Ginsenoside Compound K Assisted G-Quadruplex Folding and Regulated G-Quadruplex-Containing Transcription

Ginsenoside compound K (CK) is one of the major metabolites of the bioactive ingredients in Panax ginseng, which presents excellent bioactivity and regulates the expression of important proteins. In this work, the effects of CK on G-quadruplexes (G4s) were quantitatively analyzed in the presence and absence of their complementary sequences. CK was demonstrated to facilitate the formation of G4s, and increase the quantity of G4s in the competition with duplex. Thermodynamic experiments suggested that the electrostatic interactions were important for G4 stabilization by CK. CK was further found to regulate the transcription of G4-containing templates, reduce full-length transcripts, and decrease the transcription efficiency. Our results provide new evidence for the pharmacological study of ginsenosides at the gene level.

Internal Promoters and Their Effects on the Transcription of Operon Genes for Epothilone Production in Myxococcus xanthus

The biosynthetic genes for secondary metabolites are often clustered into giant operons with no transcription terminator before the end. The long transcripts are frangible and the transcription efficiency declines along with the process. Internal promoters might occur in operons to coordinate the transcription of individual genes, but their effects on the transcription of operon genes and the yield of metabolites have been less investigated. Epothilones are a kind of antitumor polyketides synthesized by seven multifunctional enzymes encoded by a 56-kb operon. In this study, we identified multiple internal promoters in the epothilone operon. We performed CRISPR-dCas9–mediated transcription activation of internal promoters, combined activation of different promoters, and activation in different epothilone-producing M. xanthus strains. We found that activation of internal promoters in the operon was able to promote the gene transcription, but the activation efficiency was distinct from the activation of separate promoters. The transcription of genes in the operon was influenced by not only the starting promoter but also internal promoters of the operon; internal promoters affected the transcription of the following and neighboring upstream/downstream genes. Multiple interferences between internal promoters thus changed the transcriptional profile of operon genes and the production of epothilones. Better activation efficiency for the gene transcription and the epothilone production was obtained in the low epothilone-producing strains. Our results highlight that interactions between promoters in the operon are critical for the gene transcription and the metabolite production efficiency.

Ccr4-Not complex reduces transcription efficiency in heterochromatin

Heterochromatic silencing is thought to occur through a combination of transcriptional silencing and RNA degradation, but the relative contribution of each pathway is not known. In this study we analyzed RNA Polymerase II (RNA Pol II) occupancy and levels of nascent and steady-state RNA in different strains of fission yeast, in order to quantify the contribution of each pathway to heterochromatic silencing. We found that transcriptional silencing consists of two components, reduced RNA Pol II accessibility and, unexpectedly, reduced transcriptional efficiency. Heterochromatic loci showed lower transcriptional output compared to euchromatic loci, despite the presence of comparable amounts of RNA Pol II in both types of regions. We determined that the Ccr4-Not complex and H3K9 methylation are required for reduced transcriptional efficiency in heterochromatin and that a subset of heterochromatic RNA is degraded more rapidly than euchromatic RNA. Finally, we quantified the contribution of different chromatin modifiers, RNAi and RNA degradation to each silencing pathway. Our data show that several pathways contribute to heterochromatic silencing in a locus-specific manner and reveal transcriptional efficiency as a new mechanism of silencing.

MEF2C shapes the microtranscriptome during differentiation of skeletal muscles

Myocyte enhancer factor 2C (MEF2C) is a transcription factor that regulates heart and skeletal muscle differentiation and growth. Several protein-encoding genes were identified as targets of this factor; however, little is known about its contribution to the microtranscriptome composition and dynamics in myogenic programs. In this report, we aimed to address this question. Deep sequencing of small RNAs of human muscle cells revealed a set of microRNAs (miRNAs), including several muscle-specific miRNAs, that are sensitive to MEF2C depletion. As expected, in cells with knockdown of MEF2C, we found mostly downregulated miRNAs; nevertheless, as much as one-third of altered miRNAs were upregulated. The majority of these changes are driven by transcription efficiency. Moreover, we found that MEF2C affects nontemplated 3′-end nucleotide addition of miRNAs, mainly oligouridylation. The rate of these modifications is associated with the level of TUT4 which mediates RNA 3′-uridylation. Finally, we found that a quarter of miRNAs which significantly changed upon differentiation of human skeletal myoblasts is inversely altered in MEF2C deficient cells. We concluded that MEF2C is an essential factor regulating both the quantity and quality of the microtranscriptome, leaving an imprint on the stability and perhaps specificity of many miRNAs during the differentiation of muscle cells.

Promoter engineering improves transcription efficiency in biomolecular assays

A novel T7 promoter with improved transcription efficiency has been developed. It is more suitable for diagnostic applications due to its small size and is successfully used for an RNase H activity assay with high sensitivity and selectivity.

In-Silico analysis reveals lower transcription efficiency of C241T variant of SARS-CoV-2 with host replication factors MADP1 and HNRNP-1

ABSTRACTNovel severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has claimed more than 1.5 million lives worldwide and counting. As per the GISAID database, the genomics of SARS-CoV2 is extensively studied with more than 500 genome submissions per day. Out of several hotspot mutations within the SARS-CoV-2 genome, researchers have focused a lot on missense variants but the least work is done on the UTRs. One of the most frequent 5’ UTR variants in the SARS-CoV-2 genome is the C241T with a global frequency of more than 0.9. In the present study, the effect of the C241T mutation has been studied with respect to change in RNA structure and its interaction with the host replication factors MADP1 Zinc finger CCHC-type and RNA-binding motif 1 (hnRNP1). The results obtained from molecular docking and molecular dynamics simulation indicated weaker interaction of C241T mutant stem loops with host transcription factor MADP1 indicating reduced replication efficiency. The results are also correlated with increased recovery rates and decreased death rates of global SARS-CoV-2 cases.

TATA-Like Boxes in RNA Polymerase III Promoters: Requirements for Nucleotide Sequences

tRNA and some other non-coding RNA genes are transcribed by RNA polymerase III (pol III), due to the presence of intragenic promoter, consisting of boxes A and B spaced by 30–40 bp. Such pol III promoters, called type 2, are also intrinsic to Short Interspersed Elements (SINEs). The contribution of 5′-flanking sequences to the transcription efficiency of genes containing type 2 promoters is still studied insufficiently. Here, we studied this issue, focusing on the genes of two small non-coding RNAs (4.5SH and 4.5SI), as well as B1 and B2 SINEs from the mouse genome. We found that the regions from position −31 to −24 may significantly influence the transcription of genes and SINEs. We studied the influence of nucleotide substitutions in these sites, representing TATA-like boxes, on transcription of 4.5SH and 4.5SI RNA genes. As a rule, the substitutions of A and T to G or C reduced the transcription level, although the replacement of C with A also lowered it. In 4.5SH gene, five distal nucleotides of −31/−24 box (TTCAAGTA) appeared to be the most important, while in the box −31/−24 of 4.5SI gene (CTACATGA), all nucleotides, except for the first one, contributed significantly to the transcription efficiency. Random sequences occurring at positions −31/−24 upstream of SINE copies integrated into genome, promoted their transcription with different efficacy. In the 5′-flanking sequences of 4.5SH and 4.5SI RNA genes, the recognition sites of CREB, C/EBP, and Sp1 factors were found, and their deletion decreased the transcription.