Methionine Codon Research Articles

Sequence and phylogenetic analysis of a novel alphaendornavirus, the first virus described from the oomycete plant pathogen Phytophthora heveae

Here, we report the discovery and complete genome sequence of a novel virus, designated as “Phytophthora heveae alphaendornavirus 1” (PhAEV1), from a single isolate of the plant pathogenic oomycete Phytophthora heveae (kingdom Stramenipila) isolated from a tropical evergreen lowland rainforest in northern Vietnam. PhAEV1 was detected by both cellulose affinity chromatography of dsRNA and high-throughput sequencing of total RNA, and its presence and sequence were confirmed by RT-PCR and Sanger sequencing. The PhAEV1 genome, 12,820 nucleotides (nt) in length, was predicted to encode a single large polyprotein with the catalytic core domain of viral (superfamily 1) RNA helicase (HEL, amino acid [aa] positions 1,287-1,531), glycosyltransferase (GT, aa positions ca. 2,800-3,125), and RNA-directed RNA polymerase (RdRp, aa positions 3,875-4,112). PhAEV1 is the most similar to Phytophthora cactorum alphaendornavirus 3, sharing 39.4% and 39.1% nt and aa sequence identity, respectively. In addition to the first 5′-terminal AUG codon, three additional in-frame methionine codons were found in close proximity (nt 14-16, 96-98, and 176-178), suggesting potential additional translation initiation sites. Conserved RdRp motifs (A-E) similar to those detected in related endornaviruses were identified in PhAEV1, as well as in several previously described alphaendornaviruses from other Phytophthora species in which these motifs had not been identified previously. Phylogenetic analysis showed that PhAEV1 clusters with members of the genus Alphaendornavirus in the family Endornaviridae and is basal to two other alphaendornaviruses described from another oomycete, Phytophthora cactorum. PhAEV1 is the first virus reported in P. heveae.

Analysis of Viral Promoters for Transgene Expression and of the Effect of 5'-UTRs on Alternative Translational Start Sites in Chlamydomonas.

Microalgae biotechnology has the potential to produce high quality bioproducts in a sustainable manner. Here, Chlamydomonas reinhardtii has shown great potential as a host for biotechnological exploitation. However, low expression of nuclear transgenes is still a problem and needs to be optimized. In many model organisms, viral promoters are used to drive transgene expression at high levels. However, no viruses are known to infect Chlamydomonas, and known viral promoters are not functional. Recently, two different lineages of giant viruses were identified in the genomes of Chlamydomonas reinhardtii field isolates. In this work, we tested six potentially strong promoters from these viral genomes for their ability to drive transgene expression in Chlamydomonas. We used ble, NanoLUC, and mCherry as reporter genes, and three native benchmark promoters as controls. None of the viral promoters drove expression of any reporter gene beyond background. During our study, we found that mCherry variants are produced by alternative in-frame translational start sites in Chlamydomonas. We show that this problem can be overcome by mutating the responsible methionine codons to codons for leucine and by using the 5'-UTR of βTUB2 instead of the 5'-UTRs of PSAD or RBCS2. Apparently, the βTUB2 5'-UTR promotes the use of the first start codon. This could be mediated by the formation of a stem-loop between sequences of the βTUB2 5'-UTR and sequences downstream of the first AUG in the mCherry reporter, potentially increasing the dwell time of the scanning 40S subunit on the first AUG and thus decreasing the probability of leaky scanning.

Substrate recognition and near-cognate discrimination by the wobble-base modifying enzyme tRNA-isoleucine lysidine synthase (TilS).

TilS is a bacterial exclusive wobble-base modifying enzyme that facilitates proper decoding of the rare AUA codon. Because of wobble possibilities, a traditional tRNAIle2 minor acceptor with a UAU anticodon would pair with its corresponding AUA (isoleucine) codon, but could also pair with the incorrect AUG (methionine) codon. This would result in inaccuracies during protein biosynthesis. To circumvent this problem, bacteria have mostly evolved to instead encode a tRNAIle2 that contains a CAU anticodon which is further post-transcriptionally modified by the installation of a lysine moiety at the C34 position, resulting in the final lysidine modification and the corresponding L34AU anticodon. Due to steric clashing of the lysidine modification and the C2 amine group of guanine, base pairing with AUG is prohibited, leaving AUA as the only pairing option.

