutely depend on the host's protein synthesis machinery to replicate, meaning that impeding translation is a powerful way to counteract viruses. One major approach used by cells to obstruct protein synthesis is to phosphorylate the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α). Mammals possess four different eIF2α-kinases: PKR, HRI, PEK/PERK, and GCN2. While PKR is currently considered the principal eIF2α-kinase involved in viral defence, the other eIF2α-kinases have also been found to play significant roles. Unsurprisingly, viruses have developed mechanisms to counteract the actions of eIF2α-kinases, or even to exploit them to their benefit. While some of these virulence factors are specific to one eIF2α-kinase, such as GCN2, others target all eIF2α-kinases. This review critically evaluates the current knowledge of viral mechanisms targeting the eIF2α-kinase GCN2. A detailed and in-depth understanding of the molecular mechanisms by which viruses evade host defence mechanisms will help to inform the development of powerful anti-viral measures.

Rapid yeast-based screen for Functionally Relevant Amino Acids (RS-FRAA) in a protein.

Here, we describe a fast and cost-effective procedure to generate a large array of mutant proteins and immediately screen for those with altered protein function. This protocol is a modification from three existing approaches: fusion PCR, Saccharomyces cerevisiae in-yeast recombination, and semi-quantitative growth assays. We also describe a mating step to reduce the occurrence of false positive findings due to ectopic mutations. The only requirement is that the protein elicits a phenotype in Saccharomyces cerevisiae.

A genetic approach to identify amino acids in Gcn1 required for Gcn2 activation.

The protein kinase Gcn2 is present in virtually all eukaryotic cells. It is best known for its role in helping cells cope with amino acid starvation. Under starvation, Gcn2 phosphorylates the α subunit of the eukaryotic translation initiation factor 2 (eIF2α), to stimulate a signal transduction pathway that allows cells to cope and overcome starvation. Gcn2 has been implicated in many additional biological functions. It appears that for all functions, Gcn2 must directly bind to its effector protein Gcn1, mediated via a region in Gcn1 called the RWD binding domain (RWDBD). Arg-2259 in this region is important for Gcn2 binding. Overexpression of a Gcn1 fragment only encompassing the RWDBD binds Gcn2, thereby disrupting endogenous Gcn1-Gcn2 interaction which dampens Gcn2 activation. Consequently, cells are unable to increase eIF2α phosphorylation under starvation conditions, visible by impaired growth. This dominant negative phenotype is reverted by the R2259A substitution, again allowing Gcn1-Gcn2 interaction and enhanced eIF2α phosphorylation. We have found that the amino acid substitutions, R2289A, R2297A, and K2301A, also reverted the dominant negative phenotype as well as allowed enhanced eIF2α phosphorylation, as found previously for the R2259A substitution. This suggests that the respective amino acids are relevant for the overexpressed RWDBD to disrupt Gcn1-Gcn2 interaction and impair Gcn2 activation, supporting the idea that in Gcn1 these amino acids mediate Gcn2-binding. Our findings suggest that two helices in Gcn1 constitute a Gcn2 binding site. We serendipitously found amino acid substitutions that enhanced the dominant negative phenotype that correlated with a further reduction in eIF2α-P levels, suggesting that the respective RWDBD variants are more potent in disrupting Gcn1-Gcn2 interaction.

Organisation and expression of a cluster of yolk protein genes in the Australian sheep blowfly, Lucilia cuprina.

