Assessment of genetic homogeneity of in-vitro propagated apple root stock MM 104 using ISSR and SCoT primers.

Apple is an important fruit crop that is always in demand due to its commercial and nutraceutical value. Also, the requirement for quality planting material for this fruit crop for new plantations is increasing continuously. In-vitro propagation is an alternative approach, which may help to produce genetically identical high grade planting material. In this study, for the first time, an efficient and reproducible propagation protocol has been established for apple root stock MM 104 via axillary bud. Culturing axillary buds on Murashige and Skoog apple rootstock (MM 104) resulted in better in-vitro propagation. (MS) basal medium supplemented with 3.0% (w/v) sucrose and 0.8% (w/v) agar. The axillary buds were established in MS basal medium with BA (5.0 µM), NAA (1.0 µM) and further used to establish invitro propagation protocol. Plant Growth Regulators (PGRs), BA (1.0 µM) in combination with NAA (1.0 µM) was found most efficient for shoot multiplication (100%) and produced 9.8 shoots/explants with an average shoot length of (2.4 ± cm). All the shoots produced roots in 0.1 µM IBA with a 5-day dark period. Acclimatization of in-vitro raised plantlets was obtained with vermiculite: perlite: sand: soil (2:2:1:1) resulting in 76% survival under field conditions. The study showed that the use of axillary bud is efficient for multiple-shoot production of apple rootstock (MM 104). This is the first comprehensive report on in-vitro growth of apple root stock MM 104 with an assessment of genetic stability using DNA fingerprinting profiles based on Inter Simple Sequence Repeats (ISSR) and Start Codon Targeted (SCoT). The genetic stability of in-vitro-produced plants, as determined by SCoT and ISSR primers, demonstrated genetic closeness to the mother plant.

Transcriptome-wide analysis of the function of Ded1 in translation preinitiation complex assembly in a reconstituted in vitro system.

We have developed a deep sequencing-based approach, Rec-Seq, that allows simultaneous monitoring of ribosomal 48S preinitiation complex (PIC) formation on every mRNA in the translatome in an in vitro reconstituted system. Rec-Seq isolates key early steps in translation initiation in the absence of all other cellular components and processes. Using this approach, we show that the DEAD-box ATPase Ded1 promotes 48S PIC formation on the start codons of >1000 native mRNAs, most of which have long, structured 5'-untranslated regions (5'UTRs). Remarkably, initiation measured in Rec-Seq was enhanced by Ded1 for most mRNAs previously shown to be highly Ded1-dependent by ribosome profiling of ded1 mutants in vivo, demonstrating that the core translation functions of the factor are recapitulated in the purified system. Our data do not support a model in which Ded1acts by reducing initiation at alternative start codons in 5'UTRs and instead indicate it functions by directly promoting mRNA recruitment to the 43S PIC and scanning to locate the main start codon. We also provide evidence that eIF4A, another essential DEAD-box initiation factor, is required for efficient PIC assembly on almost all mRNAs, regardless of their structural complexity, in contrast to the preferential stimulation by Ded1 of initiation on mRNAs with long, structured 5'UTRs.

Comparative mitogenomic analysis of subterranean and surface amphipods (Crustacea, Amphipoda) with special reference to the family Crangonyctidae.

