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BioRxiv : the Preprint Server For... Jun 2024Apart from its well-established role in initiation of transcription, the general transcription factor TFIIB has been implicated in the termination step as well. The...
Apart from its well-established role in initiation of transcription, the general transcription factor TFIIB has been implicated in the termination step as well. The ubiquity of TFIIB involvement in termination as well as mechanistic details of its termination function, however, remains largely unexplored. To determine the prevalence of TFIIB's role in termination, we performed GRO-seq analyses in mutant (TFIIB ) and the isogenic wild type (TFIIB ) strains of yeast. Almost a three-fold increase in readthrough of the poly(A)-termination signal was observed in TFIIB mutant compared to the TFIIB cells. Of all genes analyzed in this study, nearly 74% genes exhibited a statistically significant increase in terminator readthrough in the mutant. To gain an understanding of the mechanistic basis of TFIIB involvement in termination, we performed mass spectrometry of TFIIB, affinity purified from chromatin and soluble cellular fractions, from TFIIB and TFIIB cells. TFIIB purified from the chromatin fraction of TFIIB cells exhibited significant enrichment of CF1A and Rat1 termination complexes. There was, however, a drastic decrease in TFIIB interaction with both CF1A and Rat1 termination complexes in TFIIB mutant. ChIP assay revealed that the recruitment of Pta1 subunit of CPF complex, Rna15 subunit of CF1 complex and Rat1 subunit of Rat1 complex registered nearly 90% decline in the mutant over wild type cells. The overall conclusion of these results is that TFIIB affects termination of transcription on a genome-wide scale, and TFIIB-termination factor interaction may play a crucial role in the process.
PubMed: 38915573
DOI: 10.1101/2024.02.22.581640 -
Genome Research Jun 20243'-end cleavage and polyadenylation is an essential process for eukaryotic mRNA maturation. In yeast species, the polyadenylation signals that recruit the processing...
3'-end cleavage and polyadenylation is an essential process for eukaryotic mRNA maturation. In yeast species, the polyadenylation signals that recruit the processing machinery are degenerate and remain poorly characterized compared to the well-defined regulatory elements in mammals. Here we address this question by developing deep learning models to deconvolute degenerate -regulatory elements and quantify their positional importance in mediating yeast poly(A) site formation, cleavage heterogeneity, and strength. In , cleavage heterogeneity is promoted by the depletion of U-rich elements around poly(A) sites as well as multiple occurrences of upstream UA-rich elements. Sites with high cleavage heterogeneity show overall lower strength. The site strength and tandem site distances modulate alternative polyadenylation (APA) under the diauxic stress. Finally, we develop a deep learning model to reveal the distinct motif configuration of poly(A) sites, which show more precise cleavage than Altogether, our deep learning models provide unprecedented insights into poly(A) site formation of yeast species, and our results highlight divergent poly(A) signals across distantly related species.
PubMed: 38914436
DOI: 10.1101/gr.278606.123 -
Nature Communications Jun 2024Cytoplasmic polyadenylation plays a vital role in gametogenesis; however, the participating enzymes and substrates in mammals remain unclear. Using knockout and knock-in...
Cytoplasmic polyadenylation plays a vital role in gametogenesis; however, the participating enzymes and substrates in mammals remain unclear. Using knockout and knock-in mouse models, we describe the essential role of four TENT5 poly(A) polymerases in mouse fertility and gametogenesis. TENT5B and TENT5C play crucial yet redundant roles in oogenesis, with the double knockout of both genes leading to oocyte degeneration. Additionally, TENT5B-GFP knock-in females display a gain-of-function infertility effect, with multiple chromosomal aberrations in ovulated oocytes. TENT5C and TENT5D both regulate different stages of spermatogenesis, as shown by the sterility in males following the knockout of either gene. Finally, Tent5a knockout substantially lowers fertility, although the underlying mechanism is not directly related to gametogenesis. Through direct RNA sequencing, we discovered that TENT5s polyadenylate mRNAs encoding endoplasmic reticulum-targeted proteins essential for gametogenesis. Sequence motif analysis and reporter mRNA assays reveal that the presence of an endoplasmic reticulum-leader sequence represents the primary determinant of TENT5-mediated regulation.
Topics: Animals; Female; Male; Polyadenylation; RNA, Messenger; Mice; Mice, Knockout; Spermatogenesis; Gametogenesis; Oogenesis; Polynucleotide Adenylyltransferase; Oocytes; Fertility; Mice, Inbred C57BL
PubMed: 38909026
DOI: 10.1038/s41467-024-49479-4 -
Methods in Molecular Biology (Clifton,... 2024Single-cell RNA sequencing (scRNA-seq) enables the measurement of RNA expressed from individual cells within a tissue or population. RNA expression profiles may be used...
