mRNA Synthesis Research Articles

Enhanced gene regulation by cooperation between mRNA decay and gene transcription

It has become increasingly clear in the last few years that gene expression in eukaryotes is not a linear process from mRNA synthesis in the nucleus to translation and degradation in the cytoplasm, but works as a circular one where the mRNA level is controlled by crosstalk between nuclear transcription and cytoplasmic decay pathways. One of the consequences of this crosstalk is the approximately constant level of mRNA. This is called mRNA buffering and happens when transcription and mRNA degradation act at compensatory rates. However, if transcription and mRNA degradation act additively, enhanced gene expression regulation occurs. In this work, we analyzed new and previously published genomic datasets obtained for several yeast mutants related to either transcription or mRNA decay that are not known to play any role in the other process. We show that some, which were presumed only transcription factors (Sfp1) or only decay factors (Puf3, Upf2/3), may represent examples of RNA-binding proteins (RBPs) that make specific crosstalk to enhance the control of the mRNA levels of their target genes by combining additive effects on transcription and mRNA stability. These results were mathematically modeled to see the effects of RBPs when they have positive or negative effects on mRNA synthesis and decay rates. We found that RBPs can be an efficient way to buffer or enhance gene expression responses depending on their respective effects on transcription and mRNA stability.

An insight into the active sites of the catalytic basic protein subunit PB1 of the RNA polymerase of human influenza viruses

RNA polymerase from human influenza viruses is a heterotrimeric enzyme and performs the crucial functions of both transcription and replication for multiplication of the viruses in human cells. The heterotrimeric enzyme is made up of two basic protein subunits (PB1 and PB2) and an acidic protein subunit (PA). All the three subunits perform well-defined function(s) in the transcription and replication processes in the human cells. The basic protein subunit PB1 is shown to possess the polymerization activity, whereas the PB2 and the PA subunits are found to be involved in the cap-snatching and proofreading (PR) activities, respectively. The polymerase activity in the catalytic subunit, PB1, is found to be an RNA-dependent RNA polymerase (RdRp). Multiple sequence alignment (MSA) analysis of the PB1 subunits from all the three human influenza viruses, A, B and C shows large number of highly conserved peptides, amino acid motifs and invariant amino acids. Site-directed mutagenesis (SDM) analysis and X-ray crystallographic data have shown that two completely conserved motifs, viz. –GDN- and –SDD-, are involved in binding to the catalytic Mg2+ ion. These data are in close agreement with the MSA analysis data of the polymerases from all the three human influenza viruses. Furthermore, two highly conserved polymerase catalytic regions are identified in the PB1 subunits by sequence similarity to other DNA/RNA polymerases and hence, are proposed to function in the nucleotidyl transfer activities. Presence of the two catalytic regions suggest that the polymerase may function in a dual mode, i.e., in phase I, in association with the cap-snatching subunit PB2, it could be involved in the synthesis of mRNAs (transcription mode) and once enough proteins are made from the mRNAs, in the second phase, in association with PR exonuclease subunit PA, it could switch to the replication mode to synthesize error-free, exact copies of the viral genome. For both the activities, it could use the same invariant catalytic Mg2+-binding –GDN- and –SDD- motifs.

Active mRNA degradation by EXD2 nuclease elicits recovery of transcription after genotoxic stress

The transcriptional response to genotoxic stress involves gene expression arrest, followed by recovery of mRNA synthesis (RRS) after DNA repair. We find that the lack of the EXD2 nuclease impairs RRS and decreases cell survival after UV irradiation, without affecting DNA repair. Overexpression of wild-type, but not nuclease-dead EXD2, restores RRS and cell survival. We observe that UV irradiation triggers the relocation of EXD2 from mitochondria to the nucleus. There, EXD2 is recruited to chromatin where it transiently interacts with RNA Polymerase II (RNAPII) to promote the degradation of nascent mRNAs synthesized at the time of genotoxic attack. Reconstitution of the EXD2-RNAPII partnership on a transcribed DNA template in vitro shows that EXD2 primarily interacts with an elongation-blocked RNAPII and efficiently digests mRNA. Overall, our data highlight a crucial step in the transcriptional response to genotoxic attack in which EXD2 interacts with elongation-stalled RNAPII on chromatin to potentially degrade the associated nascent mRNA, allowing transcription restart after DNA repair.