Mitochondrial genomes play important roles in studying genome evolution, phylogenetic analyses, and species identification. Amphipods (Class Malacostraca, Order Amphipoda) are one of the most ecologically diverse crustacean groups occurring in a diverse array of aquatic and terrestrial environments globally, from freshwater streams and lakes to groundwater aquifers and the deep sea, but we have a limited understanding of how habitat influences the molecular evolution of mitochondrial energy metabolism. Subterranean amphipods likely experience different evolutionary pressures on energy management compared to surface-dwelling taxa that generally encounter higher levels of predation and energy resources and live in more variable environments. In this study, we compared the mitogenomes, including the 13 protein-coding genes involved in the oxidative phosphorylation (OXPHOS) pathway, of surface and subterranean amphipods to uncover potentially different molecular signals of energy metabolism between surface and subterranean environments in this diverse crustacean group. We compared base composition, codon usage, gene order rearrangement, conducted comparative mitogenomic and phylogenomic analyses, and examined evolutionary signals of 35 amphipod mitogenomes representing 13 families, with an emphasis on Crangonyctidae. Mitogenome size, AT content, GC-skew, gene order, uncommon start codons, location of putative control region (CR), length of rrnL and intergenic spacers differed between surface and subterranean amphipods. Among crangonyctid amphipods, the spring-dwelling Crangonyx forbesi exhibited a unique gene order, a long nad5 locus, longer rrnL and rrnS loci, and unconventional start codons. Evidence of directional selection was detected in several protein-encoding genes of the OXPHOS pathway in the mitogenomes of surface amphipods, while a signal of purifying selection was more prominent in subterranean species, which is consistent with the hypothesis that the mitogenome of surface-adapted species has evolved in response to a more energy demanding environment compared to subterranean amphipods. Overall, gene order, locations of non-coding regions, and base-substitution rates points to habitat as an important factor influencing the evolution of amphipod mitogenomes.

Proteins à la carte: riboproteogenomic exploration of bacterial N-terminal proteoform expression.

In recent years, it has become evident that the true complexity of bacterial proteomes remains underestimated. Gene annotation tools are known to propagate biases and overlook certain classes of truly expressed proteins, particularly proteoforms-protein isoforms arising from a single gene. Recent (re-)annotation efforts heavily rely on ribosome profiling by providing a direct readout of translation to fully describe bacterial proteomes. In this study, we employ a robust riboproteogenomic pipeline to conduct a systematic census of expressed N-terminal proteoform pairs, representing two isoforms encoded by a single gene raised by annotated and alternative translation initiation, in Salmonella. Intriguingly, conditional-dependent changes in relative utilization of annotated and alternative translation initiation sites (TIS) were observed in several cases. This suggests that TIS selection is subject to regulatory control, adding yet another layer of complexity to our understanding of bacterial proteomes. IMPORTANCE: With the emerging theme of genes within genes comprising the existence of alternative open reading frames (ORFs) generated by translation initiation at in-frame start codons, mechanisms that control the relative utilization of annotated and alternative TIS need to be unraveled and our molecular understanding of resulting proteoforms broadened. Utilizing complementary ribosome profiling strategies to map ORF boundaries, we uncovered dual-encoding ORFs generated by in-frame TIS usage in Salmonella. Besides demonstrating that alternative TIS usage may generate proteoforms with different characteristics, such as differential localization and specialized function, quantitative aspects of conditional retapamulin-assisted ribosome profiling (Ribo-RET) translation initiation maps offer unprecedented insights into the relative utilization of annotated and alternative TIS, enabling the exploration of gene regulatory mechanisms that control TIS usage and, consequently, the translation of N-terminal proteoform pairs.

dCas13-mediated translational repression for accurate gene silencing in mammalian cells.

Current gene silencing tools based on RNA interference (RNAi) or, more recently, clustered regularly interspaced short palindromic repeats (CRISPR)‒Cas13 systems have critical drawbacks, such as off-target effects (RNAi) or collateral mRNA cleavage (CRISPR‒Cas13). Thus, a more specific method of gene knockdown is needed. Here, we develop CRISPRδ, an approach for translational silencing, harnessing catalytically inactive Cas13 proteins (dCas13). Owing to its tight association with mRNA, dCas13 serves as a physical roadblock for scanning ribosomes during translation initiation and does not affect mRNA stability. Guide RNAs covering the start codon lead to the highest efficacy regardless of the translation initiation mechanism: cap-dependent, internal ribosome entry site (IRES)-dependent, or repeat-associated non-AUG (RAN) translation. Strikingly, genome-wide ribosome profiling reveals the ultrahigh gene silencing specificity of CRISPRδ. Moreover, the fusion of a translational repressor to dCas13 further improves the performance. Our method provides a framework for translational repression-based gene silencing in eukaryotes.

JUN mRNA translation regulation is mediated by multiple 5' UTR and start codon features.

