Episodes of Rapid Recovery of the Functional Activity of the ras85D Gene in the Evolutionary History of Phylogenetically Distant Drosophila Species

As assemblies of genomes of new species with varying degrees of relationship appear, it becomes obvious that structural rearrangements of the genome, such as inversions, translocations, and transposon movements, are an essential and often the main source of evolutionary variation. In this regard, the following questions arise. How conserved are the regulatory regions of genes? Do they have a common evolutionary origin? And how and at what rate is the functional activity of genes restored during structural changes in the promoter region? In this article, we analyze the evolutionary history of the formation of the regulatory region of the ras85D gene in different lineages of the genus Drosophila, as well as the participation of mobile elements in structural rearrangements and in the replacement of specific areas of the promoter region with those of independent evolutionary origin. In the process, we substantiate hypotheses about the selection of promoter elements from a number of frequently repeated motifs with different degrees of degeneracy in the ancestral sequence, as well as about the restoration of the minimum required set of regulatory sequences using a conversion mechanism or similar.

Functional characterization of 5′ UTR cis-acting sequence elements that modulate translational efficiency in Plasmodium falciparum and humans

Background The eukaryotic parasite Plasmodium falciparum causes millions of malarial infections annually while drug resistance to common anti-malarials is further confounding eradication efforts. Translation is an attractive therapeutic target that will benefit from a deeper mechanistic understanding. As the rate limiting step of translation, initiation is a primary driver of translational efficiency. It is a complex process regulated by both cis and trans acting factors, providing numerous potential targets. Relative to model organisms and humans, P. falciparum mRNAs feature unusual 5′ untranslated regions suggesting cis-acting sequence complexity in this parasite may act to tune levels of protein synthesis through their effects on translational efficiency. Methods Here, in vitro translation is deployed to compare the role of cis-acting regulatory sequences in P. falciparum and humans. Using parasite mRNAs with high or low translational efficiency, the presence, position, and termination status of upstream “AUG”s, in addition to the base composition of the 5′ untranslated regions, were characterized. Results The density of upstream “AUG”s differed significantly among the most and least efficiently translated genes in P. falciparum, as did the average “GC” content of the 5′ untranslated regions. Using exemplars from highly translated and poorly translated mRNAs, multiple putative upstream elements were interrogated for impact on translational efficiency. Upstream “AUG”s were found to repress translation to varying degrees, depending on their position and context, while combinations of upstream “AUG”s had non-additive effects. The base composition of the 5′ untranslated regions also impacted translation, but to a lesser degree. Surprisingly, the effects of cis-acting sequences were remarkably conserved between P. falciparum and humans. Conclusions While translational regulation is inherently complex, this work contributes toward a more comprehensive understanding of parasite and human translational regulation by examining the impact of discrete cis-acting features, acting alone or in context.

sgDI-tector: defective interfering viral genome bioinformatics for detection of coronavirus subgenomic RNAs

Coronavirus RNA-dependent RNA polymerases produce subgenomic RNAs (sgRNAs) that encode viral structural and accessory proteins. User-friendly bioinformatic tools to detect and quantify sgRNA production are urgently needed to study the growing number of next-generation sequencing (NGS) data of SARS-CoV-2. We introduced sgDI-tector to identify and quantify sgRNA in SARS-CoV-2 NGS data. sgDI-tector allowed detection of sgRNA without initial knowledge of the transcription-regulatory sequences. We produced NGS data and successfully detected the nested set of sgRNAs with the ranking M>ORF3a>N>ORF6>ORF7a>ORF8>S>E>ORF7b. We also compared the level of sgRNA production with other types of viral RNA products such as defective interfering viral genomes.

Potential Contribution of IL-27 and IL-23 Gene Polymorphisms to Multiple Sclerosis Susceptibility: An Association Analysis at Genotype and Haplotype Level