Investigating the effects of the Geobacillus kaustophilus TilS lysidine modification on accurate mRNA decoding and aminoacylation.

Accurate protein biosynthesis requires translational fidelity and decoding efficiency. Mistranslated proteins are susceptible to misfolding, aggregation, or degradation; often resulting in disease. Accurate protein biosynthesis is expedited by attaching the correct amino acid to the 3′ end of the corresponding tRNA. Aminoacyl-tRNA synthetase (aaRS) enzymes catalyze the covalent linkage of amino acids to the cognate tRNA during translation. Due to the importance of accuracy, these enzymes must be specific in which tRNA substrates they utilize. Wobble-base pairing can occur between tRNAIle2UAU and the AUG methionine codon. Thus, evolution has dictated tRNAIle2 minor isoacceptors that are transcribed with a CAU codon. Typically, tRNAIle2 is encoded with the CAU anticodon; however, the same codon is utilized by tRNAMet when decoding the AUG codon.In bacterial systems, a post-transcriptional modification of C34 of tRNAIle2CAU facilitates accurate protein biosynthesis as aaRS's use the lysidine modification as an identity element for recognition and proper charging. The tRNA isoleucine lysidine synthetase enzyme (TilS) modifies the wobble position of tRNAIle2 precursors generating a LAU codon. This prevents methionyl tRNA synthetase (MetRS) mischarging via steric hindrances and modified tRNAIle2LAU is ultimately charged by IleRS. This work attempts to further characterize Geobacillus kaustophilus TilS by exploring the effects of distal residue mutations on lysidinylation function and investigating the recognition capacity of GkMetRS and GkIleRS for both cognate and noncognate tRNA substrates. Exploring the impact of the lysidine modification on charging efficiency will provide an increased understanding of aaRS discrimination and codon recognition mechanisms.

Deletion of the initial methionine codon of the Tmem95 gene causes subfertility, but not complete infertility, in male mice.

Dear Editor,In mammals, sperm izumo sperm-egg fusion 1 (IZUMO1) and oocyte IZUMO1 receptor (JUNO) interaction plays a pivotal role in the recognition of gametes prior to membrane fusion through multiple intermolecular reactions [1–3]. Although many of the factors involved in gamete fusion have recently been identified, their specific contributions remain largely unknown [1]. Among them, transmembrane protein 95 (TMEM95) was initially identified as a responsible gene for male subfertility in Fleckvieh bulls by a genome-wide association study [4, 5]. Furthermore, in 2020, two research teams reported that CRISPR/Cas9-mediated Tmem95 disruption caused complete sterility in male mice due to gamete fusion failure [6, 7]. In order to analyze the phenotype, we newly produced a TMEM95 knockout mouse line. We designed a single guide RNA to target the initial methionine codon for the Tmem95 gene since this gene has some alternative splicing...

VEXAS syndrome : when do we have to consider it ?

VEXAS syndrome was recently discovered in patients who developed late in adulthood an inflammatory syndrome with fever, cytopenias, dysplastic bone marrow, cutaneous and pulmonary neutrophilic inflammation, arthritis, chondritis, or vasculitis. It is the result of an inactivating somatic mutation affecting methionine codon 41 of the UBA1 gene which encodes an ubiquitin activating enzyme (E1). Systemic corticosteroids generally reduce symptoms, while other immunosuppressive drugs only have limited long-term effects. Azacitidine is a promising treatment, but further studies are warranted. Here, we describe 2 new cases including one associated with pyoderma gangrenosum and cryoglobulinemia.

A computational screen for alternative genetic codes in over 250,000 genomes.

The genetic code has been proposed to be a 'frozen accident,' but the discovery of alternative genetic codes over the past four decades has shown that it can evolve to some degree. Since most examples were found anecdotally, it is difficult to draw general conclusions about the evolutionary trajectories of codon reassignment and why some codons are affected more frequently. To fill in the diversity of genetic codes, we developed Codetta, a computational method to predict the amino acid decoding of each codon from nucleotide sequence data. We surveyed the genetic code usage of over 250,000 bacterial and archaeal genome sequences in GenBank and discovered five new reassignments of arginine codons (AGG, CGA, and CGG), representing the first sense codon changes in bacteria. In a clade of uncultivated Bacilli, the reassignment of AGG to become the dominant methionine codon likely evolved by a change in the amino acid charging of an arginine tRNA. The reassignments of CGA and/or CGG were found in genomes with low GC content, an evolutionary force that likely helped drive these codons to low frequency and enable their reassignment.

