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Nucleic Acids Research Apr 2024LINE-1 (L1) retrotransposons are mobile genetic elements that create new genomic insertions by a copy-paste mechanism involving L1 RNA/RNP intermediates. L1 encodes two...
LINE-1 (L1) retrotransposons are mobile genetic elements that create new genomic insertions by a copy-paste mechanism involving L1 RNA/RNP intermediates. L1 encodes two ORFs, of which L1-ORF2p nicks genomic DNA and reverse transcribes L1 mRNA using the nicked DNA as a primer which base-pairs with poly(A) tail of L1 mRNA. To better understand the importance of non-templated L1 3' ends' dynamics and the interplay between L1 3' and 5' ends, we investigated the effects of genomic knock-outs and temporal knock-downs of XRN1, DCP2, and other factors. We hypothesized that in the absence of XRN1, the major 5'→3' exoribonuclease, there would be more L1 mRNA and retrotransposition. Conversely, we observed that loss of XRN1 decreased L1 retrotransposition. This occurred despite slight stabilization of L1 mRNA, but with decreased L1 RNP formation. Similarly, loss of DCP2, the catalytic subunit of the decapping complex, lowered retrotransposition despite increased steady-state levels of L1 proteins. In both XRN1 and DCP2 depletions we observed shortening of L1 3' poly(A) tails and their increased uridylation by TUT4/7. We explain the observed reduction of L1 retrotransposition by the changed qualities of non-templated L1 mRNA 3' ends demonstrating the important role of L1 3' end dynamics in L1 biology.
Topics: Humans; HeLa Cells; Long Interspersed Nucleotide Elements; Retroelements; RNA; RNA, Messenger
PubMed: 38197223
DOI: 10.1093/nar/gkad1251 -
Translational Cancer Research Dec 2023Breast cancer is one of the main causes of death among women. RNA binding proteins (RBPs) play a crucial role in the progression of breast cancer, with increasingly...
BACKGROUND
Breast cancer is one of the main causes of death among women. RNA binding proteins (RBPs) play a crucial role in the progression of breast cancer, with increasingly detailed understanding of RBP functional molecular mechanisms in breast cancer, the functional research of RBPs may help elucidate the potential mechanisms of tumor occurrence, development, invasion, metastasis and prognosis. DDB1- and CUL4-associated factor 13 (DCAF13) is an RBPs has been identified as a substrate receptor for the CUL4-DDB1 E3 ligase complex. Its expression is related to the prognosis of certain cancer. We tried to explore both co-expressed network and biological functions of DCAF13 in breast cancer.
METHODS
The Cancer Genome Atlas (TCGA) database was used to analyze the different expression of DCAF13 messenger RNA (mRNA) between normal breast tissue and breast carcinoma tissue, and the clinical data about 960 samples were downloaded from the cBio Cancer Genomics Portal (cBioPortal). The expression level of DCAF13, co-expression network, and survival were analyzed. Those with a fold change ≥1 and FDR <0.05 were considered to have statistical significance. Unsupervised clustering of differentially expressed RBPs was performed based on log2-transformed FPKM values using the "pheatmap" package in R. Genes with a Spearman score >0.55 were regarded as moderately co-expressed genes. The Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database was used to construct a co-expression network. Meanwhile, the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were used to identify the biological process cluster and pathway cluster, respectively.
RESULTS
Compared with normal breast tissue, DCAF13 mRNA expression was significantly increased in breast cancer tissue (P<0.01). The Database for Annotation, Visualization and Integrated Discovery (DAVID) was used to identify the functions of the co-expressed network. These genes were mainly enriched in mitosis, nuclear division, metabolic process, recombination, replication and repair of DNA, double-strand break repair, posttranscriptional regulation of gene expression, regulation of cell cycle, division and proliferation, regulation of protein stability and also participation in in regulation of poly(A) RNA binding, mRNA binding, tRNA binding, adenosine triphosphate (ATP) binding. KEGG pathway analysis revealed that the genes were mainly enriched in cell cycle, oocyte meiosis and oxidative phosphorylation. According to survival analysis, upregulation of DCAF13 mRNA was significant for overall survival (OS) (P=0.0163).