Enhancer–promoter entanglement explains their transcriptional interdependence

Enhancers not only activate target promoters to stimulate messenger RNA (mRNA) synthesis, but they themselves also undergo transcription to produce enhancer RNAs (eRNAs), the significance of which is not well understood. Transcription at the participating enhancer-promoter pair appears coordinated, but it is unclear why and how. Here, we employ cell-free transcription assays using constructs derived from the human GREB1 locus to demonstrate that transcription at an enhancer and its target promoter is interdependent. This interdependence is observable under conditions where direct enhancer-promoter contact (EPC) takes place. We demonstrate that transcription activation at a participating enhancer-promoter pair is dependent on i) the mutual availability of the enhancer and promoter, ii) the state of transcription at both the enhancer and promoter, iii) local abundance of both eRNA and mRNA, and iv) direct EPC. Our results suggest transcriptional interdependence between the enhancer and the promoter as the basis of their transcriptional concurrence and coordination throughout the genome. We propose a model where transcriptional concurrence, coordination and interdependence are possible if the participating enhancer and promoter are entangled in the form of EPC, reside in a proteinaceous bubble, and utilize shared transcriptional resources and regulatory inputs.

Mimicking Loading-Induced Cartilage Self-Heating in Vitro Promotes Matrix Formation in Chondrocyte-Laden Constructs with Different Mechanical Properties.

Articular cartilage presents a mechanically sensitive tissue. Chondrocytes, the sole cell type residing in the tissue, perceive and react to physical cues as signals that significantly modulate their behavior. Hyaline cartilage is a connective tissue with high dissipative capabilities, able to increase its temperature during daily activities, thus providing a dynamic thermal milieu for the residing chondrocytes. This condition, self-heating, which is still chiefly ignored among the scientific community, adds a new thermal dimension in cartilage mechanobiology. Motivated by the lack of studies exploring this dynamic temperature increase as a potential stimulus in cartilage-engineered constructs, we aimed to elucidate whether loading-induced evolved temperature serves as an independent or complementary regulatory cue for chondrocyte function. In particular, we evaluated the chondrocytes' response to thermal and/or mechanical stimulation in two types of scaffolds exhibiting dissipation levels close to healthy and degenerated articular cartilage. It was found, in both scaffold groups, that the combination of dynamic thermal and mechanical stimuli induced superior effects in the expression of major chondrogenic genes, such as SOX9 and LOXL2, compared to either signal alone. Similar effects were also observed in proteoglycan accumulation over time, along with increased mRNA transcription and synthesis of TRPV4, and for the first time demonstrated in chondrocytes, TREK1 ion channels. Conversely, the chondrogenic response of cells to isolated thermal or mechanical cues was generally scaffold-type dependent. Nonetheless, the significance of thermal stimulus as a chondro-inductive signal was better supported in both studied groups. Our data indicates that the temperature evolution is necessary for chondrocytes to more effectively perceive and translate applied mechanical loading.