The Australian sheep blowfly Lucilia cuprina is a major pest for the Australian and New Zealand sheep industries. With the long-term aim of making a strain of L. cuprina suitable for a genetic control program, we previously developed a tetracycline-repressible female lethal genetic system in Drosophila. A key part of this system is a female-specific promoter from a yolk protein (yp) gene controlling expression of the tetracycline-dependent transactivator (tTA). Here we report the sequence of a 14.2 kb genomic clone from L. cuprina that contains a cluster of three complete yp genes and one partial yp gene. The Lcyp genes are specifically expressed in females that have received a protein meal. A bioinformatic analysis of the promoter of one of the yp genes (LcypA) identified several putative binding sites for DSX, a known regulator of yp gene expression in other Diptera. A transgenic strain of L. cuprina was made that contained the LcypA promoter driving the expression of the Escherichia coli lacZ reporter gene. Transgenic females express high levels of ß-galactosidase after a protein meal. Thus the LcypA promoter could be used to obtain female-specific expression of tTA in transgenic L. cuprina.

Sex-biased transcription enhancement by a 5' tethered Gal4-MOF histone acetyltransferase fusion protein in Drosophila.

BACKGROUND: In male Drosophila melanogaster, the male specific lethal (MSL) complex is somehow responsible for a two-fold increase in transcription of most X-linked genes, which are enriched for histone H4 acetylated at lysine 16 (H4K16ac). This acetylation requires MOF, a histone acetyltransferase that is a component of the MSL complex. MOF also associates with the non-specific lethal or NSL complex. The MSL complex is bound within active genes on the male X chromosome with a 3' bias. In contrast, the NSL complex is enriched at promoter regions of many autosomal and X-linked genes in both sexes. In this study we have investigated the role of MOF as a transcriptional activator. RESULTS: MOF was fused to the DNA binding domain of Gal4 and targeted to the promoter region of UAS-reporter genes in Drosophila. We found that expression of a UAS-red fluorescent protein (DsRed) reporter gene was strongly induced by Gal4-MOF. However, DsRed RNA levels were about seven times higher in female than male larvae. Immunostaining of polytene chromosomes showed that Gal4-MOF co-localized with MSL1 to many sites on the X chromosome in male but not female nuclei. However, in female nuclei that express MSL2, Gal4-MOF co-localized with MSL1 to many sites on polytene chromosomes but DsRed expression was reduced. Mutation of conserved active site residues in MOF (Glu714 and Cys680) reduced HAT activity in vitro and UAS-DsRed activation in Drosophila. In the presence of Gal4-MOF, H4K16ac levels were enriched over UAS-lacZ and UAS-arm-lacZ reporter genes. The latter utilizes the constitutive promoter from the arm gene to drive lacZ expression. In contrast to the strong induction of UAS-DsRed expression, UAS-arm-lacZ expression increased by about 2-fold in both sexes. CONCLUSIONS: Targeting MOF to reporter genes led to transcription enhancement and acetylation of histone H4 at lysine 16. Histone acetyltransferase activity was required for the full transcriptional response. Incorporation of Gal4-MOF into the MSL complex in males led to a lower transcription enhancement of UAS-DsRed but not UAS-arm-lacZ genes. We discuss how association of Gal4-MOF with the MSL or NSL proteins could explain our results.

The importance of location and orientation of male specific lethal complex binding sites of differing affinities on reporter gene dosage compensation in Drosophila.

The male specific lethal (MSL) complex is required for X chromosome dosage compensation in Drosophila. The complex binds to most actively transcribed X-linked genes in males and upregulates expression. High resolution chromatin immunoprecipitation assays have identified over one hundred high affinity binding sites on the X chromosome. One of the first high affinity sites discovered is at cytological location 18D11. The MSL complex binds weakly to a single copy of a 510bp fragment from 18D11 but strongly to a tetramer of the fragment. Here we have investigated the effect of insertion of sites of differing affinities, either upstream or within the transcribed gene, on complex binding and transcription upregulation. Insertion of four copies of the 18D11 fragment upstream or at the 3' end of a reporter gene led to strong MSL complex binding and increased expression in males. In contrast, the MSL complex did not bind consistently to autosomal transgenes that contained a single copy of the 18D11 site upstream of the gene promoter. However, MSL complex binding was observed in all lines if the single 18D11 fragment was inserted into the 3' end of the reporter gene in either orientation. This is consistent with previous studies that showed gene transcription facilitates MSL complex binding. Surprisingly, transcription elevation in males was only observed if the 18D11 fragment was in the forward orientation and only in some lines. Our results suggest that MSL complex binding to weaker sites and transcription enhancement is influenced by gene transcription, binding site orientation and the local chromatin environment. In contrast, strong binding sites do not need to be transcribed to recruit sufficient complex to cause transcription elevation of nearby genes.

