Reverse transcription priming methods affect normalisation choices for gene expression levels in oocytes and early embryos.
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ABSTRACT:
SUBMITTER: Yu B
PROVIDER: S-EPMC8314208 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundIntergenic transcription, either failure to terminate at the transcription end site (TES), or transcription initiation at other intergenic regions, is present in cultured cells and enhanced in the presence of stressors such as viral infection. Transcription termination failure has not been characterized in natural biological samples such as pre-implantation embryos which express more than 10,000 genes and undergo drastic changes in DNA methylation.ResultsUsing Automatic Readthrough Transcription Detection (ARTDeco) and data of in vivo developed bovine oocytes and embryos, we found abundant intergenic transcripts that we termed as read-outs (transcribed from 5 to 15 kb after TES) and read-ins (transcribed 1 kb up-stream of reference genes, extending up to 15 kb up-stream). Read-throughs (continued transcription from TES of expressed reference genes, 4-15 kb in length), however, were much fewer. For example, the numbers of read-outs and read-ins ranged from 3,084 to 6,565 or 33.36-66.67% of expressed reference genes at different stages of embryo development. The less copious read-throughs were at an average of 10% and significantly correlated with reference gene expression (P < 0.05). Interestingly, intergenic transcription did not seem to be random because many intergenic transcripts (1,504 read-outs, 1,045 read-ins, and 1,021 read-throughs) were associated with common reference genes across all stages of pre-implantation development. Their expression also seemed to be regulated by developmental stages because many were differentially expressed (log2 fold change ≥ 2, P < 0.05). Additionally, while gradual but un-patterned decreases in DNA methylation densities 10 kb both up- and down-stream of the intergenic transcribed regions were observed, the correlation between intergenic transcription and DNA methylation was insignificant. Finally, transcription factor binding motifs and polyadenylation signals were found in 27.2% and 12.15% of intergenic transcripts, respectively, suggesting considerable novel transcription initiation and RNA processing.ConclusionIn summary, in vivo developed oocytes and pre-implantation embryos express large numbers of intergenic transcripts, which are not related to the overall DNA methylation profiles either up- or down-stream.
| S-EPMC10246250 | biostudies-literature
Project description:L1 retrotransposons have a prominent role in reshaping mammalian genomes. To replicate, the L1 ribonucleoprotein particle (RNP) first uses its endonuclease (EN) to nick the genomic DNA. The newly generated DNA end is subsequently used as a primer to initiate reverse transcription within the L1 RNA poly(A) tail, a process known as target-primed reverse transcription (TPRT). Prior studies demonstrated that most L1 insertions occur into sequences related to the L1 EN consensus sequence (degenerate 5'-TTTT/A-3' sites) and frequently preceded by imperfect T-tracts. However, it is currently unclear whether--and to which degree--the liberated 3'-hydroxyl extremity on the genomic DNA needs to be accessible and complementary to the poly(A) tail of the L1 RNA for efficient priming of reverse transcription. Here, we employed a direct assay for the initiation of L1 reverse transcription to define the molecular rules that guide this process. First, efficient priming is detected with as few as 4 matching nucleotides at the primer 3' end. Second, L1 RNP can tolerate terminal mismatches if they are compensated within the 10 last bases of the primer by an increased number of matching nucleotides. All terminal mismatches are not equally detrimental to DNA extension, a C being extended at higher levels than an A or a G. Third, efficient priming in the context of duplex DNA requires a 3' overhang. This suggests the possible existence of additional DNA processing steps, which generate a single-stranded 3' end to allow L1 reverse transcription. Based on these data we propose that the specificity of L1 reverse transcription initiation contributes, together with the specificity of the initial EN cleavage, to the distribution of new L1 insertions within the human genome.