Multidirectional Instability of Shoulder Joint: A Review

Introduction: The biomechanics of the shoulder joint are complex. To allow a functional range of motion, stability is sacrificed. Glenohumeral joint stability is maintained by static and dynamic stabilizers. Static stabilizers are non-contractile structures including capsulolabral structures and bony glenoid while dynamic stabilizers are contractile structures including rotator cuff, conjoint tendon, and long head of the bicep. The objective of this review is to elaborate on multidirectional instability (MDI) of the shoulder joint and the management of this disorder. Review: Laxity can be affected by hereditary and genetic factors. This can be seen for example in generalized joint laxity which often manifests in the shoulder as MDI. Mutations in collagen genes can cause altered collagen structure, mostly in collagen Type I, resulting in smaller and longer collagen cells. Abnormalities could be found in mRNA synthesis or amino acid transcription for collagen protein, resulting in amino acid content variation. Alterations in fine-tuning mechanism, cleavage process, ionic interaction, cross-linking and metabolism could also result in collagen quality variation. Genetic factors could also cause elastin overproduction rate or amount. Elastin amino acid content alterations could also be affected genetically. These alterations produced a looser static stabilizer. Even so, shoulder joint have multiple static stabilizers and the individual having hereditary laxity does not always have problems related to joint laxity. If the laxity caused problems, it is usually in the form of pain, discomfort, or disruption in shoulder joint function. To compensate for the loose static stabilizer, rehabilitation should be optimized on the contractile, musculature structure of the glenohumeral joint. Besides rehabilitation, capsular shift procedure could also be performed as management of the affected joint by open surgery, arthroscopic capsular plication, or arthroscopic thermal capsulorrhaphy. Conclusion: Based on the review conducted, reoperation rates for open surgery, arthroscopic technique, and thermal technique were approximately 10%, 5%, and 15% subsequently. This should be considered when choosing the appropriate surgery method for MDI management if rehabilitation on contractile structures does not improve patients’ condition.

Spatiotemporal Epigenetic Control of the Histone Gene Chromatin Landscape during the Cell Cycle.

Higher-order genomic organization supports the activation of histone genes in response to cell cycle regulatory cues that epigenetically mediates stringent control of transcription at the G1/S-phase transition. Histone locus bodies (HLBs) are dynamic, non-membranous, phase-separated nuclear domains where the regulatory machinery for histone gene expression is organized and assembled to support spatiotemporal epigenetic control of histone genes. HLBs provide molecular hubs that support synthesis and processing of DNA replication-dependent histone mRNAs. These regulatory microenvironments support long-range genomic interactions among non-contiguous histone genes within a single topologically associating domain (TAD). HLBs respond to activation of the cyclin E/CDK2/NPAT/HINFP pathway at the G1/S transition. HINFP and its coactivator NPAT form a complex within HLBs that controls histone mRNA transcription to support histone protein synthesis and packaging of newly replicated DNA. Loss of HINFP compromises H4 gene expression and chromatin formation, which may result in DNA damage and impede cell cycle progression. HLBs provide a paradigm for higher-order genomic organization of a subnuclear domain that executes an obligatory cell cycle-controlled function in response to cyclin E/CDK2 signaling. Understanding the coordinately and spatiotemporally organized regulatory programs in focally defined nuclear domains provides insight into molecular infrastructure for responsiveness to cell signaling pathways that mediate biological control of growth, differentiation phenotype, and are compromised in cancer.

Apelin promotes osteosarcoma metastasis by upregulating PLOD2 expression via the Hippo signaling pathway and hsa_circ_0000004/miR-1303 axis.

Osteosarcoma is a highly mortal bone tumor, with a high metastatic potential, promoted in part by the enzyme procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (PLOD2). Increasing level of PLOD2 in osteosarcoma tissue correlates with lymphatic and distant metastasis. The adipokine apelin (APLN) is also found in different cancers and APLN upregulation promotes angiogenesis and metastasis, but its effects on osteosarcoma metastasis are uncertain. We explored APLN functioning in metastatic osteosarcoma. An analysis of records from the Gene Expression Omnibus (GEO) database showed higher levels of APLN expression in osteosarcoma tissue than in normal tissue. Similarly, levels of APLN and PLOD2 mRNA synthesis were upregulated in osteosarcoma tissue. Levels of APLN and PLOD2 protein correlated positively with osteosarcoma clinical stages. APLN increased PLOD2 expression in human osteosarcoma cell lines and cell migration via the mammalian Sterile 20-like kinase 1 (MST1), monopolar spindle-one-binder protein (MOB)1, and YAP cascades, and through hsa_circ_0000004 functioning as a sponge of miR-1303. We also found that knockdown of APLN antagonized lung metastasis in mice with osteosarcoma. APLN may be a therapeutic target in osteosarcoma metastasis.