Single-cell RNA sequencing (scRNA-seq) enables the measurement of RNA expressed from individual cells within a tissue or population. RNA expression profiles may be used to draw conclusions about cellular states, cell subtypes within the population, responses to perturbations, and cellular behavior in the context of disease. Here we describe a method for scRNA-seq via single-cell encapsulation and capture of the polyadenosine tails at the 3' end of mRNA transcripts combined with cell and molecular barcoding, allowing for the sequencing of 3' untranslated regions in order to identify expressed genes from a cell.
Topics: Single-Cell Analysis; Humans; 3' Untranslated Regions; RNA, Messenger; Sequence Analysis, RNA; Gene Expression Profiling; Animals; High-Throughput Nucleotide Sequencing; Poly A; Transcriptome
PubMed: 38907922
DOI: 10.1007/978-1-0716-3918-4_16 -
Journal of Advanced Pharmaceutical... 2024We developed innovative self-amplifying mRNA (sa-mRNA) vaccine based on the derivative of S and Nsp3 proteins, which are considered crucial adhering to human host cells....
We developed innovative self-amplifying mRNA (sa-mRNA) vaccine based on the derivative of S and Nsp3 proteins, which are considered crucial adhering to human host cells. We performed B-cell, Major histocompatibility complex (MHC) I, and II epitope which were merged with the KK and GPGPG linker. We also incorporated 5' cap sequence, Kozak sequence, replicase sequence, 3'/5' UTR, and poly A tail within the vaccine structure. The vaccine structure was subsequently docked and run the molecular dynamic simulation with TLR7 molecules. As the results of immune response simulation, the immune response was accelerated drastically up to >10-fold for immunoglobulin, interferon-γ, interleukin-2, immunoglobulin M (IgM) + immunoglobulin G (IgG) isotype, IgM isotype, and IgG1 isotype in secondary and tertiary dose, whereas natural killer cells, macrophages, and dendritic cells showed relatively high concentrations after the first dose. As our finding, the IgM + IgG, IgG1 + IgG2, and IgM level (induced by sa-mRNA vaccine) ensued three times with two-fold increase in days 25, and 50, then decreased after days 70-150. However, 150-350 days demonstrated constantly in the range of 20,000-21,000.
PubMed: 38903554
DOI: 10.4103/JAPTR.JAPTR_424_23 -
BioRxiv : the Preprint Server For... Apr 2024Poly(A)-binding protein (Pab1 in yeast) is involved in mRNA decay and translation initiation, but its molecular functions are incompletely understood. We found that...
Poly(A)-binding protein (Pab1 in yeast) is involved in mRNA decay and translation initiation, but its molecular functions are incompletely understood. We found that auxin-induced degradation of Pab1 reduced bulk mRNA and polysome abundance in a manner suppressed by deleting the catalytic subunit of decapping enzyme (), demonstrating that enhanced decapping/degradation is the major driver of reduced mRNA abundance and protein synthesis at limiting Pab1 levels. An increased median poly(A) tail length conferred by Pab1 depletion was also nullified by , suggesting that mRNA isoforms with shorter tails are preferentially decapped/degraded at limiting Pab1. In contrast to findings on mammalian cells, the translational efficiencies (TEs) of many mRNAs were altered by Pab1 depletion; however, these changes were broadly diminished by , suggesting that reduced mRNA abundance is a major driver of translational reprogramming at limiting Pab1. Thus, assembly of the closed-loop mRNP via PABP-eIF4G interaction appears to be dispensable for normal translation of most yeast mRNAs in vivo. Interestingly, histone mRNAs and proteins are preferentially diminished on Pab1 depletion dependent on Dcp2, accompanied by activation of internal cryptic promoters in the manner expected for reduced nucleosome occupancies, revealing a new layer of post-transcriptional control of histone gene expression.
PubMed: 38903079
DOI: 10.1101/2024.04.19.590253 -
Cell Systems Jun 2024Poly(A) tails are crucial for mRNA translation and degradation, but the exact relationship between tail length and mRNA kinetics remains unclear. Here, we employ a small...
Poly(A) tails are crucial for mRNA translation and degradation, but the exact relationship between tail length and mRNA kinetics remains unclear. Here, we employ a small library of identical mRNAs that differ only in their poly(A)-tail length to examine their behavior in human embryonic kidney cells. We find that tail length strongly correlates with mRNA degradation rates but is decoupled from translation. Interestingly, an optimal tail length of ∼100 nt displays the highest translation rate, which is identical to the average endogenous tail length measured by nanopore sequencing. Furthermore, poly(A)-tail length variability-a feature of endogenous mRNAs-impacts translation efficiency but not mRNA degradation rates. Stochastic modeling combined with single-cell tracking reveals that poly(A) tails provide cells with an independent handle to tune gene expression fluctuations by decoupling mRNA degradation and translation. Together, this work contributes to the basic understanding of gene expression regulation and has potential applications in nucleic acid therapeutics.