An Engineered Escherichia coli Strain with Synthetic Metabolism for in-Cell Production of Translationally Active Methionine Derivatives.

In the last decades, it has become clear that the canonical amino acid repertoire codified by the universal genetic code is not up to the needs of emerging biotechnologies. For this reason, extensive genetic code re‐engineering is essential to expand the scope of ribosomal protein translation, leading to reprogrammed microbial cells equipped with an alternative biochemical alphabet to be exploited as potential factories for biotechnological purposes. The prerequisite for this to happen is a continuous intracellular supply of noncanonical amino acids through synthetic metabolism from simple and cheap precursors. We have engineered an Escherichia coli bacterial system that fulfills these requirements through reconfiguration of the methionine biosynthetic pathway and the introduction of an exogenous direct trans‐sulfuration pathway. Our metabolic scheme operates in vivo, rescuing intermediates from core cell metabolism and combining them with small bio‐orthogonal compounds. Our reprogrammed E. coli strain is capable of the in‐cell production of l‐azidohomoalanine, which is directly incorporated into proteins in response to methionine codons. We thereby constructed a prototype suitable for economic, versatile, green sustainable chemistry, pushing towards enzyme chemistry and biotechnology‐based production.

Alternately Translated Product of Cx37 Fails to Arrest Growth in Rin Cells but Alters Cx37 Hemichannel Function when Co‐Expressed

Connexin proteins facilitate transmembrane and intercellular communication via channel formation (hemichannel) and intercellular docking of apposed hemichannels to form gap junction channels, respectively. Additionally, they play a role in determining growth phenotype (e.g., arrest, proliferation, death). Connexin 37 (Cx37), expressed in connexin‐deficient rat insulinoma (Rin) cells induces growth arrest or cell death in a manner dependent on the phosphorylation of specific carboxyl terminus (CT) residues. Notably, phosphorylation at serine 319 (S319) is both necessary and sufficient for growth arrest, and mimicking phosphorylation with aspartate substitution at either S275 or S321 is sufficient to induce cell death. 13 of the 21 members of the connexin gene family have a conserved methionine at position 213; in Cx43 mRNA, this methionine codon serves as an alternate translation start site that produces a CT‐spanning translation product. This 20kDa (20k) product interacts with Cx43, chaperoning its trafficking to the membrane. Here we test the hypotheses that 1) this conserved codon in Cx37 mRNA also serves as an alternate translation start site to produce a 13kDa product (13k) that, when expressed alone, regulates growth phenotype of Rin cells and 2) when co‐expressed with Cx37, regulates its channel function. To test these hypotheses, we expressed V5‐tagged 13k or one of two phosphomimetic mutants, 13k‐S321D and 13k‐S275D, in Rin cells or in Rin cells expressing Cx37. Despite a similar phosphorylation profile to Cx37, as determined by mass spectrometry, 13k did not arrest Rin cell growth and neither phosphomimetic mutant induced cell death nor growth arrest. In co‐expressing cells, immunoprecipitation (IP) of 13k failed to co‐IP full‐length Cx37, suggesting no direct interaction between these isoforms; however, 13k and the phosphomimetic mutants altered Cx37 hemichannel conductance and open probability properties, suggesting an indirect interaction of 13k isoforms with Cx37. In conclusion, these data suggest that while 13k and phosphomimetic mutants alone are insufficient to alter the growth phenotype of Rin cells toward growth arrest or death, they nevertheless lead to altered channel properties of full‐length Cx37. The findings herein will inform further research in understanding how connexins are regulated and, in turn, the role they play in regulating cell growth phenotypes.Support or Funding InformationR01 HL131712 | Undergraduate Biology Research Program | University Funds

Dissecting Contributions to Efficient Catalysis in the tRNA Modifying Enzyme TilS