Identification and Functional Analysis of RYR1 Variants in a Family with a Suspected Myopathy and Associated Malignant Hyperthermia.

BACKGROUND: The ryanodine receptor 1 (RyR1) is a major skeletal muscle calcium release channel located in the sarcoplasmic reticulum and involved in excitation-contraction coupling. Variants in the gene encoding RyR1 have been linked to a range of neuromuscular disorders including myopathies and malignant hyperthermia (MH). OBJECTIVE: We have identified three RYR1 variants (c.1983 G>A, p.Trp661*; c.7025A>G, p.Asn2342Ser and c.2447 C>T, p.Pro816Leu) in a family with a suspected myopathy and associated malignant hyperthermia susceptibility. We used calcium release assays to functionally characterise these variants in a recombinant system. METHODS: Site-directed mutagenesis was used to introduce each variant separately into the human RYR1 cDNA. HEK293-T cells were transfected with the recombinant constructs and calcium release assays were carried out using 4-chloro-m-cresol (4-CmC) as the RyR1 agonist to investigate the functional consequences of each variant. RESULTS: RYR1 c.1983 G>A, p.Trp661* resulted in a non-functional channel, c.7025A>G, p.Asn2342Ser in a hypersensitive channel and c.2447 C>T, p.Pro816Leu in a hypersensitive channel at higher concentrations of 4-CmC. CONCLUSIONS: The p.Trp661* RYR1 variant should be considered as a risk factor for myopathies. The p.Asn2342Ser RYR1 variant, when expressed as a compound heterozygote with a nonsense mutation on the second allele, is likely to result in MH-susceptibility. The role of the p.Pro816Leu variant in MH remains unclear.

Masseter muscle rigidity and the role of DNA analysis to confirm malignant hyperthermia susceptibility.

Malignant hyperthermia (MH) is an uncommon, autosomal dominant disorder of skeletal muscle, triggered by inhalational anaesthetics or depolarizing muscle relaxants. Masseter muscle rigidity (MMR) can be regarded as potentially a preceding sign for an MH reaction. Susceptibility to MH can be determined by the in vitro contracture test (IVCT) or DNA analysis where a familial variant is known. Our aims were to review patients with MMR, where IVCT and DNA analysis had been undertaken, to determine if DNA analysis could be used as an initial screening tool for MH susceptibility, and, by reviewing standard monitored variables (SMVs), to determine if any clinical characteristics could be used to differentiate between MMR patients who are MH susceptible (MHS) and those who are not. Patients with MMR were identified from the Palmerston North Hospital MH Reactions Database. IVCT and DNA analysis results were documented. DNA testing was performed retrospectively in the majority of patients as many patients had presented before DNA analysis was available. Forty-one patients were analysed. Fourteen were DNA positive/IVCT positive and six DNA positive only (48% in total), seven were IVCT positive/DNA negative and 14 were IVCT normal. Increased creatine kinase (>18,000 units/L) was consistent with MH susceptibility. Severity of MMR was not linked to MH susceptibility. This study confirmed that DNA analysis can be used as a first-line test for MH susceptibility in patients presenting with MMR (consistent with European MH Group recommendations). Creatine kinase was the only SMV that was significantly different between MHS and MH normal individuals.

Functional Characterization of C-terminal Ryanodine Receptor 1 Variants Associated with Central Core Disease or Malignant Hyperthermia.