Regulation of mRNA translation by eukaryotic initiation factors (eIFs) is crucial for cell survival. In humans, eIF3 stimulates translation of the JUN mRNA which encodes the transcription factor JUN, an oncogenic transcription factor involved in cell cycle progression, apoptosis, and cell proliferation. Previous studies revealed that eIF3 activates translation of the JUN mRNA by interacting with a stem loop in the 5' untranslated region (5' UTR) and with the 5' -7-methylguanosine cap structure. In addition to its interaction site with eIF3, the JUN 5' UTR is nearly one kilobase in length, and has a high degree of secondary structure, high GC content, and an upstream start codon (uAUG). This motivated us to explore the complexity of JUN mRNA translation regulation in human cells. Here we find that JUN translation is regulated in a sequence and structure-dependent manner in regions adjacent to the eIF3-interacting site in the JUN 5' UTR. Furthermore, we identify contributions of an additional initiation factor, eIF4A, in JUN regulation. We show that enhancing the interaction of eIF4A with JUN by using the compound Rocaglamide A (RocA) represses JUN translation. We also find that both the upstream AUG (uAUG) and the main AUG (mAUG) contribute to JUN translation and that they are conserved throughout vertebrates. Our results reveal additional layers of regulation for JUN translation and show the potential of JUN as a model transcript for understanding multiple interacting modes of translation regulation.

The first mitochondrial genome of Calophyllum soulattri Burm.f.

Calophyllum soulattri Burm.f. is traditionally used to treat skin infections and reduce rheumatic pain, yet genetic and genomic studies are still limited. Here, we present the first complete mitochondrial genome of C. soulattri. It is 378,262 bp long with 43.97% GC content, containing 55 genes (30 protein-coding, 5 rRNA, and 20 tRNA). Repeat analysis of the mitochondrial genome revealed 194 SSRs, mostly mononucleotides, and 266 pairs of dispersed repeats ( ≥ 30 bp) that were predominantly palindromic. There were 23 homologous fragments found between the mitochondrial and plastome genomes. We also predicted 345 C-to-U RNA editing sites from 30 protein-coding genes (PCGs) of the C. soulatrii mitochondrial genome. These RNA editing events created the start codon of nad1 and the stop codon of ccmFc. Most PCGs of the C. soulattri mitochondrial genome underwent negative selection, but atp4 and ccmB experienced positive selection. Phylogenetic analyses showed C. soulattri is a sister taxon of Garcinia mangostana. This study has shed light on C. soulattri's evolution and Malpighiales' phylogeny. As the first complete mitochondrial genome in Calophyllaceae, it can be used as a reference genome for other medicinal plant species within the family for future genetic studies.

Modeling alternative translation initiation sites in plants reveals evolutionarily conserved cis-regulatory codes in eukaryotes.

mRNA translation relies on identifying translation initiation sites (TISs) in mRNAs. Alternative TISs are prevalent across plant transcriptomes, but the mechanisms for their recognition are unclear. Using ribosome profiling and machine learning, we developed models for predicting alternative TISs in the tomato (Solanum lycopersicum). Distinct feature sets were predictive of AUG and nonAUG TISs in 5' untranslated regions and coding sequences, including a novel CU-rich sequence that promoted plant TIS activity, a translational enhancer found across dicots and monocots, and humans and viruses. Our results elucidate the mechanistic and evolutionary basis of TIS recognition, whereby cis-regulatory RNA signatures affect start site selection. The TIS prediction model provides global estimates of TISs to discover neglected protein-coding genes across plant genomes. The prevalence of cis-regulatory signatures across plant species, humans, and viruses suggests their broad and critical roles in reprogramming the translational landscape.

The First Complete Mitochondrial Genome of the Genus Litostrophus: Insights into the Rearrangement and Evolution of Mitochondrial Genomes in Diplopoda.