(1) Background: interleukin 23 (IL-23) and interleukin 27 (IL-27) modulate the activity of T helper 17 cells (Th17) with critical roles in autoimmune diseases and multiple sclerosis (MS). The genes responsible for cytokine generation are highly influenced by the presence of single nucleotide polymorphisms (SNP) in main regions such as regulatory sequences or in promoter regions, contributing to disease susceptibility and evolution. The present study analyzed the associations of IL-23 and IL-27 SNPs with susceptibility to multiple sclerosis. (2) Methods: We performed a case-control study including 252 subjects: 157 patients diagnosed with MS and 95 controls. We used polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to determine the genotypes for IL-27 T4730С (rs 181206), IL-27 A964G (rs 153109), and IL-23 receptor gene (IL-23R) G1142A (rs 11209026). (3) Results: The IL27-T4730С gene polymorphism was significantly associated with an increased odds of MS under the dominant genetic model (TC + CC variant genotypes, adjusted odds ratio OR = 4.06, 95% CI: 2.14–7.83, p-value = 0.000007, Q-value = 0.000063). Individuals carrying the IL-27 A924G variant (AG + GG) genotype presented higher odds of MS compared to non-carriers under the dominant model (adjusted OR = 1.93, 95% CI: 1.05–3.51, p-value = 0.0324, Q-value = 0.05832) and the allelic genetic model (unadjusted p-value = 0.015, OR = 1.58, 95% CI: 1.09–2.28), while IL-23-R381Q SNP conferred a decreased odds of MS under a codominant model of inheritance (adjusted OR = 0.26, 95% CI: 0.08–0.92, p-value = 0.0276, Q-value = 0.058) and an allelic model (unadjusted p-value = 0.008, OR = 0.23, 95% CI: 0.07–0.75). In an additive model with adjustment for age group (≤40 years vs. >40 years), sex and smoking, patients carrying the G-C (A964G, T4730C) haplotype had a 3.18 increased risk (95% CI: 1.74–5.81, p < 0.001) to develop multiple sclerosis. (4) Conclusions: The results of the current study showed a significant relationship of IL-27-A964G and IL-27-T4730C polymorphisms with increased risk of MS, and also the protective role of the IL-23-R381Q polymorphism. Moreover, the haplotype-based analysis proposed the mutant G-C (A924G, T4730C) as a significant risk haplotype for the development of MS.

Transcriptome Analysis of Anther Wall Reveals Novel Insights Into the Regulatory Mechanisms Underlying Anther Wall Development in Rice

Rice is an important food staple that is consumed by half of the human population. Therefore, understanding the regulatory mechanism of male fertility in rice can improve production by enhancing the efficiency of hybrid seed production. However, information on the control mechanism of male fertility by anther dehiscence or wall development in rice is very limited. To further understand the regulatory mechanism for anther dehiscence in rice, we carried out transcriptome analysis for two tissues: the anther wall and pollen at the anthesis stage. With the anatomical meta-expression data, in addition to these tissues, the differentially expressed genes (DEGs) between the two tissues were further refined to identify 1,717 pollen-preferred genes and 534 anther wall-preferred genes. A GUS transgenic line and RT-qPCR analysis for anther wall-preferred genes supported the fidelity of our gene candidates for further analysis. The refined DEGs were functionally classified through Gene Ontology (GO) enrichment and MapMan analyses. Through the analysis of cis-acting elements and alternative splicing variants, we also suggest the feature of regulatory sequences in promoter regions for anther wall-preferred expression and provide information of the unique splicing variants in anther walls. Subsequently, it was found that hormone signaling and the resulting transcriptional regulation pathways may play an important role in anther dehiscence and anther wall development. Our result could provide useful insight for future research to broaden the molecular mechanism of anther dehiscence or anther wall development in rice.

Transposable elements mediate genetic effects altering the expression of nearby genes in colorectal cancer

Transposable elements (TEs) are interspersed repeats that contribute to more than half of the human genome, and TE-embedded regulatory sequences are increasingly recognized as major components of the human regulome. Perturbations of this system can contribute to tumorigenesis, but the impact of TEs on gene expression in cancer cells remains to be fully assessed. Here, we analyzed 275 normal colon and 276 colorectal cancer (CRC) samples from the SYSCOL colorectal cancer cohort and discovered 10,111 and 5,152 TE expression quantitative trait loci (eQTLs) in normal and tumor tissues, respectively. Amongst the latter, 376 were exclusive to CRC, likely driven by changes in methylation patterns. We identified that transcription factors are more enriched in tumor-specific TE-eQTLs than shared TE-eQTLs, indicating that TEs are more specifically regulated in tumor than normal. Using Bayesian Networks to assess the causal relationship between eQTL variants, TEs and genes, we identified that 1,758 TEs are mediators of genetic effect, altering the expression of 1,626 nearby genes significantly more in tumor compared to normal, of which 51 are cancer driver genes. We show that tumor-specific TE-eQTLs trigger the driver capability of TEs subsequently impacting expression of nearby genes. Collectively, our results highlight a global profile of a new class of cancer drivers, thereby enhancing our understanding of tumorigenesis and providing potential new candidate mechanisms for therapeutic target development.

DNA methylation in transposable elements disrupts the connection between three-dimensional chromatin organization and gene expression upon rice genome duplication.