BACKGROUND: Central core disease and malignant hyperthermia are human disorders of skeletal muscle resulting from aberrant Ca2+ handling. Most malignant hyperthermia and central core disease cases are associated with amino acid changes in the type 1 ryanodine receptor (RyR1), the skeletal muscle Ca2+-release channel. Malignant hyperthermia exhibits a gain-of-function phenotype, and central core disease results from loss of channel function. For a variant to be classified as pathogenic, functional studies must demonstrate a correlation with the pathophysiology of malignant hyperthermia or central core disease. OBJECTIVE: We assessed the pathogenicity of four C-terminal variants of the ryanodine receptor using functional analysis. The variants were identified in families affected by either malignant hyperthermia or central core disease. METHODS: Four variants were introduced separately into human cDNA encoding the skeletal muscle ryanodine receptor. Following transient expression in HEK-293T cells, functional studies were carried out using calcium release assays in response to an agonist. Two previously characterized variants and wild-type skeletal muscle ryanodine receptor were used as controls. RESULTS: The p.Met4640Ile variant associated with central core disease showed no difference in calcium release compared to wild-type. The p.Val4849Ile variant associated with malignant hyperthermia was more sensitive to agonist than wild-type but did not reach statistical significance and two variants (p.Phe4857Ser and p.Asp4918Asn) associated with central core disease were completely inactive. CONCLUSIONS: The p.Val4849Ile variant should be considered a risk factor for malignant hyperthermia, while the p.Phe4857Ser and p.Asp4918Asn variants should be classified as pathogenic for central core disease.

Functional analysis of RYR1 variants linked to malignant hyperthermia.

Malignant hyperthermia manifests as a rapid and sustained rise in temperature in response to pharmacological triggering agents, e.g. inhalational anesthetics and the muscle relaxant suxamethonium. Other clinical signs include an increase in end-tidal CO2, increased O2 consumption, as well as tachycardia, and if untreated a malignant hyperthermia episode can result in death. The metabolic changes are caused by dysregulation of skeletal muscle Ca2+ homeostasis, resulting from a defective ryanodine receptor Ca2+ channel, which resides in the sarcoplasmic reticulum and controls the flux of Ca2+ ions from intracellular stores to the cytoplasm. Most genetic variants associated with susceptibility to malignant hyperthermia occur in the RYR1 gene encoding the ryanodine receptor type 1. While malignant hyperthermia susceptibility can be diagnosed by in vitro contracture testing of skeletal muscle biopsy tissue, it is advantageous to use DNA testing. Currently only 35 of over 400 potential variants in RYR1 have been classed as functionally causative of malignant hyperthermia and thus can be used for DNA diagnostic tests. Here we describe functional analysis of 2 RYR1 variants (c. 7042_7044delCAG, p.ΔGlu2348 and c.641C>T, p.Thr214Met) that occur in the same malignant hyperthermia susceptible family. The p.Glu2348 deletion, causes hypersensitivity to ryanodine receptor agonists using in vitro analysis of cloned human RYR1 cDNA expressed in HEK293T cells, while the Thr214Met substitution, does not appear to significantly alter sensitivity to agonist in the same system. We suggest that the c. 7042_7044delCAG, p.ΔGlu2348 RYR1 variant could be added to the list of diagnostic mutations for susceptibility to malignant hyperthermia.

Efficient germ-line transformation of the economically important pest species Lucilia cuprina and Lucilia sericata (Diptera, Calliphoridae).

