ABSTRACT
tRNA-derived small RNAs (tsRNAs) from spermatozoa could act as acquired epigenetic factors and contribute to offspring phenotypes. However, the roles of specific tsRNAs in early embryo development remain to be elucidated. Here, using pigs as a research model, we probed the tsRNA dynamics during spermatogenesis and sperm maturation and demonstrated the delivery of tsRNAs from semen-derived exosomes to spermatozoa. By microinjection of antisense sequences into in vitro fertilized oocytes and subsequent single-cell RNA-seq of embryos, we identified a specific functional tsRNA group (termed here Gln-TTGs) that participate in the early cleavage of porcine preimplantation embryos, probably by regulating cell cycle-associated genes and retrotransposons. We conclude that specific tsRNAs present in mature spermatozoa play significant roles in preimplantation embryo development.
Subject(s)
Blastocyst , Cell Division , RNA, Transfer, Gln/physiology , RNA/metabolism , Spermatozoa/metabolism , Animals , Embryonic Development , Female , Male , Microinjections , Pregnancy , Sperm Maturation , Spermatogenesis , SwineABSTRACT
In eukaryotes, translation termination is dependent on the availability of both release factors, eRF1 and eRF3; however, the precise mechanisms involved remain poorly understood. In particular, the fact that the phenotype of release factor mutants is pleiotropic could imply that other factors and interactions are involved in translation termination. To identify unknown elements involved in this process, we performed a genetic screen using a reporter strain in which a leaky stop codon is inserted in the lacZ reporter gene, attempting to isolate factors modifying termination efficiency when overexpressed. Twelve suppressors and 11 antisuppressors, increasing or decreasing termination readthrough, respectively, were identified and analyzed for three secondary phenotypes often associated with translation mutations: thermosensitivity, G418 sensitivity, and sensitivity to osmotic pressure. Interestingly, among these candidates, we identified two genes, SSO1 and STU2, involved in protein transport and spindle pole body formation, respectively, suggesting puzzling connections with the translation termination process.
Subject(s)
Protein Biosynthesis/genetics , Saccharomyces cerevisiae Proteins/physiology , Saccharomyces cerevisiae/genetics , Cytoskeleton/physiology , Fungal Proteins/genetics , Fungal Proteins/physiology , Gene Expression , Genetic Vectors , Membrane Proteins/genetics , Membrane Proteins/physiology , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/physiology , Protein Biosynthesis/physiology , Protein Transport , Qa-SNARE Proteins , RNA, Transfer, Gln/genetics , RNA, Transfer, Gln/physiology , Saccharomyces cerevisiae/physiology , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
Base insertion mutations in the anticodons of two different Escherichia coli tRNAs have been isolated that allow suppression of a series of +1 frameshift mutations. Insertion of a U between positions 34 and 35 of tRNAGln1 or addition of a G between positions 36 and 37 of tRNA(Lys) expand the anticodons of both tRNAs similarly to 3'-GUUU(-5') and allow decoding of complementary 5'-CAAA(-3') quadruplets. Analysis of the suppressed mRNA sequences suggests that suppression occurs by pairing of the expanded anticodons to all four bases of the complementary, quadruplet codon. The tRNA Gln mutants are identical to the sufG class of frameshift suppressors isolated both in Salmonella enterica serovar Typhimurium and E. coli by Kohno and Roth and previously thought to affect tRNA(Lys).
Subject(s)
Escherichia coli/genetics , Frameshift Mutation , RNA, Transfer, Gln/chemistry , RNA, Transfer, Gln/physiology , RNA, Transfer, Lys/chemistry , RNA, Transfer, Lys/physiology , Anticodon , Base Sequence , Chromosome Mapping , Codon , Genes, Bacterial , Genes, Suppressor , Models, Genetic , Molecular Sequence Data , Mutagenesis, Insertional , Nucleic Acid Conformation , RNA, Bacterial/chemistry , RNA, Bacterial/genetics , RNA, Transfer, Gln/genetics , RNA, Transfer, Lys/genetics , Salmonella/geneticsABSTRACT
A number of ciliated protozoa are known to read the stop codons UAA and UAG as sense codons that specify glutamine during protein synthesis. In considering evolutionary mechanisms for this curious divergence from the standard genetic code, we propose the existence of progenitor tRNAs for glutamine that can weakly suppress UAA and UAG codons. It has been previously shown that multicopy plasmids that overexpress normal tRNA(CAAGln) and tRNA(CAGGln) genes from the yeast Saccharomyces cerevisiae can partially suppress a number of yeast ochre and amber mutations, respectively. In the present study we show that the tRNA(CAGGln) gene can also function as a weak amber suppressor when expressed in cells at physiological levels. This observation is consistent with a role of tRNA(CAGGln) as an evolutionary progenitor of tRNAs that strongly decode UAG codons.