| S-EPMC3649969 | biostudies-literature
Project description:BackgroundReverse transcription (RT) of HIV and SIV is initiated by the binding of the acceptor stem of tRNALys3 to the primer binding site (PBS) of the viral RNA genome. Previous studies have suggested that this tRNALys3 is not the only molecule capable of priming reverse transcription, and that at least one other lysyl tRNA, tRNALys5, which has an acceptor stem sequence varying from tRNALys3 by only a single transition mutation resulting in the integration of a thymine (T) at position 8 of the PBS in the viral genome, can prime reverse transcription.ResultsWe undertook an unbiased approach, evaluating the primer binding site by deep-sequencing of HIV and SIV directly from the plasma of 15 humans and 11 macaques. We found that in humans there are low but measurable levels of viral RNA genomes harboring a PBS containing the noncanonical T at position 8 (PBS-Lys5) corresponding to the tRNAlys5 sequence and representing an average of 0.52% (range 0.07-1.6%) of the total viral population. This value is remarkably consistent with the proportion of PBS-Lys5 we identified in a cross-sectional assessment of the LANL HIV database (0.51%). In macaques chronically infected with SIVmac239, the PBS-Lys5 was also detected but at a frequency 1-log less than seen for HIV, with an average of 0.056% (range 0.01-0.09%). At this proportion, PBS-Lys5 was comparable to other transition mutations, making it impossible to determine whether the mutation observed is a result of use of tRNALys5 as an RT primer at very low levels or merely the product of in vitro cDNA synthesis/PCR error. We also identified two novel PBS sequences in HIV and SIV at low levels in vivo corresponding to tRNALys6 and tRNALys1,2, suggesting that these tRNAs may rarely also be used to prime RT. In vivo reversion of the PBS-Lys5 found in SIVmac239 was rapid and reached background levels by 30 days post-infection.ConclusionsWe conclude that while alternative tRNAs can initiate reverse transcription of HIV and SIV in vivo, their overall contributions to the replicating viral population are small.
| S-EPMC6450007 | biostudies-literature
Project description:Porcine epidemic diarrhea virus (PEDV) is a highly transmissible coronavirus that causes a severe enteric disease particularly in neonatal piglets. In this study, a rapid method for detecting PEDV was developed based on cross-priming amplification and nucleic acid test strip(CPA-NATS). Five primers specific for the N gene sequence of PEDV were used for the cross-priming amplification. Detection of amplification products based on labeled probe primers was conducted with strip binding antibody of labeled markers. The CPA method was evaluated and compared with a PCR method. The reverse transcription CPA system was further optimized for detecting PEDV RNA in clinical specimens. Results showed that the method was highly specific for the detection of PEDV, and had the same sensitivity as PCR, with detection limit of 10(-6) diluted plasmid containing the target gene of PEDV. It was also successfully applied to detecting PEDV in clinical specimens. The reverse transcription CPA-NATS detection system established in this study offers a specific, sensitive, rapid, and simple detection tool for screening PEDV, which can contribute to strategies in the effective control of PEDV in swine.
| S-EPMC4835727 | biostudies-literature
Project description:Unraveling molecular pathways responsible for regulation of early embryonic development is crucial for our understanding of female infertility. Maternal determinants that control the transition from oocyte to embryo are crucial molecules that govern developmental competence of the newly conceived zygote. We describe a series of defects that are triggered by a disruption of maternal lethal effect gene, Nlrp5. Previous studies have shown that Nlrp5 hypomorph embryos fail to develop beyond the two-cell stage. Despite its importance in preimplantation development, the mechanism by which the embryo arrest occurs remains unclear. We confirmed that Nlrp5 mutant and wild-type females possess comparable ovarian germ pool and follicular recruitment rates. However, ovulated oocytes lacking Nlrp5 have abnormal mitochondrial localization and increased activity in order to sustain physiological ATP content. This results in an accumulation of reactive oxygen species and increased cellular stress causing mitochondrial depletion. Compromised cellular state is also accompanied by increased expression of cell death inducer Bax and depletion of cytochrome c. However, neither genetic deletion (Bax/Nlrp5 double knockout) nor mimetic interference (BH4 domain or Bax inhibitory peptide) were sufficient to alleviate embryo demise caused by depletion of Nlrp5. We therefore conclude that lack of Nlrp5 in oocytes triggers premature activation of the mitochondrial pool, causing mitochondrial damage that cannot be rescued by inactivation of Bax.
