Read-through Transcription Research Articles

The MTR4/hnRNPK complex surveils aberrant polyadenylated RNAs with multiple exons

RNA surveillance systems degrade aberrant RNAs that result from defective transcriptional termination, splicing, and polyadenylation. Defective RNAs in the nucleus are recognized by RNA-binding proteins and MTR4, and are degraded by the RNA exosome complex. Here, we detect aberrant RNAs in MTR4-depleted cells using long-read direct RNA sequencing and 3′ sequencing. MTR4 destabilizes intronic polyadenylated transcripts generated by transcriptional read-through over one or more exons, termed 3′ eXtended Transcripts (3XTs). MTR4 also associates with hnRNPK, which recognizes 3XTs with multiple exons. Moreover, the aberrant protein translated from KCTD13 3XT is a target of the hnRNPK-MTR4-RNA exosome pathway and forms aberrant condensates, which we name KCTD13 3eXtended Transcript-derived protein (KeXT) bodies. Our results suggest that RNA surveillance in human cells inhibits the formation of condensates of a defective polyadenylated transcript-derived protein.

TOMM40-APOE chimera linking Alzheimer's highest risk genes: a new pathway for mitochondria regulation and APOE4 pathogenesis.

The patho-mechanism of apolipoprotein variant, APOE4, the strongest genetic risk for late-onset Alzheimer's disease (AD) and longevity, remains unclear. APOE's neighboring gene, TOMM40 (mitochondria protein transport channel), is associated with brain trauma outcome and aging-related cognitive decline, however its role in AD APOE4-independently is controversial. We report that TOMM40 is prone to transcription readthrough into APOE that can generate spliced TOMM40-APOE mRNA chimera (termed T9A2) detected in human neurons and other cells and tissues. T9A2 translation tethers APOE (normal APOE3 or APOE4) to near-full-length TOM40 that is targeted to mitochondria. Importantly, T9A2-APOE3 boosts mitochondrial bioenergetic capacity and decreases oxidative stress significantly more than T9A2-APOE4 and APOE3, and lacking in APOE4. We describe detailed interactomes of these actors that may inform about the activities and roles in pathogenesis. T9A2 uncovers a new candidate pathway for mitochondria regulation and oxidative stress-protection that are impaired in APOE4 genotypes and could initiate neurodegeneration.

Complex sporulation-specific expression of transcription termination factor Rho highlights its involvement in Bacillus subtilis cell differentiation

Termination factor Rho, responsible for the main factor-dependent pathway of transcription termination and the major inhibitor of antisense transcription, is an emerging regulator of various physiological processes in microorganisms. In Gram-positive bacterium Bacillus subtilis, Rho is involved in the control of cell adaptation to starvation and, in particular, in the control of sporulation, a complex differentiation program leading to formation of a highly resistant dormant spore. While the initiation of sporulation requires a decrease in Rho protein levels during the transition to stationary phase, the mechanisms regulating the expression of rho gene throughout the cell cycle remain largely unknown. Here we show that a drop in the activity of the vegetative SigA-dependent rho promoter causes the inhibition of rho expression in stationary phase. However, after the initiation of sporulation, rho gene is specifically reactivated in two compartments of the sporulating cell using distinct mechanisms. In the mother cell, rho expression occurs by read-through transcription initiated at the SigH-dependent promoter of the distal spo0F gene. In the forespore, rho gene is transcribed from the intrinsic promoter recognized by the alternative sigma factor SigF. These regulatory elements ensure the activity of Rho during sporulation, which appears important for the proper formation of spores. We provide experimental evidence that disruption of the spatiotemporal expression of rho during sporulation affects the resistance properties of spores, their morphology, and the ability to return to vegetative growth under favorable growth conditions.

Rli51 Attenuates Transcription of the Listeria Pathogenicity Island 1 Gene mpl and Functions as a Trans-Acting sRNA in Intracellular Bacteria.