This study presents the complete mitochondrial genome (mitogenome) of Litostrophus scaber, which is the first mitogenome of the genus Litostrophus. The mitogenome is a circular molecule with a length of 15,081 bp. The proportion of adenine and thymine (A + T) was 69.25%. The gene ND4L used TGA as the initiation codon, while the other PCGs utilized ATN (A, T, G, C) as the initiation codons. More than half of the PCGs used T as an incomplete termination codon. The transcription direction of the L. scaber mitogenome matched Spirobolus bungii, in contrast to most millipedes. Novel rearrangements were found in the L. scaber mitogenome: trnQ -trnC and trnL1- trnP underwent short-distance translocations and the gene block rrnS-rrnL-ND1 moved to a position between ND4 and ND5, resulting in the formation of a novel gene order. The phylogenetic analysis showed that L. scaber is most closely related to S. bungii, followed by Narceus magnum. These findings enhance our understanding of the rearrangement and evolution of Diplopoda mitogenomes.

CdsA, a CDP-diacylglycerol synthase involved in phospholipid and glycolipid MPIase biosynthesis, possesses multiple initiation codons.

CdsA is a CDP-diacylglycerol synthase essential for phospholipid and glycolipid MPIase biosynthesis, and therefore for growth. The initiation codon of CdsA has been assigned as "TTG," while methionine at the 37th codon was reported to be an initiation codon in the original report. Since a vector containing the open reading frame starting with "TTG" under a controllable promoter complemented the cdsA knockout, "TTG" could function as an initiation codon. However, no evidence supporting that this "TTG" is the sole initiation codon has been reported. We determined the initiation codon by examining the ability of mutants around the N-terminal region to complement cdsA mutants. Even if the "TTG" was substituted with a stop codon, the clear complementation was observed. Moreover, the clones with multiple mutations of stop codons complemented the cdsA mutant up to the 37th codon, indicating that cdsA possesses multiple codons that can function as initiation codons. We constructed an experimental system in which the chromosomal expression of cdsA can be analyzed. By means of this system, we found that the cdsA mutant with substitution of "TTG" with a stop codon is fully functional. Thus, we concluded that CdsA contains multiple initiation codons.

N6-methyladenosine in 5' UTR does not promote translation initiation.

The most abundant N6-methyladenosine (m6A) modification on mRNAs is installed non-stoichiometrically across transcripts, with 5' untranslated regions (5' UTRs) being the least conductive. 5' UTRs are essential for translation initiation, yet the molecular mechanisms orchestrated by m6A remain poorly understood. Here, we combined structural, biochemical, and single-molecule approaches and show that at the most common position, a single m6A does not affect translation yields, the kinetics of translation initiation complex assembly, or start codon recognition both under permissive growth and following exposure to oxidative stress. Cryoelectron microscopy (cryo-EM) structures of the late preinitiation complex reveal that m6A purine ring established stacking interactions with an arginine side chain of the initiation factor eIF2α, although with only a marginal energy contribution, as estimated computationally. These findings provide molecular insights into m6A interactions with the initiation complex and suggest that the subtle stabilization is unlikely to affect the translation dynamics under homeostatic conditions or stress.

The structure of a human translation initiation complex reveals two independent roles for the helicase eIF4A.

Eukaryotic translation initiation involves recruitment of the 43S pre-initiation complex to the 5' end of mRNA by the cap-binding complex eIF4F, forming the 48S translation initiation complex (48S), which then scans along the mRNA until the start codon is recognized. We have previously shown that eIF4F binds near the mRNA exit channel of the 43S, leaving open the question of how mRNA secondary structure is removed as it enters the mRNA channel on the other side of the 40S subunit. Here we report the structure of a human 48S that shows that, in addition to the eIF4A that is part of eIF4F, there is a second eIF4A helicase bound at the mRNA entry site, which could unwind RNA secondary structures as they enter the 48S. The structure also reveals conserved interactions between eIF4F and the 43S, probaby explaining how eIF4F can promote mRNA recruitment in all eukaryotes.

Yeast eIF2A has a minimal role in translation initiation and uORF-mediated translational control in vivo.