Oil-Based Hysterosalpingography ?mproves Leukaemia ?nhibitory Factor Expression ?n Endometrial Flushing Samples

Objective: The biological mechanism of the fertility-enhancing effect of hysterosalpingography (HSG) is not fully known. Leukemia inhibitory factor (LIF) is an important cytokine involved in implantation. This study was planned to investigate the effect of HSG on LIF mRNA expression in endometrial flushing samples. Methods: Forty infertile patients scheduled for HSG were included in the study. Before HSG, endometrial flushing was performed in the mid-luteal phase, followed by contrast-medium infusion into the cavity. Second flushing was done in the mid-luteal phase of the next cycle. Ten patients who underwent diagnostic hysteroscopy were taken as the second control group. LIF mRNA levels were measured by RT-PCR in endometrial flushing samples collected from each group. Results: The pre-HSG LIF mRNA levels of the infertile group were significantly lower than those of the fertile group. In infertile patients, LIF mRNA expression increased 3.2 times after HSG compared to baseline levels. LIF values reached fertile levels after HSG. While the LIF levels measured in the hysteroscopy group were significantly lower than in fertile patients, they were similar to the pre-HSG values of infertile patients. LIF levels of infertile patients after HSG were found to be significantly higher than those of the hysteroscopy group. Conclusions: In infertile patients, HSG contributes to receptivity by increasing LIF mRNA synthesis.

Separation of long-stranded RNAs by RP-HPLC using an octadecyl-based column with super-wide pores.

Messenger ribonucleic acids (mRNAs) have been used in vaccines for various diseases and are attracting attention as a new pharmaceutical paradigm. The purification of mRNAs is necessary because various impurities, such as template DNAs and transcription enzymes, remain in the crude product after mRNA synthesis. Among the various purification methods, reversed-phase high-performance liquid chromatography (RP-HPLC) is currently attracting attention. Herein, we optimized the pore size of the packing materials, the mobile phase composition, and the temperature of the process; we also evaluated changes in the separation patterns of RNA strands of various lengths via RP-HPLC. Additionally, single-stranded (50-1000 nucleotides in length) and double-stranded (80-500 base pairs in length) RNAs were separated while their non-denatured states were maintained by performing the analysis at 60°C using triethylammonium acetate as the mobile phase and octadecyl-based RNA-RP1 with super-wide pores (> 30nm) as the column. Furthermore, impurities in a long-stranded RNA of several thousand nucleotides synthesized by in vitro transcription were successfully separated using an RNA-RP1 column. The columns used in this study are expected to separate various RNA strands and the impurities contained in them.

The multi-faceted nature of 15 CFTR exonic variations: Impact on their functional classification and perspectives for therapy

BackgroundThe majority of variants of unknown clinical significance (VUCS) in the CFTR gene are missense variants. While change on the CFTR protein structure or function is often suspected, impact on splicing may be neglected. Such undetected splicing default of variants may complicate the interpretation of genetic analyses and the use of an appropriate pharmacotherapy. MethodsWe selected 15 variants suspected to impact CFTR splicing after in silico predictions on 319 missense variants (214 VUCS), reported in the CFTR-France database. Six specialized laboratories assessed the impact of nucleotide substitutions on splicing (minigenes), mRNA expression levels (quantitative PCR), synthesis and maturation (western blot), cellular localization (immunofluorescence) and channel function (patch clamp) of the CFTR protein. We also studied maturation and function of the truncated protein, consecutive to in-frame aberrant splicing, on additional plasmid constructs. ResultsSix of the 15 variants had a major impact on CFTR splicing by in-frame (n = 3) or out-of-frame (n = 3) exon skipping. We reclassified variants into: splicing variants; variants causing a splicing defect and the impairment of CFTR folding and/or function related to the amino acid substitution; deleterious missense variants that impair CFTR folding and/or function; and variants with no consequence on the different processes tested. ConclusionThe 15 variants have been reclassified by our comprehensive approach of in vitro experiments that should be used to properly interpret very rare exonic variants of the CFTR gene. Targeted therapies may thus be adapted to the molecular defects regarding the results of laboratory experiments.