CONCLUSIONS
DCAF13 is up-regulated in breast cancer, the OS of patients with DCAF13 up-regulation was obviously reduced. DCAF13 was used as a diagnostic marker and therapeutic target for breast cancer. By building a co-expression network of DCAF13 and conducting bioinformatics analysis, it is possible to find the biomarker to evaluate patient prognosis. This finding provides a new target in mechanism and cell research of breast cancer.
PubMed: 38197079
DOI: 10.21037/tcr-23-1923 -
Doklady. Biochemistry and Biophysics Feb 2024The TREX-2-ORC protein complex of D. melanogaster is necessary for the export of the bulk of synthesized poly(A)-containing mRNA molecules from the nucleus to the...
The TREX-2-ORC protein complex of D. melanogaster is necessary for the export of the bulk of synthesized poly(A)-containing mRNA molecules from the nucleus to the cytoplasm through the nuclear pores. However, the role of this complex in the export of other types of RNA remains unknown. We have shown that TREX-2-ORC participates in the nuclear export of histone mRNAs: it associates with histone mRNPs, binds to histone H3 mRNA at the 3'-terminal part of the coding region, and participates in the export of histone mRNAs from the nucleus to the cytoplasm.
Topics: Animals; Active Transport, Cell Nucleus; Histones; Drosophila melanogaster; RNA, Messenger; Nuclear Proteins; Cell Nucleus
PubMed: 38189888
DOI: 10.1134/S160767292370059X -
RSC Advances Jan 2024As one of the most promising types of label-free nanopores has great potential for DNA sequencing fast detection of different DNA bases. As one of the most promising...
As one of the most promising types of label-free nanopores has great potential for DNA sequencing fast detection of different DNA bases. As one of the most promising types of label-free nanopores, two-dimensional nanopore materials have been developed over the past two decades. However, how to detect different DNA bases efficiently and accurately is still a challenging problem. In the present work, the translocation of four homogeneous DNA strands (, poly(A), poly(C), poly(G), and poly(T)) through two-dimensional transition-metal carbide (MXene) membrane nanopores with different surface terminal groups is investigated all-atom molecular dynamics simulations. Interestingly, it is found that the four types of bases can be distinguished by different ion currents and dwell times when they are transported through the TiC(OH) nanopore. This is mainly attributed to the different orientation and position distributions of the bases, the hydrogen bonding inside the MXene nanopore, and the interaction of the ssDNA with the nanopore. The present study enhances the understanding of the interaction between DNA strands and MXene nanopores with different functional groups, which may provide useful guidelines for the design of MXene-based devices for DNA sequencing in the future.
PubMed: 38188982
DOI: 10.1039/d3ra05432b -
Microbial Cell Factories Jan 2024The 5´ untranslated region (5´ UTR) plays a key role in regulating translation efficiency and mRNA stability, making it a favored target in genetic engineering and...
BACKGROUND
The 5´ untranslated region (5´ UTR) plays a key role in regulating translation efficiency and mRNA stability, making it a favored target in genetic engineering and synthetic biology. A common feature found in the 5´ UTR is the poly-adenine (poly(A)) tract. However, the effect of 5´ UTR poly(A) on protein production remains controversial. Machine-learning models are powerful tools for explaining the complex contributions of features, but models incorporating features of 5´ UTR poly(A) are currently lacking. Thus, our goal is to construct such a model, using natural 5´ UTRs from Kluyveromyces marxianus, a promising cell factory for producing heterologous proteins.
RESULTS
We constructed a mini-library consisting of 207 5´ UTRs harboring poly(A) and 34 5´ UTRs without poly(A) from K. marxianus. The effects of each 5´ UTR on the production of a GFP reporter were evaluated individually in vivo, and the resulting protein abundance spanned an approximately 450-fold range throughout. The data were used to train a multi-layer perceptron neural network (MLP-NN) model that incorporated the length and position of poly(A) as features. The model exhibited good performance in predicting protein abundance (average R = 0.7290). The model suggests that the length of poly(A) is negatively correlated with protein production, whereas poly(A) located between 10 and 30 nt upstream of the start codon (AUG) exhibits a weak positive effect on protein abundance. Using the model as guidance, the deletion or reduction of poly(A) upstream of 30 nt preceding AUG tended to improve the production of GFP and a feruloyl esterase. Deletions of poly(A) showed inconsistent effects on mRNA levels, suggesting that poly(A) represses protein production either with or without reducing mRNA levels.