Accurate protein synthesis is essential for cell viability, as mistranslation events can affect protein structure, function, and regulation. Bacteria do not translate the rare AUA isoleucine codon with a tRNAIleUAU, as this would wobble pair with the AUG methionine codon and lead to translational ambiguity. Instead, most bacteria use a lysidine modification at the tRNAIle2 wobble position. tRNA isoleucine lysidine synthetase (TilS) installs a lysidine moiety at the wobble position of tRNAIle2CAU, resulting in a tRNAIle2LAU that preferentially pairs with the AUA codon, resulting in accurate charging of the tRNA with isoleucine and decoding at the ribosome. Evolved mutants of Burkholderia cenocepacia TilS have been observed that contribute to a gain of fitness in nutrient-depleted media. Despite the gain of fitness observed, the mutants display a loss of lysidinylation function both in vitro and in vivo. We target catalytically relevant regions of TilS for mutagenesis and functional assays, and model enzyme dynamics in silico. Using all of this knowledge in correspondence, the contribution of these evolutionary mutants can be elucidated.

The Translational Switch of MYC Protein Aliases in Myeloma Tumor Cells

Our systematic investigation on MYC (C-Myc) - a crucial pro-regeneration transcription factor - has determined that genetic aberrations (chromosomal rearrangements) and overexpression of the gene are highly prevalent in multiple myeloma (MM). At diagnosis, the rearrangements of MYC locus (8q24) were detected in 51% of MM patients using karyotyping and interphase FISH with DNA probe sets to MYC and the flank genomic sequences. The abnormalities of 8q24 in MM include monosomy (5%), amplification (≥3 copies; 13%), break-apart (14%), jumping (11%), and others (8%). Such aberrations were present in 49% of low-risk MM (183/376) and in 62% of high-risk MM (51/82) defined by the gene expression profiling cancer-risk model (GEP-70). The GEP also indicated that 74% of MM patients had excessively high levels of MYC overexpression. Nonetheless, there was no significant correlation between the MYC cytogenetic abnormalities and the levels of MYC expression in myeloma tumor cells - unlike the primary IGH translocations that drive transcriptional spikes of its partner genes. MYC expression by GEP was unable to precisely predict the presence of MYC chromosomal rearrangements in MM. In fact, the clinical outcomes of MM patients with MYC aberrations vs. the patients without detectable 8q24 events showed that neither the numerical variations nor the structural rearrangements of 8q24 had significant impact on the overall survival of patients who were treated on Total Therapy protocols. Further, we have shown that chromosomal rearrangements and the levels of MYC overexpression were not intrinsically relevant to the translation of MYC protein in myeloma. Importantly, the fate of myeloma cells was associated with the presence of two endogenous polypeptide-isoforms of MYC - the canonical MYC protein alias (P01106; 48.8kD) and an alternative MYC protein alias (alt-MYC; > 50kD). In newly diagnosed MM, P01106 was absent in most of the patients; whereas, it was a constitutive alias well maintained across the myeloma tumor cells from the patients with relapsed and progressive diseases and in all of the self-renewable myeloma cell lines. In contrast, alt-MYC alias was present as a stand-alone alias or co-existing with P01106 alias in newly diagnosed myeloma. Most importantly, both MYC aliases were absent in some cell lines and primary tumor cells suggesting cancer cell growth may not be dependent on any of MYC aliases despite of high levels of MYC transcription. Since the MYC gene originated from a viral integration, MYC mRNA has dual-translation features due to an internal ribosome entry segment (IRES) at 5'UTR. Previous studies have determined that the translations can be alternatively initiated from an in-frame CUG (Leucine) codon to generate NP_002458 alias or at a downstream canonical cap-dependent AUG (Methionine) codon to generate P01106 alias. Although these two aliases share identical C-termini, the translational switch can extend the N-terminal in NP_002458 alias. In vitro, the switch of P01106 to alt-MYC has resulted in cell growth arrest. Such alternation was induced by a transient gene transfection of P53 tumor suppressor (TP53); seemingly, the alternative MYC alias acts as an antagonist to P01106 in cell proliferation. Overall, it was generally unpredictable which of the MYC aliases were the dominant driver; therefore, MYC cytogenetic rearrangements and overexpression may have unknown indirect impacts on the outcomes. We are currently examining the precise amino acid sequences on both N-terminal and C-terminal ends of MYC aliases to study the regulatory mechanisms of the translational switching. This may indicate a novel anti-cancer strategy to cease cancer proliferation by deliberately inducing alternative translation of the antagonistic alias of an oncogene, and eventually overcome cooperation of the chromosomal rearrangements and dysregulated oncogene transcriptions in progressive cancer cells. Disclosures van Rhee: Adicet Bio: Consultancy; Karyopharm Therapeutics: Consultancy; EUSA: Consultancy; Castleman Disease Collaborative Network: Consultancy; Takeda: Consultancy; Sanofi Genzyme: Consultancy; Kite Pharma: Consultancy.