Polyploidy serves as a major force in plant evolution and domestication of cultivated crops. However, the relationship and underlying mechanism between three-dimensional (3D) chromatin organization and gene expression upon rice genome duplication is largely unknown. Here we compared the 3D chromatin structures between diploid (2C) and autotetraploid (4C) rice by high-throughput chromosome conformation capture analysis, and found that 4C rice presents weakened intra-chromosomal interactions compared to its 2C progenitor. Moreover, we found that changes of 3D chromatin organizations including chromatin compartments, topologically associating domain (TAD) and loops uncouple from gene expression. Moreover, DNA methylations in the regulatory sequences of genes in compartment A/B switched regions and TAD boundaries are not related to their expressions. Importantly, in contrast to that there was no significant difference of methylation levels in TEs in promoters of differentially expressed genes (DEGs) and non-DEGs between 2C and 4C rice, we found that the hypermethylated transposable elements across genes in compartment A/B switched regions and TAD boundaries suppress the expression of these genes. We propose that the rice genome doubling might modulate TE methylation which results in the disconnection between the alteration of 3D chromatin structure and gene expression.

Nanopore-Based Direct RNA-Sequencing Reveals a High-Resolution Transcriptional Landscape of Porcine Reproductive and Respiratory Syndrome Virus

The TRS-mediated discontinuous transcription process is a hallmark of Arteriviruses. Precise assessment of the intricate subgenomic RNA (sg mRNA) populations is required to understand the kinetics of viral transcription. It is difficult to reconstruct and comprehensively quantify splicing events using short-read sequencing, making the identification of transcription-regulatory sequences (TRS) particularly problematic. Here, we applied long-read direct RNA sequencing to characterize the recombined RNA molecules produced in porcine alveolar macrophages during early passage infection of porcine reproductive and respiratory syndrome virus (PRRSV). Based on sequencing two PRRSV isolates, namely XM-2020 and GD, we revealed a high-resolution and diverse transcriptional landscape in PRRSV. The data revealed intriguing differences in subgenomic recombination types between the two PRRSVs while also demonstrating TRS-independent heterogeneous subpopulation not previously observed in Arteriviruses. We find that TRS usage is a regulated process and share the common preferred TRS in both strains. This study also identified a substantial number of TRS-mediated transcript variants, including alternative-sg mRNAs encoding the same annotated ORF, as well as putative sg mRNAs encoded nested internal ORFs, implying that the genetic information encoded in PRRSV may be more intensively expressed. Epigenetic modifications have emerged as an essential regulatory layer in gene expression. Here, we gained a deeper understanding of m5C modification in poly(A) RNA, elucidating a potential link between methylation and transcriptional regulation. Collectively, our findings provided meaningful insights for redefining the transcriptome complexity of PRRSV. This will assist in filling the research gaps and developing strategies for better control of the PRRS.

Profiling of open chromatin in developing pig (Sus scrofa) muscle to identify regulatory regions

There is very little information about how the genome is regulated in domestic pigs (Sus scrofa). This lack of knowledge hinders efforts to define and predict the effects of genetic variants in pig breeding programmes. In order to address this knowledge gap, we need to identify regulatory sequences in the pig genome starting with regions of open chromatin. We used the ‘Improved Protocol for the Assay for Transposase-Accessible Chromatin (Omni-ATAC-Seq)’ to identify putative regulatory regions in flash frozen semitendinosus muscle from 24 male piglets. We collected samples from the smallest, average, and largest sized male piglets from each litter through five developmental time points. Of the 4,661 ATAC-Seq peaks identified that represent regions of open chromatin, &gt;50% were within 1 kb of known transcription start sites. Differential read count analysis revealed 377 ATAC-Seq defined genomic regions where chromatin accessibility differed significantly across developmental time points. We found regions of open chromatin associated with down regulation of genes involved in muscle development that were present in small sized foetal piglets but absent in large foetal piglets at day 90 of gestation. The dataset that we have generated provides: a resource for studies of genome regulation in pigs, and contributes valuable functional annotation information to filter genetic variants for use in genomic selection in pig breeding programmes.

Identification of Novel Genomic Variations in Susceptibility to Nonsyndromic Cleft Lip and Palate Patients

Background: Nonsyndromic cleft lip with or without palate (NSCL/P) is a multifactorial and common birth malformation caused by genetic and environmental factors, as well as by teratogens. Genome-wide association studies found genetic variations with modulatory effects of NSCL/P formation in Chinese and Iranian populations. We aimed to identify the susceptibility of single-nucleotide polymorphisms (SNPs) to nonsyndromic cleft lip with or without palate in the Indian population. Material and Methods: The present study was conducted on NSCL/P cases and controls. Genomic DNA was extracted from peripheral blood and Axiom- Precision Medicine Research Array (PMRA) was performed. The Axiom-PMRA covers 902,527 markers and several thousand novel risk variants. Quality control-passed samples were included for candidate genetic variation identification, gene functional enrichment, and pathway and network analysis. Results: The genome-wide association study identified fourteen novel candidate gene SNPs that showed the most significant association with the risk of NSCL/P, and eight were predicted to have regulatory sequences. Conclusion: The GWAS study showed novel candidate genetic variations in NSCL/P formations. These findings contribute to the understanding of genetic predisposition to nonsyndromic cleft lip with or without palate.