Initiating translation of most eukaryotic mRNAs depends on recruitment of methionyl initiator tRNA (Met-tRNAi) in a ternary complex (TC) with GTP-bound eukaryotic initiation factor 2 (eIF2) to the small (40S) ribosomal subunit, forming a 43S preinitiation complex (PIC) that attaches to the mRNA and scans the 5'-untranslated region (5' UTR) for an AUG start codon. Previous studies have implicated mammalian eIF2A in GTP-independent binding of Met-tRNAi to the 40S subunit and its recruitment to specialized mRNAs that do not require scanning, and in initiation at non-AUG start codons, when eIF2 function is attenuated by phosphorylation of its α-subunit during stress. The role of eIF2A in translation in vivo is poorly understood however, and it was unknown whether the conserved ortholog in budding yeast can functionally substitute for eIF2. We performed ribosome profiling of a yeast deletion mutant lacking eIF2A and isogenic wild-type (WT) cells in the presence or absence of eIF2α phosphorylation induced by starvation for amino acids isoleucine and valine. Whereas starvation of WT confers changes in translational efficiencies (TEs) of hundreds of mRNAs, the eIF2AΔ mutation conferred no significant TE reductions for any mRNAs in non-starved cells, and it reduced the TEs of only a small number of transcripts in starved cells containing phosphorylated eIF2α. We found no evidence that eliminating eIF2A altered the translation of mRNAs containing putative internal ribosome entry site (IRES) elements, or harboring uORFs initiated by AUG or near-cognate start codons, in non-starved or starved cells. Thus, very few mRNAs (possibly only one) appear to employ eIF2A for Met-tRNAi recruitment in yeast cells, even when eIF2 function is attenuated by stress.

CAG repeat expansions create splicing acceptor sites and produce aberrant repeat-containing RNAs.

Expansions of CAG trinucleotide repeats cause several rare neurodegenerative diseases. The disease-causing repeats are translated in multiple reading frames and without an identifiable initiation codon. The molecular mechanism of this repeat-associated non-AUG (RAN) translation is not known. We find that expanded CAG repeats create new splice acceptor sites. Splicing of proximal donors to the repeats produces unexpected repeat-containing transcripts. Upon splicing, depending on the sequences surrounding the donor, CAG repeats may become embedded in AUG-initiated open reading frames. Canonical AUG-initiated translation of these aberrant RNAs may account for proteins that have been attributed to RAN translation. Disruption of the relevant splice donors or the in-frame AUG initiation codons is sufficient to abrogate RAN translation. Our findings provide a molecular explanation for the abnormal translation products observed in CAG trinucleotide repeat expansion disorders and add to the repertoire of mechanisms by which repeat expansion mutations disrupt cellular functions.

Modulations in the host cell proteome by the hantavirus nucleocapsid protein.

Hantaviruses have evolved a unique translation strategy to boost the translation of viral mRNA in infected cells. Hantavirus nucleocapsid protein (NP) binds to the viral mRNA 5' UTR and the 40S ribosomal subunit via the ribosomal protein S19. NP associated ribosomes are selectively loaded on viral transcripts to boost their translation. Here we demonstrate that NP expression upregulated the steady-state levels of a subset of host cell factors primarily involved in protein processing in the endoplasmic reticulum. Detailed investigation of Valosin-containing protein (VCP/p97), one of the upregulated host factors, in both transfected and virus infected cells revealed that NP with the assistance of VCP mRNA 5' UTR facilitates the translation of downstream VCP ORF. The VCP mRNA contains a 5' UTR of 987 nucleotides harboring six unusual start codons upstream of the correct start codon for VCP which is located at 988th position from the 5' cap. In vitro translation of a GFP reporter transcript harboring the VCP mRNA 5' UTR generated both GFP and a short polypeptide of ~14 KDa by translation initiation from start codon located in the 5' UTR at 542nd position from the 5' cap. The translation initiation from 542nd AUG in the UTR sequence was confirmed in cells using a dual reporter construct expressing mCherry and GFP. The synthesis of 14KDa polypeptide dramatically inhibited the translation of the ORF from the downstream correct start codon at 988th position from the 5' cap. We report that purified NP binds to the VCP mRNA 5' UTR with high affinity and NP binding site is located close to the 542ndAUG. NP binding shuts down the translation of 14KDa polypeptide which then facilitates the translation initiation at the correct AUG codon. Knockdown of VCP generated lower levels of poorly infectious hantavirus particle in the cellular cytoplasm whose egress was dramatically inhibited in human umbilical vein endothelial cells. We demonstrated that VCP binds to the hantavirus glycoprotein Gn before its incorporation into assembled virions and facilitates viral spread to neighboring cells during infection. Our results suggest that ribosome engagement at the 542nd AUG codon in the 5' UTR likely regulates the endogenous steady state levels of VCP in cells. Hantaviruses interrupt this regulatory mechanism to enhance the steady state levels of VCP in virus infected cells. This augmentation facilitates virus replication, supports the transmission of the virus to adjacent cells, and promotes the release of infectious virus particles from the host cell.