In-Cell Synthesis of Bioorthogonal Alkene Tag S-Allyl-Homocysteine and Its Coupling with Reprogrammed Translation.

In this study, we report our initial results on in situ biosynthesis of S-allyl-l-homocysteine (Sahc) by simple metabolic conversion of allyl mercaptan in Escherichia coli, which served as the host organism endowed with a direct sulfhydration pathway. The intracellular synthesis we describe in this study is coupled with the direct incorporation of Sahc into proteins in response to methionine codons. Together with O-acetyl-homoserine, allyl mercaptan was added to the growth medium, followed by uptake and intracellular reaction to give Sahc. Our protocol efficiently combined the in vivo synthesis of Sahc via metabolic engineering with reprogrammed translation, without the need for a major change in the protein biosynthesis machinery. Although the system needs further optimisation to achieve greater intracellular Sahc production for complete protein labelling, we demonstrated its functional versatility for photo-induced thiol-ene coupling and the recently developed phosphonamidate conjugation reaction. Importantly, deprotection of Sahc leads to homocysteine-containing proteins—a potentially useful approach for the selective labelling of thiols with high relevance in various medical settings.

Complexity and Conservation of Thermospermine-Responsive uORFs of SAC51 Family Genes in Angiosperms.

ACAULIS5 (ACL5) encodes thermospermine synthase in Arabidopsis and its loss-of-function mutant acl5 shows excess xylem differentiation and severe dwarfism. SAC51 encodes a basic helix-loop-helix (bHLH) protein and was identified from sac51-d, a dominant suppressor mutant of acl5, which restores the wild-type phenotype without thermospermine. The 5′ leader of the SAC51 mRNA contains multiple upstream open-reading frames (uORFs) and sac51-d has a premature stop codon in the fourth uORF. This uORF is conserved among SAC51 family genes in vascular plants. According to the GUS reporter assay, the SAC51 promoter was not responsive to thermospermine but the SAC51 5′ leader fused to the constitutive 35S promoter enhanced the GUS activity in response to thermospermine. Disruption experiments of each start codon of the SAC51 uORFs revealed that uORF4 and uORF6 whose start codon corresponds to the second methionine codon of uORF4 had an inhibitory effect on the main ORF translation while the other four uORFs rather had a stimulatory effect. The response of the 5′ leader to thermospermine was retained after disruption of each one of six start codons of these uORFs but abolished by mutating both uORF4 and uORF6 start codons, suggesting the importance of the C-terminal sequence shared by these uORFs in the action of thermospermine. We introduced GUS fusions with 5′ leaders of SAC51 family genes from other angiosperm species into Arabidopsis and found that all 5′ leaders responsive to thermospermine, so far examined, contained these two conserved, and overlapping uORFs.

To Code or Not to Code: What Is the Linc?

The annotation of the mammalian protein coding genome is incomplete. Arbitrary open reading frame (ORF) size restriction and the absolute requirement for a methionine codon as the sole initiator of translation, have constrained identification of potentially important transcripts with non‐canonical protein coding potential. Using unbiased transcriptomic approaches in macrophages responding to bacterial infection, we show widespread ribosome association with a large number of RNAs that were previously annotated as “non‐protein coding”. Although the ability of such non‐canonical ORFs to encode functional protein is controversial, we identify a plethora of novel short and non‐ATG initiated ORFs with the ability to generate stable and spatially distinct proteins. Importantly, we show that the translation of a novel ORF ‘hidden’ within the long non‐coding RNA Aw112010 is essential for the orchestration of mucosal immunity during both bacterial infection and colitis. Together this work expands our interpretation of the protein coding genome and demonstrates the critical nature of proteinaceous products generated from non‐canonical ORFs to the immune response in vivo. We therefore propose that the misannotation of non‐canonical ORF‐containing genes as non‐coding RNAs may obscure the essential role of a multitude of previously undiscovered protein coding genes in immunity and disease.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Alternative splicing of the Izumo1 gene ensures triggering gamete fusion in mice