Low Resource Integrated Platform for Production and Analysis of Capped mRNA.

The existing platform for large-scale mRNA production is fast, but consumable costs, process technicality, and complexity represent key bottlenecks limiting global mRNA biologics manufacturing. Another challenge is the lack of a consolidated platform for mRNA product characterization and assays that meet regulatory requirements. Bridging these innovation gaps to simplify processes and reduce cost would improve mRNA biologics manufacturability, especially in low-resource settings. This study develops a "cotranscriptional" capping strategy that utilizes T7 RNA polymerase, and the Vaccinia Capping System to synthesize and cap mRNA. We created an "integrated reaction buffer" that supports both capping enzymes for catalytic and in vitro transcription processes, enabling one-pot, two-step capped mRNA synthesis. Additionally, we report a novel, one-step analytic platform for rapid, quantitative, capped mRNA analysis. The assay involves target mRNA segment protection with cheap DNA primers and RNase digest of non-hybridized or non-target sequences before analysis by single nucleotide-resolving urea-polyacrylamide gel electrophoresis (PAGE). The integrated approach simplifies production processes and saves costs. Moreover, this assay has potential applications for mRNA analyses and post-transcriptional modification detection in biological samples. Finally, we propose a strategy that may enable unparalleled sequence coverage in RNase mass mapping by adapting the developed assay and replacing urea-PAGE with mass spectrometry.

TFIIS Is Crucial During Early Transcript Elongation for Transcriptional Reprogramming in Response to Heat Stress

In addition to the stage of transcriptional initiation, the production of mRNAs is regulated during elongation. Accordingly, the synthesis of mRNAs by RNA polymerase II (RNAPII) in the chromatin context is modulated by various transcript elongation factors. TFIIS is an elongation factor that stimulates the transcript cleavage activity of RNAPII to reactivate stalled elongation complexes at barriers to transcription including nucleosomes. Since Arabidopsis tfIIs mutants grow normally under standard conditions, we have exposed them to heat stress (HS), revealing that tfIIs plants are highly sensitive to elevated temperatures. Transcriptomic analyses demonstrate that particularly HS-induced genes are expressed at lower levels in tfIIs than in wildtype. Mapping the distribution of elongating RNAPII uncovered that in tfIIs plants RNAPII accumulates at the +1 nucleosome of genes that are upregulated upon HS. The promoter-proximal RNAPII accumulation in tfIIs under HS conditions conforms to that observed upon inhibition of the RNAPII transcript cleavage activity. Further analysis of the RNAPII accumulation downstream of transcriptional start sites illustrated that RNAPII stalling occurs at +1 nucleosomes that are depleted in the histone variant H2A.Z upon HS. Therefore, assistance of early transcript elongation by TFIIS is required for reprogramming gene expression to establish plant thermotolerance.

Coupling gene expression dynamics to cell size dynamics and cell cycle events: Exact and approximate solutions of the extended telegraph model

The standard model describing the fluctuations of mRNA numbers in single cells is the telegraph model which includes synthesis and degradation of mRNA, and switching of the gene between active and inactive states. While commonly used, this model does not describe how fluctuations are influenced by the cell cycle phase, cellular growth and division, and other crucial aspects of cellular biology. Here, we derive the analytical time-dependent solution of an extended telegraph model that explicitly considers the doubling of gene copy numbers upon DNA replication, dependence of the mRNA synthesis rate on cellular volume, gene dosage compensation, partitioning of molecules during cell division, cell-cycle duration variability, and cell-size control strategies. Based on the time-dependent solution, we obtain the analytical distributions of transcript numbers for lineage and population measurements in steady-state growth and also find a linear relation between the Fano factor of mRNA fluctuations and cell volume fluctuations. We show that generally the lineage and population distributions in steady-state growth cannot be accurately approximated by the steady-state solution of extrinsic noise models, i.e. a telegraph model with parameters drawn from probability distributions. This is because the mRNA lifetime is often not small enough compared to the cell cycle duration to erase the memoryof division and replication. Accurate approximations are possible when this memory is weak, e.g. for genes with bursty expression and for which there is sufficient gene dosage compensation when replication occurs.