The green blowfly species Lucilia cuprina and Lucilia sericata are economically important pests for the sheep industries of Australia and New Zealand. L. cuprina has long been considered a good target for a genetic pest management program. In addition, L. sericata maggots are used in the cleaning of wounds and necrotic tissue of patients suffering from ulcers that are difficult to treat by other methods. Development of efficient transgenesis methods would greatly facilitate the development of strains ideal for genetic control programs or could potentially improve "maggot therapy". We have previously reported the germ-line transformation of L. cuprina and the design of a "female killing system" that could potentially be applied to this species. However, the efficiency of transformation obtained was low and transformed lines were difficult to detect due to the low expression of the EGFP marker used. Here we describe an efficient and reliable method for germ-line transformation of L. cuprina using new piggyBac vector and helper plasmids containing the strong promoter from the L. cuprina hsp83 gene to drive expression of the transposase and fluorescent protein marker gene. We also report, for the first time, the germ-line transformation of L. sericata using the new piggyBac vector/helper combination.

Incorporation of the noncoding roX RNAs alters the chromatin-binding specificity of the Drosophila MSL1/MSL2 complex.

The male-specific lethal (MSL) protein-RNA complex is required for X chromosome dosage compensation in Drosophila melanogaster. The MSL2 and MSL1 proteins form a complex and are essential for X chromosome binding. In addition, the MSL complex must integrate at least one of the noncoding roX RNAs for normal X chromosome binding. Here we find the amino-terminal RING finger domain of MSL2 binds as a complex with MSL1 to the heterochromatic chromocenter and a few sites on the chromosome arms. This binding required the same amino-terminal basic motif of MSL1 previously shown to be essential for binding to high-affinity sites on the X chromosome. While the RING finger domain of MSL2 is sufficient to increase the expression of roX1 in females, activation of roX2 requires motifs in the carboxyl-terminal domain. Binding to hundreds of sites on the X chromosome and efficient incorporation of the roX RNAs into the MSL complex require proline-rich and basic motifs in the carboxyl-terminal domain of MSL2. We suggest that incorporation of the roX RNAs into the MSL complex alters the binding specificity of the chromatin-binding module formed by the amino-terminal domains of MSL1 and MSL2.

Essentiality of the early transcript in the replication origin of the lactococcal prolate phage c2.

The genome of the prolate-headed lytic lactococcal bacteriophage c2 is organized into two divergently oriented blocks consisting of the early genes and the late genes. These blocks are separated by the noncoding origin of DNA replication. We examined the functional role of transcription of the origin in a plasmid model system. Deletion of the early promoter P(E)1 abolished origin function. Introduction of mutations into P(E)1 which did not eliminate promoter activity or replacement of P(E)1 with an unrelated but functional promoter did not abolish replication. The A-T-rich region upstream of P(E)1, which is conserved in prolate phages, was not required for plasmid replication. Replacement of the P(E)1 transcript template sequence with an unrelated sequence with a similar G+C content abolished replication, showing that the sequence encoding the transcript is essential for origin function. Truncated transcript and internal deletion constructs did not support replication except when the deletion was at the very 3' end of the DNA sequence coding for the transcript. The P(E)1 transcript could be detected for all replication-proficient constructs. Recloning in a plasmid vector allowed detection of P(E)1 transcripts from some fragments that did not support replication, indicating that stability of the transcript alone was not sufficient for replication. The data suggest that production of a transcript of a specific length and with a specific sequence or structure is essential for the function of the phage c2 origin in this model system.

Sequence diversity and functional conservation of the origin of replication in lactococcal prolate phages.

Prolate or c2-like phages are a large homologous group of viruses that infect the bacterium Lactococcus lactis. In a collection of 122 prolate phages, three distinct, non-cross-hybridizing groups of origins of DNA replication were found. The nonconserved sequence was confined to the template for an untranslated transcript, P(E)1-T, 300 to 400 nucleotides in length, while the flanking sequences were conserved. All three origin types, despite the low sequence homology, have the same functional characteristics: they express abundant P(E)1-T transcripts and can function as origins of plasmid replication in the absence of phage proteins. Using chimeric constructs, we showed that hybrids of two nonhomologous origin sequences failed to function as replication origins, suggesting that preservation of a particular secondary structure of the P(E)1-T transcript is required for replication. This is the first systematic survey of the sequence and function of origins of replication in a group of lactococcal phages.