IZUMO1 is a sperm acrosomal membrane protein that is essential for mammalian fertilization through recognition of JUNO on the oocyte surface and accompanying IZUMO1-JUNO complex formation. Here, we report a new Izumo1 gene splicing variant (IZUMO1_v2) with a unique 52-amino-acid-long signal sequence transcribed from Exon 1b. Although the mRNA amount of Izumo1_v2 is 76 times lower than that of the original Izumo1 (IZUMO1_v1) in the testis, the cell-oocyte assay indicates that IZUMO1_v2-expressing COS-7 cells have the ability to attach to the oocyte equivalent of IZUMO1_v1. To clarify the physiological function of IZUMO1_v2, we produced an IZUMO1_v1-specific knockout mouse line with a nine-base deletion adjacent to the initial methionine codon of IZUMO1_v1 by the CRISPR/Cas9 system. The IZUMO1_v1 knockout male mice carry 0.19-fold lower level of IZUMO1 protein in the spermatozoon; however, reduction in fertility was only minimally affected compared to the wild-type mice, suggesting that only a small fraction of IZUMO1 is sufficient for triggering sperm-egg fusion. We propose that the alternative splicing generating IZUMO1_v2 might function as a fail-safe in mouse for when splicing is disturbed.

The translation of non-canonical open reading frames controls mucosal immunity.

The annotation of the mammalian protein coding genome is incomplete. Arbitrary open reading frame (ORF) size restriction and the absolute requirement for a methionine codon as the sole initiator of translation, have constrained identification of potentially important transcripts with non-canonical protein coding potential1,2. Using unbiased transcriptomic approaches in macrophages responding to bacterial infection, we show widespread ribosome association with a large number of RNAs that were previously annotated as “non-protein coding”. Although the ability of such non-canonical ORFs to encode functional protein is controversial3,4, we identify a plethora of novel short and non-ATG initiated ORFs with the ability to generate stable and spatially distinct proteins. Importantly, we show that the translation of a novel ORF ‘hidden’ within the long non-coding RNA Aw112010 is essential for the orchestration of mucosal immunity during both bacterial infection and colitis. Together this work expands our interpretation of the protein coding genome and demonstrates the critical nature of proteinaceous products generated from non-canonical ORFs to the immune response in vivo. We therefore propose that the misannotation of non-canonical ORF-containing genes as non-coding RNAs may obscure the essential role of a multitude of previously undiscovered protein coding genes in immunity and disease.

Phenotypic Assessment Suggests Multiple Start Codons for HetN, an Inhibitor of Heterocyst Differentiation, in Anabaena sp. Strain PCC 7120.

Multicellular organisms must carefully regulate the timing, number, and location of specialized cellular development. In the filamentous cyanobacterium Anabaena sp. strain PCC 7120, nitrogen-fixing heterocysts are interspersed between vegetative cells in a periodic pattern to achieve an optimal exchange of bioavailable nitrogen and reduced carbon. The spacing between heterocysts is regulated by the activity of two developmental inhibitors, PatS and HetN. PatS functions to create a de novo pattern from a homogenous field of undifferentiated cells, while HetN maintains the pattern throughout subsequent growth. Both PatS and HetN harbor the peptide motif ERGSGR, which is sufficient to inhibit development. While the small size of PatS makes the interpretation of inhibitory domains relatively simple, HetN is a 287-amino-acid protein with multiple functional regions. Previous work suggested the possibility of a truncated form of HetN containing the ERGSGR motif as the source of the HetN-derived inhibitory signal. In this work, we present evidence that the glutamate of the ERGSGR motif is required for proper HetN inhibition of heterocysts. Mutational analysis and subcellular localization indicate that the gene encoding HetN uses two methionine start codons (M1 and M119) to encode two protein forms: M1 is required for protein localization, while M119 is primarily responsible for inhibitory function. Finally, we demonstrate that patS and hetN are not functionally equivalent when expressed from the other gene's regulatory sequences. Taken together, these results help clarify the functional forms of HetN and will help refine future work defining a HetN-derived inhibitory signal in this model of one-dimensional periodic patterning.IMPORTANCE The proper placement of different cell types during a developmental program requires the creation and maintenance of a biological pattern to define the cells that will differentiate. Here we show that the HetN inhibitor, responsible for pattern maintenance of specialized nitrogen-fixing heterocyst cells in the filamentous cyanobacterium Anabaena, may be produced from two different start methionine codons. This work demonstrates that the two start sites are individually involved in a different HetN function, either membrane localization or inhibition of cellular differentiation.