Construction and Application of Orthogonal T7 Expression System in Eukaryote: An Overview.

The T7 system is an orthogonal transcription-system, which is characterized by simplicity, higher efficiency, and higher processivity, and it is used for protein or mRNA synthesis in various biological-systems. In comparison with prokaryotes, the construction of the T7 expression system is still on-going in eukaryotes, but it shows greatly applicable prospects. In the present paper, development of T7 expression system construction in eukaryotes is reviewed, including its construction in animal (mammalian cells, trypanosomatid protozoa, Xenopus oocytes, zebrafish), plant, and microorganism and its application in vaccine production and gene therapy. In addition, the innate challenges of T7 expression system construction in eukaryote and its potential application in vaccine production and gene therapy are discussed.

Recent Advances in Lipid Nanoparticles for Delivery of mRNA.

Messenger RNA (mRNA), which is composed of ribonucleotides that carry genetic information and direct protein synthesis, is transcribed from a strand of DNA as a template. On this basis, mRNA technology can take advantage of the body's own translation system to express proteins with multiple functions for the treatment of various diseases. Due to the advancement of mRNA synthesis and purification, modification and sequence optimization technologies, and the emerging lipid nanomaterials and other delivery systems, mRNA therapeutic regimens are becoming clinically feasible and exhibit significant reliability in mRNA stability, translation efficiency, and controlled immunogenicity. Lipid nanoparticles (LNPs), currently the leading non-viral delivery vehicles, have made many exciting advances in clinical translation as part of the COVID-19 vaccines and therefore have the potential to accelerate the clinical translation of gene drugs. Additionally, due to their small size, biocompatibility, and biodegradability, LNPs can effectively deliver nucleic acids into cells, which is particularly important for the current mRNA regimens. Therefore, the cutting-edge LNP@mRNA regimens hold great promise for cancer vaccines, infectious disease prevention, protein replacement therapy, gene editing, and rare disease treatment. To shed more lights on LNP@mRNA, this paper mainly discusses the rational of choosing LNPs as the non-viral vectors to deliver mRNA, the general rules for mRNA optimization and LNP preparation, and the various parameters affecting the delivery efficiency of LNP@mRNA, and finally summarizes the current research status as well as the current challenges. The latest research progress of LNPs in the treatment of other diseases such as oncological, cardiovascular, and infectious diseases is also given. Finally, the future applications and perspectives for LNP@mRNA are generally introduced.

RNA-controlled nucleocytoplasmic shuttling of mRNA decay factors regulates mRNA synthesis and a novel mRNA decay pathway

mRNA level is controlled by factors that mediate both mRNA synthesis and decay, including the 5’ to 3’ exonuclease Xrn1. Here we show that nucleocytoplasmic shuttling of several yeast mRNA decay factors plays a key role in determining both mRNA synthesis and decay. Shuttling is regulated by RNA-controlled binding of the karyopherin Kap120 to two nuclear localization sequences (NLSs) in Xrn1, location of one of which is conserved from yeast to human. The decaying RNA binds and masks NLS1, establishing a link between mRNA decay and Xrn1 shuttling. Preventing Xrn1 import, either by deleting KAP120 or mutating the two Xrn1 NLSs, compromises transcription and, unexpectedly, also cytoplasmic decay, uncovering a cytoplasmic decay pathway that initiates in the nucleus. Most mRNAs are degraded by both pathways - the ratio between them represents a full spectrum. Importantly, Xrn1 shuttling is required for proper responses to environmental changes, e.g., fluctuating temperatures, involving proper changes in mRNA abundance and in cell proliferation rate.

