Identification of Hammerhead-variant ribozyme sequences in SARS-CoV-2.

The SARS-CoV-2 RNA virus and variants, responsible for the COVID-19 pandemic has become endemic, raised a need for further understanding of the viral genome and biology. Despite vast research on SARS-CoV-2, no ribozymes have been found in the virus genome. Here we report the identification of 39 Hammerhead-variant ribozyme sequences (CoV-HHRz) in SARS-CoV-2. These sequences are highly conserved within SARS-CoV-2 variants but show large diversity among other coronaviruses. In vitro CoV-HHRz sequences possess the characteristics of typical ribozymes; cleavage is pH and ion dependent, although their activity is relatively low and Mn2+ is required for cleavage. The cleavage sites of four CoV-HHRz coincide with the breakpoint of expressed subgenomic RNA (sgRNAs) in SARS-CoV-2 transcriptome data suggesting in vivo activity. The CoV-HHRz are involved in processing sgRNAs for ORF7b, ORF 10 and ORF1ab nsp13 which are essential for viral packaging and life cycle.

Padi6 expression patterns in buffalo oocytes and preimplantation embryos.

The subcortical maternal complex, which consists of maternal-effect genes, plays a crucial role in the development of oocytes and preimplantation embryo until the activation of the zygote genome. One such gene, known as peptidyl-arginine deiminase VI (Padi6), is involved in the oocyte maturation, fertilization and embryonic development. However, the precise function of Padi6 gene in buffalo is still unclear and requires further investigation. In this study, the sequence, mRNA and protein expression patterns of Padi6 gene were analyzed in oocytes, preimplantation embryos and somatic tissues of buffalo. The coding sequence of gene was successfully cloned and characterized. Real-time quantitative PCR results indicated an absence of Padi6 transcripts in somatic tissues. Notably, the expression levels of Padi6 in oocytes showed an increased from the germinal vesicle stage to metaphase II stage, followed by a rapid decrease during the morula and blastocyst stages. Immunofluorescence analysis confirmed these findings, revealing a noticeable decline in protein expression levels. Our research provides the initial comprehensive expression profile of Padi6 in buffalo oocytes and preimplantation embryos, serving as a solid foundation for further investigations into the functionality of maternal-effect genes in buffalo.