| S-EPMC3364921 | biostudies-literature
Project description:Maternal obesity impairs oocyte quality and embryo development. However, the potential molecular pathways remain to be explored. In the present study, we examined the effects of obesity on telomere status in oocytes and embryos obtained from mice fed with high-fat diet (HFD). Of note, telomere shortening was observed in both oocytes and early embryos from obese mice, as evidenced by the reduced expression of telomerase reverse transcriptase and activity of telomerase. Moreover, quantitative analysis of telomere dysfunction-induced foci (TIFs) revealed that maternal obesity induces the defective telomeres in oocytes and embryos. Meanwhile, the high frequency of aneuploidy was detected in HFD oocytes and embryos as compared to controls, accompanying with the increased incidence of apoptotic blastocysts. In conclusion, these results indicate that telomere dysfunction might be a molecular pathway mediating the effects of maternal obesity on oocyte quality and embryo development.
| S-EPMC7858262 | biostudies-literature
Project description: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.
| S-EPMC10984561 | biostudies-literature
Project description:CRISPR/Cas9, a highly versatile genome-editing tool, has garnered significant attention in recent years. Despite the unique characteristics of oocytes and early embryos compared to other cell types, this technology has been increasing used in mammalian reproduction. In this comprehensive review, we elucidate the fundamental principles of CRISPR/Cas9-related methodologies and explore their wide-ranging applications in deciphering molecular intricacies during oocyte and early embryo development as well as in addressing associated diseases. However, it is imperative to acknowledge the limitations inherent to these technologies, including the potential for off-target effects, as well as the ethical concerns surrounding the manipulation of human embryos. Thus, a judicious and thoughtful approach is warranted. Regardless of these challenges, CRISPR/Cas9 technology undeniably represents a formidable tool for genome and epigenome manipulation within oocytes and early embryos. Continuous refinements in this field are poised to fortify its future prospects and applications.
| S-EPMC10594749 | biostudies-literature
Project description:Twenty-six imprinted genes were quantified in bovine in vivo produced oocytes and embryos using RNA-seq. Eighteen were detectable and their transcriptional patterns were: largely decreased (MEST and PLAGL1); first decreased and then increased (CDKN1C and IGF2R); peaked at a specific stage (PHLDA2, SGCE, PEG10, PEG3, GNAS, MEG3, DGAT1, ASCL2, NNAT, and NAP1L5); or constantly low (DIRAS3, IGF2, H19 and RTL1). These patterns reflect mRNAs that are primarily degraded, important at a specific stage, or only required at low quantities. The mRNAs for several genes were surprisingly abundant. For instance, transcripts for the maternally imprinted MEST and PLAGL1, were high in oocytes and could only be expressed from the maternal allele suggesting that their genomic imprints were not yet established/recognized. Although the mRNAs detected here were likely biallelically transcribed before the establishment of imprinted expression, the levels of mRNA during these critical stages of development have important functional consequences. Lastly, we compared these genes to their counterparts in mice, humans and pigs. Apart from previously known differences in the imprinting status, the mRNA levels were different among these four species. The data presented here provide a solid reference for expression profiles of imprinted genes in embryos produced using assisted reproductive biotechnologies.
| S-EPMC4671149 | biostudies-literature
Project description:Rapid onsite whole-genome sequencing of two suspected severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) N gene diagnostic escape samples revealed a previously unreported N gene point mutation at genome position 29195. Because the G29195T mutation occurs within a region probed by a commonly referenced U.S. CDC N gene reverse transcription (RT)-PCR assay, we hypothesize that the G29195T mutation rendered the N gene target of a proprietary commercial assay undetectable. The putative diagnostic escape G29195T mutation demonstrates the need for nearly real-time surveillance, as emergence of a novel SARS-CoV-2 variant with the potential to escape diagnostic tests continues to be a threat. IMPORTANCE Accurate diagnostic detection of SARS-CoV-2 currently depends on the large-scale deployment of RT-PCR assays. SARS-CoV-2 RT-PCR assays target predetermined regions in the viral genomes by complementary binding of primers and probes to nucleic acid sequences in the clinical samples. Potential diagnostic escapes, such as those of clinical samples harboring the G29195T mutation, may result in false-negative SARS-CoV-2 RT-PCR results. The rapid detection and sharing of potential diagnostic escapes are essential for diagnostic laboratories and manufacturers around the world, to optimize their assays as SARS-CoV-2 continues to evolve.
| S-EPMC8754125 | biostudies-literature