An initiator codon mutation in SDE2 causes recessive embryonic lethality in Holstein cattle

Researching depletions in homozygous genotypes for specific haplotypes among the large cohorts of animals genotyped for genomic selection is a very efficient strategy to map recessive lethal mutations. In this study, by analyzing real or imputed Illumina BovineSNP50 (Illumina Inc., San Diego, CA) genotypes from more than 250,000 Holstein animals, we identified a new locus called HH6 showing significant negative effects on conception rate and nonreturn rate at 56 d in at-risk versus control mating. We fine-mapped this locus in a 1.1-Mb interval and analyzed genome sequence data from 12 carrier and 284 noncarrier Holstein bulls. We report the identification of a strong candidate mutation in the gene encoding SDE2 telomere maintenance homolog (SDE2), a protein essential for genomic stability in eukaryotes. This A-to-G transition changes the initiator ATG (methionine) codon to ACG because the gene is transcribed on the reverse strand. Using RNA sequencing and quantitative reverse-transcription PCR, we demonstrated that this mutation does not significantly affect SDE2 splicing and expression level in heterozygous carriers compared with control animals. Initiation of translation at the closest in-frame methionine codon would truncate the SDE2 precursor by 83 amino acids, including the cleavage site necessary for its activation. Finally, no homozygote for the G allele was observed in a large population of nearly 29,000 individuals genotyped for the mutation. The low frequency (1.3%) of the derived allele in the French population and the availability of a diagnostic test on the Illumina EuroG10K SNP chip routinely used for genomic evaluation will enable rapid and efficient selection against this deleterious mutation.

Improvement of a yeast self-excising integrative vector by prevention of expression leakage of the intronated Cre recombinase gene during plasmid maintenance in Escherichia coli.

The use of plasmids possessing a regulatable gene coding for a site-specific recombinase together with its recognition sequences significantly facilitates genome manipulations since it allows self-excision of the portion of the genetic construct integrated into the host genome. Stable maintenance of such plasmids in Escherichia coli, which is used for plasmid preparation, requires prevention of recombinase synthesis in this host, which can be achieved by interrupting the recombinase gene with an intron. Based on this approach, Saccharomyces cerevisiae and Hansenula polymorpha self-excising vectors possessing intronated gene for Cre recombinase and its recognition sites (LoxP) were previously constructed. However, this work shows instability of the H. polymorpha vectors during plasmid maintenance in E. coli cells. This could be due to recombination between the loxP sites caused by residual expression of the cre gene. Prevention of translation reinitiation on an internal methionine codon completely solved this problem. A similar modification was made in a self-excising vector designed for S. cerevisiae. Apart from substantial improvement of yeast self-excising vectors, the obtained results also narrow down the essential part of Cre sequence.

DealingwithanUnconventionalGeneticCodein Mitochondria:TheBiogenesisandPathogenic Defectsofthe5-FormylcytosineModificationin MitochondrialtRNAMet.

Human mitochondria contain their own genome, which uses an unconventional genetic code. In addition to the standard AUG methionine codon, the single mitochondrial tRNA Methionine (mt-tRNAMet) also recognises AUA during translation initiation and elongation. Post-transcriptional modifications of tRNAs are important for structure, stability, correct folding and aminoacylation as well as decoding. The unique 5-formylcytosine (f5C) modification of position 34 in mt-tRNAMet has been long postulated to be crucial for decoding of unconventional methionine codons and efficient mitochondrial translation. However, the enzymes responsible for the formation of mitochondrial f5C have been identified only recently. The first step of the f5C pathway consists of methylation of cytosine by NSUN3. This is followed by further oxidation by ABH1. Here, we review the role of f5C, the latest breakthroughs in our understanding of the biogenesis of this unique mitochondrial tRNA modification and its involvement in human disease.