The envelope proteins from SARS-CoV-2 and SARS-CoV potently reduce the infectivity of human immunodeficiency virus type 1 (HIV-1)

BackgroundViroporins are virally encoded ion channels involved in virus assembly and release. Human immunodeficiency virus type 1 (HIV-1) and influenza A virus encode for viroporins. The human coronavirus SARS-CoV-2 encodes for at least two viroporins, a small 75 amino acid transmembrane protein known as the envelope (E) protein and a larger 275 amino acid protein known as Orf3a. Here, we compared the replication of HIV-1 in the presence of four different β-coronavirus E proteins.ResultsWe observed that the SARS-CoV-2 and SARS-CoV E proteins reduced the release of infectious HIV-1 yields by approximately 100-fold while MERS-CoV or HCoV-OC43 E proteins restricted HIV-1 infectivity to a lesser extent. Mechanistically, neither reverse transcription nor mRNA synthesis was involved in the restriction. We also show that all four E proteins caused phosphorylation of eIF2-α at similar levels and that lipidation of LC3-I could not account for the differences in restriction. However, the level of caspase 3 activity in transfected cells correlated with HIV-1 restriction in cells. Finally, we show that unlike the Vpu protein of HIV-1, the four E proteins did not significantly down-regulate bone marrow stromal cell antigen 2 (BST-2).ConclusionsThe results of this study indicate that while viroporins from homologous viruses can enhance virus release, we show that a viroporin from a heterologous virus can suppress HIV-1 protein synthesis and release of infectious virus.

Development of a Novel Platform for in-Vitro Immune-Monitoring of Antigen-Specific T Cells and Discovery of Novel Antigen-Specific TCRs Using Immortalized B Cells Transfected with mRNA of Antigen Fused to Mitd (IBMAM)

Introduction Immunomonitoring of antigen (Ag)-specific T cells is critical in evaluating cellular immune function. While peripheral mononuclear blood cells (PBMCs) are the most widely used study materials for Ag-specific T cells, low-frequency Ag-specific T cells in PBMCs remain as one unsolved technical limitation. While increasing the epitope expression level serves as an effective method in expanding low-frequency Ag-specific T cells, we propose to establish a novel platform where immortalized B cells processing the mRNA of antigens linked to MHC class-I trafficking domain (MITD) [IBMAM] are used as the means for increasing epitope expression levels. We optimized the B cell immortalization, tested epitope minigene arrangement, and compared wild-type (WT)-MITD to MITD-phosphorylational variants in antigen presentation. To determine if IBMAM platform can successfully detect and expand Ag-specific T cells, we applied this platform to HCMV-specific T cell detection and expansion using HCMV-seropositive donors’ PBMC samples. In addition, we hypothesized that the IBMAM platform would be an extremely effective tool in facilitating novel TCR discovery and validation, given the powerful high-throughput TCR sequencing nowadays. Materials and Methods To establish immortalized B cells in vitro, B cells were isolated from the HCMV-seropositive normal healthy donors (NHDs), stimulated, and transduced with a retrovirus containing BCL-6 and BCL-XL. In vitro transcription constructs for mRNA synthesis were based on the pMRNA-120bp-vector. HCMV-MITD vectors were generated containing the HCMV pp65-full length gene with either MITD-WT, various MITD mutations mimicking phosphorylation, or no MITD. HCMV specific CD8+ T cells (asteria comp) and TCRαβ-KO Jurkat cells (Promega Corporation) were used for T cell activation. Purified CD4+ T lymphocytes obtained from the NHDs PBMC were co-cultured with autologous immortalized B cells transfected with HCMV-MITD-WT mRNA and then measured for activation using IFNγ ELISA. We developed two approaches for IBMAM mediated Ag-specific T cell activation and detection based on whether IFNγ is detectable from the PBMC. For the first approach, if IFNγ is not detected, NHDs PBMC is stimulated twice using IBMAM and subsequently Ag-specific T cells are detected using IBMAM again. For the second approach, if IFNγ is detected, T cells are isolated again from NHDs PBMC and expanded using rapid expansion procedures (REP). After the first REP, Ag-specific T cells are detected using IBMAM, sorted, and a second REP is performed for further study. Results The three key components of the IBMAM system are the mRNA of antigen, MITD conjugation, and antigen presenting cells (APCs). Our data shows that B cells serving as potent APCs can be successfully immortalized by transfecting at least 5 x 105 cells with retrovirus (1MOI) and co-culturing with CD40L-IL21 expressing human fibroblasts. After coincubation with anti-HCMV T cells, B cells transfected with HCMV-MITD-WT mRNA led to a significantly higher IFNγ secretion in comparison to using HCMV mRNA containing no MITD. While previous studies indicate phosphorylation on serine/tyrosine in MITD can potentially increase MITD's efficacy in antigen presentation, our results suggest a relatively comparable antigen-presentation ability of MITD-WT with the other MITD phosphorylational variants. Our results support a mRNA concentration of 7.5-30ug per 106 B cell dose for B cell transfection, and T:B cell ratios of 1:1 or 1:2 for efficient induction of Ag-specific T cell responses. For approaches 1 and 2, successful detection, activation and expansion of HCMV-specific T cells were observed from low frequency NHDs samples using the IBMAM platform. A panel of TCRs theoretically complexed to NY-ESO-1-60-72-HLA-B*07:02 were knocked in the TCRαβ-KO Jurkat cells for expression, followed by coincubation with IBMAM platform involving B cells processing the mRNA of NY-ESO-1-MITD. Via measuring Jurkat activation, we have shown that the IBMAM platform is effective in detecting the reactive TCRs. Conclusions We have established a highly sensitive method of presenting naturally processed epitopes for immuno-monitoring that we will use for the detection of Ag-specific T cells that can be applied to patient samples with low frequency of Ag-specific T cells. This platform can also serve as an effective tool in novel antigen specific TCR discovery.