Unraveling the plasticity of translation initiation in prokaryotes: Beyond the invariant Shine-Dalgarno sequence.

Translation initiation in prokaryotes is mainly defined, although not exclusively, by the interaction between the anti-Shine-Dalgarno sequence (antiSD), located at the 3'-terminus of the 16S ribosomal RNA, and a complementary sequence, the ribosome binding site, or Shine-Dalgarno (SD), located upstream of the start codon in prokaryotic mRNAs. The antiSD has a conserved 5'-CCUCC-3' core, but inter-species variations have been found regarding the participation of flanking bases in binding. These variations have been described for certain bacteria and, to a lesser extent, for some archaea. To further analyze these variations, we conducted binding-energy prediction analyses on over 6,400 genomic sequences from both domains. We identified 15 groups of antiSD variants that could be associated with the organisms' phylogenetic origin. Additionally, our findings revealed that certain organisms exhibit variations in the core itself. Importantly, an unaltered core is not necessarily required for the interaction between the 3'-terminus of the rRNA and the region preceding the AUG of the mRNA. In our study, we classified organisms into four distinct categories: i) those possessing a conserved core and demonstrating binding; ii) those with a conserved core but lacking evidence of binding; iii) those exhibiting binding in the absence of a conserved core; and iv) those lacking both a conserved core and evidence of binding. Our results demonstrate the flexibility of organisms in evolving different sequences involved in translation initiation beyond the traditional Shine-Dalgarno sequence. These findings are discussed in terms of the evolution of translation initiation in prokaryotic organisms.

The molecular basis of translation initiation and its regulation in eukaryotes.

The regulation of gene expression is fundamental for life. Whereas the role of transcriptional regulation of gene expression has been studied for several decades, it has been clear over the past two decades that post-transcriptional regulation of gene expression, of which translation regulation is a major part, can be equally important. Translation can be divided into four main stages: initiation, elongation, termination and ribosome recycling. Translation is controlled mainly during its initiation, a process which culminates in a ribosome positioned with an initiator tRNA over the start codon and, thus, ready to begin elongation of the protein chain. mRNA translation has emerged as a powerful tool for the development of innovative therapies, yet the detailed mechanisms underlying the complex process of initiation remain unclear. Recent studies in yeast and mammals have started to shed light on some previously unclear aspects of this process. In this Review, we discuss the current state of knowledge on eukaryotic translation initiation and its regulation in health and disease. Specifically, we focus on recent advances in understanding the processes involved in assembling the 43S pre-initiation complex and its recruitment by the cap-binding complex eukaryotic translation initiation factor 4F (eIF4F) at the 5' end of mRNA. In addition, we discuss recent insights into ribosome scanning along the 5' untranslated region of mRNA and selection of the start codon, which culminates in joining of the 60S large subunit and formation of the 80S initiation complex.