Topics: Humans; RNA, Messenger; Poly A; Protein Biosynthesis; RNA Stability; HEK293 Cells; Gene Expression Regulation
PubMed: 38901403
DOI: 10.1016/j.cels.2024.05.004 -
Molecules (Basel, Switzerland) May 2024mRNA vaccines are entering a period of rapid development. However, their synthesis is still plagued by challenges related to mRNA impurities and fragments (incomplete...
mRNA vaccines are entering a period of rapid development. However, their synthesis is still plagued by challenges related to mRNA impurities and fragments (incomplete mRNA). Most impurities of mRNA products transcribed in vitro are mRNA fragments. Only full-length mRNA transcripts containing both a 5'-cap and a 3'-poly(A) structure are viable for in vivo expression. Therefore, RNA fragments are the primary product-related impurities that significantly hinder mRNA efficacy and must be effectively controlled; these species are believed to originate from either mRNA hydrolysis or premature transcriptional termination. In the manufacturing of commercial mRNA vaccines, T7 RNA polymerase-catalyzed in vitro transcription (IVT) synthesis is a well-established method for synthesizing long RNA transcripts. This study identified a pivotal domain on the T7 RNA polymerase that is associated with erroneous mRNA release. By leveraging the advantageous properties of a T7 RNA polymerase mutant and precisely optimized IVT process parameters, we successfully achieved an mRNA integrity exceeding 91%, thereby further unlocking the immense potential of mRNA therapeutics.
Topics: RNA, Messenger; DNA-Directed RNA Polymerases; Transcription, Genetic; Viral Proteins; mRNA Vaccines
PubMed: 38893337
DOI: 10.3390/molecules29112461 -
International Journal of Molecular... Jun 2024RNA sequencing (RNA-Seq) is a powerful technique and is increasingly being used in clinical research and drug development. Currently, several RNA-Seq methods have been... (Comparative Study)
Comparative Study
RNA sequencing (RNA-Seq) is a powerful technique and is increasingly being used in clinical research and drug development. Currently, several RNA-Seq methods have been developed. However, the relative advantage of each method for degraded RNA and low-input RNA, such as RNA samples collected in the field of clinical setting, has remained unknown. The Standard method of RNA-Seq captures mRNA by poly(A) capturing using Oligo dT beads, which is not suitable for degraded RNA. Here, we used three commercially available RNA-Seq library preparation kits (SMART-Seq, xGen Broad-range, and RamDA-Seq) using random primer instead of Oligo dT beads. To evaluate the performance of these methods, we compared the correlation, the number of detected expressing genes, and the expression levels with the Standard RNA-Seq method. Although the performance of RamDA-Seq was similar to that of Standard RNA-Seq, the performance for low-input RNA and degraded RNA has decreased. The performance of SMART-Seq was better than xGen and RamDA-Seq in low-input RNA and degraded RNA. Furthermore, the depletion of ribosomal RNA (rRNA) improved the performance of SMART-Seq and xGen due to increased expression levels. SMART-Seq with rRNA depletion has relative advantages for RNA-Seq using low-input and degraded RNA.
Topics: Sequence Analysis, RNA; Humans; RNA Stability; Gene Expression Profiling; High-Throughput Nucleotide Sequencing; RNA; RNA, Ribosomal; RNA, Messenger; RNA-Seq
PubMed: 38892331
DOI: 10.3390/ijms25116143 -
Advanced Science (Weinheim,... Jun 2024Helicobacter pylori (H. pylori) infection is the primary risk factor for the pathogenesis of gastric cancer (GC). N6-methyladenosine (mA) plays pivotal roles in mRNA...
Helicobacter pylori (H. pylori) infection is the primary risk factor for the pathogenesis of gastric cancer (GC). N6-methyladenosine (mA) plays pivotal roles in mRNA metabolism and hnRNPA2B1 as an mA reader is shown to exert mA-dependent mRNA stabilization in cancer. This study aims to explore the role of hnRNPA2B1 in H. pylori-associated GC and its novel molecular mechanism. Multiple datasets and tissue microarray are utilized for assessing hnRNPA2B1 expression in response to H. pylori infection and its clinical prognosis in patients with GC. The roles of hnRNPA2B1 are investigated through a variety of techniques including glucose metabolism analysis, mA-epitranscriptomic microarray, Ribo-seq, polysome profiling, RIP-seq. In addition, hnRNPA2B1 interaction with poly(A) binding protein cytoplasmic 1 (PABPC1) is validated using mass spectrometry and co-IP. These results show that hnRNPA2B1 is upregulated in GC and correlated with poor prognosis. H. pylori infection induces hnRNPA2B1 upregulation through recruiting NF-κB to its promoter. Intriguingly, cytoplasm-anchored hnRNPA2B1 coordinated PABPC1 to stabilize its relationship with cap-binding eIF4F complex, which facilitated the translation of CIP2A, DLAT and GPX1 independent of mA modification. In summary, hnRNPA2B1 facilitates the non-mA translation of epigenetic mRNAs in GC progression by interacting with PABPC1-eIF4F complex and predicts poor prognosis for patients with GC.
PubMed: 38887155
DOI: 10.1002/advs.202309712