RNA polymerase II transcription attenuation at the yeast DNA repair gene DEF1 is biologically significant and dependent on the Hrp1 RNA-recognition motif.

Premature transcription termination (i.e. attenuation) is a potent gene regulatory mechanism that represses mRNA synthesis. Attenuation of RNA polymerase II is more prevalent than once appreciated, targeting 10-15% of mRNA genes in yeast through higher eukaryotes, but its significance and mechanism remain obscure. In the yeast Saccharomyces cerevisiae, polymerase II attenuation was initially shown to rely on Nrd1-Nab3-Sen1 termination, but more recently our laboratory characterized a hybrid termination pathway involving Hrp1, an RNA-binding protein in the 3'-end cleavage factor. One of the hybrid attenuation gene targets is DEF1, which encodes a repair protein that promotes degradation of polymerase II stalled at DNA lesions. In this study, we characterized the chromosomal DEF1 attenuator and the functional role of Hrp1. DEF1 attenuator mutants overexpressed Def1 mRNA and protein, exacerbated polymerase II degradation, and hindered cell growth, supporting a biologically significant DEF1 attenuator function. Using an auxin-induced Hrp1 depletion system, we identified new Hrp1-dependent attenuators in MNR2, SNG1, and RAD3 genes. An hrp1-5 mutant (L205S) known to impair binding to cleavage factor protein Rna14 also disrupted attenuation, but surprisingly no widespread defect was observed for an hrp1-1 mutant (K160E) located in the RNA-recognition motif. We designed a new RNA recognition motif mutant (hrp1-F162W) that altered a highly conserved residue and was lethal in single copy. In a heterozygous strain, hrp1-F162W exhibited dominant-negative readthrough defects at several gene attenuators. Overall, our results expand the hybrid RNA polymerase II termination pathway, confirming that Hrp1-dependent attenuation controls multiple yeast genes and may function through binding cleavage factor proteins and/or RNA.