CONCLUSION
The effects of poly(A) on protein production depend on its length and position. Integrating poly(A) features into machine-learning models improves simulation accuracy. Deleting or reducing poly(A) upstream of 30 nt preceding AUG tends to enhance protein production. This optimization strategy can be applied to enhance the yield of K. marxianus and other microbial cell factories.
Topics: 5' Untranslated Regions; Base Sequence; Kluyveromyces; RNA, Messenger
PubMed: 38172836
DOI: 10.1186/s12934-023-02271-3 -
Cell Research Jan 2024Here, we present a gene regulation strategy enabling programmable control over eukaryotic translational initiation. By excising the natural poly-adenylation (poly-A)...
Here, we present a gene regulation strategy enabling programmable control over eukaryotic translational initiation. By excising the natural poly-adenylation (poly-A) signal of target genes and replacing it with a synthetic control region harboring RNA-binding protein (RBP)-specific aptamers, cap-dependent translation is rendered exclusively dependent on synthetic translation initiation factors (STIFs) containing different RBPs engineered to conditionally associate with different eIF4F-binding proteins (eIFBPs). This modular design framework facilitates the engineering of various gene switches and intracellular sensors responding to many user-defined trigger signals of interest, demonstrating tightly controlled, rapid and reversible regulation of transgene expression in mammalian cells as well as compatibility with various clinically applicable delivery routes of in vivo gene therapy. Therapeutic efficacy was demonstrated in two animal models. To exemplify disease treatments that require on-demand drug secretion, we show that a custom-designed gene switch triggered by the FDA-approved drug grazoprevir can effectively control insulin expression and restore glucose homeostasis in diabetic mice. For diseases that require instantaneous sense-and-response treatment programs, we create highly specific sensors for various subcellularly (mis)localized protein markers (such as cancer-related fusion proteins) and show that translation-based protein sensors can be used either alone or in combination with other cell-state classification strategies to create therapeutic biocomputers driving self-sufficient elimination of tumor cells in mice. This design strategy demonstrates unprecedented flexibility for translational regulation and could form the basis for a novel class of programmable gene therapies in vivo.
Topics: Animals; Mice; Diabetes Mellitus, Experimental; Eukaryotic Initiation Factor-4F; Protein Processing, Post-Translational; Gene Expression Regulation; Carrier Proteins; Mammals
PubMed: 38172533
DOI: 10.1038/s41422-023-00896-y -
Frontiers in Microbiology 2023A novel virga-like virus, provisionally named Rubber tree latent virus 2 (RTLV2), was identified from rubber tree (). It is a close relative of the previously reported...
A novel virga-like virus, provisionally named Rubber tree latent virus 2 (RTLV2), was identified from rubber tree (). It is a close relative of the previously reported Rubber tree latent virus 1 (RTLV1). The complete genomes of RTLV1 and RTLV2 were sequenced and comparatively analyzed in terms of genome organization, putative gene products and phylogenetic relationship. Both RTLV1 and RTLV2 have positive-sense single-stranded RNA genomes that encode seven open reading frames (ORFs), forming a similar genomic layout. In phylogenetic analyses based on replicase and coat protein amino acid sequences, RTLV1 and RTLV2 were clustered with unclassified virga-like viruses. They are distinct from currently recognized plant virus families. RTLV1 and RTLV2 can be distinguished from members of by the presence of a putative coat protein duplex and a poly(A) tail at the 3'-terminus. The authenticity of RTLV1 and RTLV2 as infectious viruses was confirmed through field investigations and transmissibility assays. In conclusion, RTLV1 and RTLV2 represent a novel plant virus group that does not readily fit into current virus families.
PubMed: 38156006
DOI: 10.3389/fmicb.2023.1286369 -
Frontiers in Molecular Biosciences 2023Exogeneous messenger ribonucleic acid (mRNA) can be used as therapeutic and preventive medication. However, during the enzymatic production process, commonly called...