Functional analysis of the AUG initiator codon context reveals novel conserved sequences that disfavor mRNA translation in eukaryotes.

mRNA translation is a fundamental process for life. Selection of the translation initiation site (TIS) is crucial, as it establishes the correct open reading frame for mRNA decoding. Studies in vertebrate mRNAs discovered that a purine at -3 and a G at +4 (where A of the AUG initiator codon is numbered + 1), promote TIS recognition. However, the TIS context in other eukaryotes has been poorly experimentally analyzed. We analyzed in vitro the influence of the -3, -2, -1 and + 4 positions of the TIS context in rabbit, Drosophila, wheat, and yeast. We observed that -3A conferred the best translational efficiency across these species. However, we found variability at the + 4 position for optimal translation. In addition, the Kozak motif that was defined from mammalian cells was only weakly predictive for wheat and essentially non-predictive for yeast. We discovered eight conserved sequences that significantly disfavored translation. Due to the big differences in translational efficiency observed among weak TIS context sequences, we define a novel category that we termed 'barren AUG context sequences (BACS)', which represent sequences disfavoring translation. Analysis of mRNA-ribosomal complexes structures provided insights into the function of BACS. The gene ontology of the BACS-containing mRNAs is presented.

The interplay between cis- and trans-acting factors drives selective mRNA translation initiation in eukaryotes.

Translation initiation consists in the assembly of the small and large ribosomal subunits on the start codon. This important step directly modulates the general proteome in living cells. Recently, genome wide studies revealed unexpected translation initiation events from unsuspected novel open reading frames resulting in the synthesis of a so-called 'dark proteome'. Indeed, the identification of the start codon by the translation machinery is a critical step that defines the translational landscape of the cell. Therefore, translation initiation is a highly regulated process in all organisms. In this review, we focus on the various cis- and trans-acting factors that rule the regulation of translation initiation in eukaryotes. Recent discoveries have shown that the guidance of the translation machinery for the choice of the start codon require sophisticated molecular mechanisms. In particular, the 5'UTR and the coding sequences contain cis-acting elements that trigger the use of AUG codons but also non-AUG codons to initiate protein synthesis. The use of these alternative start codons is also largely influenced by numerous trans-acting elements that drive selective mRNA translation in response to environmental changes.

Effects of CRISPR/Cas9-mediated dnd1 knockout impairs gonadal development in striped catfish.

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology allows for the generation of loss-of-function mutations to enable efficient gene targeting to produce desired phenotypes, such as the production of germ cell-free fish. This technology could provide several applications for aquaculture and conservation of fisheries resources, such as the prevention of overpopulation in fish culture and gene flow from escaped farmed fish into wild populations and the production of germ cell-free recipient larvae for germ cell transplantation. This study aimed to develop CRISPR/Cas9 mediated dead-end 1 (dnd1) knockout techniques for striped catfish (Pangasianodon hypophthalmus). To optimise CRISPR/Cas9-induced dnd1 knockout, three single-guide RNAs (sgRNAs) were designed to target upstream sequences of start codon of the dnd1 gene. A combination of two concentrations of each sgRNA (100 and 200 ng/µl) and three concentrations of Cas9 (100, 250, and 500 ng/µl) was microinjected into fertilised striped catfish eggs. These sgRNAs/Cas9 could induce indel mutations and lower the primordial germ cell (PGC) numbers. Histological analyses indicated that sgRNA3 targeting upstream and nearest to the start codon at 200 ng/µL and Cas9 at 500 ng/µL showed the lowest PGC number. The reduction in PGC number was confirmed by in situ hybridisation using antisense dnd1 and vasa probes. All sgRNA/Cas9 combinations reduced the expression of dnd1, cxcr4b, dazl, nanos1, nanos2, and vasa, and the lowest expression levels were observed in gonads obtained from fish injected with 200 ng/µL sgRNA3 and 500 ng/µL Cas9 (P < 0.05). In addition, at 1 year of age, a significantly lower gonadosomatic index was observed in fish injected with all sgRNA and Cas9 at 500 ng/µL. Moreover, compared to the control fish, the ovaries and testes presented different morphologies in the sgRNA/Cas9-injected fish, that is, few previtellogenic oocytes in the ovary and spermatogonial cell-less testes. In conclusion, CRISPR/Cas 9 targeting dnd1 knockout at the upstream sequences of start codon was achieved, which resulted in the downregulation of dnd1 and lowered PGC number.