Exogeneous messenger ribonucleic acid (mRNA) can be used as therapeutic and preventive medication. However, during the enzymatic production process, commonly called transcription, by-products occur which can reduce the therapeutic efficacy of mRNA. One such by-product is double-stranded RNA (dsRNA). We therefore sought to limit the generation of dsRNA by-products during transcription. transcription was performed with a DNA template including a poly(A)-tail-encoding region, dinucleotide or trinucleotide cap analogs for cotranscriptional capping, and relevant nucleoside triphosphates. Concentrations of UTP or modified UTP (m1ΨTP) and GTP were reduced and fed over the course of the reaction. mRNA was analyzed for dsRNA contamination, yield of the reaction, RNA integrity, and capping efficiency before translational activity was assessed. Limiting the steady-state level of UTP or m1ΨTP during the enzymatic reaction reduced dsRNA formation, while not affecting mRNA yield or RNA integrity. Capping efficiency was optimized with the use of a combined GTP and UTP or m1ΨTP feed, while still reducing dsRNA formation. Lower dsRNA levels led to higher protein expression from the corresponding mRNAs. Low steady-state concentrations of UTP and GTP, fed in combination over the course of the transcription reaction, produce mRNA with high capping and low levels of dsRNA formation, resulting in high levels of protein expression. This novel approach may render laborious purification steps to remove dsRNA unnecessary.
PubMed: 38146535
DOI: 10.3389/fmolb.2023.1291045 -
International Journal of Molecular... Dec 2023The self-assembly of porphyrins onto polyelectrolytes could lead to interesting changes in their reactivity with respect to the bulk solution. Here, we investigated the...
The self-assembly of porphyrins onto polyelectrolytes could lead to interesting changes in their reactivity with respect to the bulk solution. Here, we investigated the kinetics of Zn incorporation into tetra-cationic water-soluble 5,10,15,20-tetrakis-(N-methylpyridinium-4-yl)porphyrin (TMpyP(4)) in the presence of poly(L-glutamic acid) (PGA) in a pH range from 4 to 6.5. Under these conditions, the porphyrin electrostatically interacted with the polymer, which gradually switched from an α-helical to a random coil structure. The profile of the logarithm of the observed rate constant () versus the pH was sigmoidal with an inflection point close to the pH of the conformation transition for PGA. At a pH of 5.4, when PGA was in its highly charged random coil conformation, an almost 1000-fold increase in the reaction rates was observed. An increase in the ionic strength of the bulk solution led to a decrease in the metal insertion rates. The role of the charged matrix was explained in terms of its ability to assemble both reagents in proximity, in agreement with the theory of counter-ion condensation around polyelectrolytes in an aqueous solution.
Topics: Glutamic Acid; Zinc; Polyelectrolytes; Porphyrins; Cations; Poly A; Catalysis
PubMed: 38139200
DOI: 10.3390/ijms242417371 -
Cells Dec 2023Immunotherapy using checkpoint inhibitors targeting the interaction between PD-1 on T cells and PD-L1 on cancer cells has shown significant results in non-small-cell...
Immunotherapy using checkpoint inhibitors targeting the interaction between PD-1 on T cells and PD-L1 on cancer cells has shown significant results in non-small-cell lung cancer (NSCLC). Not all patients respond to the therapy, and PD-L1 expression heterogeneity is proposed to be one determinant for this. The alternative processing of RNA, which depends on an alternative poly-A site in intron 4, generates a shorter mRNA variant () encoding soluble PD-L1 (sPD-L1), relative to the canonical mRNA encoding membrane-associated PD-L1 (mPD-L1). This study aimed to identify factors influencing the ratio between these two mRNAs in NSCLC cells. First, we verified the existence of the alternative RNA processing in NSCLC cells, and from in silico analyses, we identified a candidate list of regulatory factors. Examining selected candidates showed that CRISPR/Cas9-generated loss-of-function mutations in increased the / mRNA ratio and, accordingly, the sPD-L1/mPD-L1 balance. The CDK12/13 inhibitor THZ531 could also increase the / mRNA ratio and impact the transcriptional response to IFN-γ stimulation. The fact that CDK12 regulates transcript variant formation in NSCLC cells is consistent with CDK12's role in promoting transcriptional elongation over intron-located poly-A sites. This study lays the groundwork for clinical investigations to delineate the implications of the CDK12-mediated balancing of sPD-L1 relative to mPD-L1 for immunotherapeutic responses in NSCLC.
Topics: Humans; B7-H1 Antigen; Carcinoma, Non-Small-Cell Lung; Cyclin-Dependent Kinases; Lung Neoplasms; RNA Processing, Post-Transcriptional; RNA, Messenger
PubMed: 38132164
DOI: 10.3390/cells12242844