Listeria pathogenicity island 1 (LIPI-1) is a genetic region containing a cluster of genes essential for virulence of the bacterial pathogen Listeria monocytogenes. Main virulence factors in LIPI-1 include long 5' untranslated regions (5'UTRs), among which is Rli51, a small RNA (sRNA) in the 5'UTR of the Zn-metalloprotease-coding mpl. So far, Rli51 function and molecular mechanisms have remained obscure. Here, we show that Rli51 exhibits a dual mechanism of regulation, functioning as a cis- and as a trans-acting sRNA. Under nutrient-rich conditions, rli51-mpl transcription is prematurely terminated, releasing a short 121-nucleotide-long sRNA. Rli51 is predicted to function as a transcription attenuator that can fold into either a terminator or a thermodynamically more stable antiterminator. We show that the sRNA Rli21/RliI binds to a single-stranded RNA loop in Rli51, which is essential to mediate premature transcription termination, suggesting that sRNA binding could stabilize the terminator fold. During intracellular infection, rli51 transcription is increased, which generates a higher abundance of the short Rli51 sRNA and allows for transcriptional read-through into mpl. Comparative intracellular bacterial transcriptomics in rli51-null mutants and the wild-type reference strain EGD-e suggests that Rli51 upregulates iron-scavenging proteins and downregulates virulence factors from LIPI-1. MS2 affinity purification confirmed that Rli51 binds transcripts of the heme-binding protein Lmo2186 and Lmo0937 in vivo. These results prove that Rli51 functions as a trans-acting sRNA in intracellular bacteria. Our research shows a growth condition-dependent mechanism of regulation for Rli51, preventing unintended mpl transcription in extracellular bacteria and regulating genes important for virulence in intracellular bacteria.

Dynamic Changes in Histone Modifications Are Associated with Differential Chromatin Interactions.

Eukaryotic genomes are organized into chromatin domains through long-range chromatin interactions which are mediated by the binding of architectural proteins, such as CTCF and cohesin, and histone modifications. Based on the published Hi-C and ChIP-seq datasets in human monocyte-derived macrophages, we identified 206 and 127 differential chromatin interactions (DCIs) that were not located within transcription readthrough regions in influenza A virus- and interferon β-treated cells, respectively, and found that the binding positions of CTCF and RAD21 within more than half of the DCI sites did not change. However, five histone modifications, H3K4me3, H3K27ac, H3K36me3, H3K9me3, and H3K27me3, showed significantly more dramatic changes than CTCF and RAD21 within the DCI sites. For H3K4me3, H3K27ac, H3K36me3, and H3K27me3, significantly more dramatic changes were observed outside than within the DCI sites. We further applied a motif scanning approach to discover proteins that might correlate with changes in histone modifications and chromatin interactions and found that PRDM9, ZNF384, and STAT2 frequently bound to DNA sequences corresponding to 1 kb genomic intervals with gains or losses of a histone modification within the DCI sites. This study explores the dynamic regulation of chromatin interactions and extends the current knowledge of the relationship between histone modifications and chromatin interactions.

Misexpression of inactive genes in whole blood is associated with nearby rare structural variants

Gene misexpression is the aberrant transcription of a gene in a context where it is usually inactive. Despite its known pathological consequences in specific rare diseases, we have a limited understanding of its wider prevalence and mechanisms in humans. To address this, we analyzed gene misexpression in 4,568 whole-blood bulk RNA sequencing samples from INTERVAL study blood donors. We found that while individual misexpression events occur rarely, in aggregate they were found in almost all samples and a third of inactive protein-coding genes. Using 2,821 paired whole-genome and RNA sequencing samples, we identified that misexpression events are enriched in cis for rare structural variants. We established putative mechanisms through which a subset of SVs lead to gene misexpression, including transcriptional readthrough, transcript fusions, and gene inversion. Overall, we develop misexpression as a type of transcriptomic outlier analysis and extend our understanding of the variety of mechanisms by which genetic variants can influence gene expression.

Context-dependent antisense transcription from a neighboring gene interferes with the expression of mNeonGreen as a functional in vivo fluorescent reporter in the chloroplast of Chlamydomonas reinhardtii.

Advancing chloroplast genetic engineering in Chlamydomonas reinhardtii remains challenging, decades after its first successful transformation. This study introduces the development of a chloroplast-optimized mNeonGreen fluorescent reporter, enabling in vivo observation through a sixfold increase in fluorescence via context-aware construct engineering. Our research highlights the influence of transcriptional readthrough and antisense mRNA pairing on post-transcriptional regulation, pointing to novel strategies foroptimizing heterologous gene expression. We further demonstrate the applicability of these insights using an accessible experimentation system using glass-bead transformation and reestablishment of photosynthesis using psbH mutants, focusing on the mitigation of transcriptional readthrough effects. By characterizing heterologous expression using regulatory elements such as PrrnS, 5'atpA, and 3' rbcL in a sense-transcriptional context, we further documented up to twofold improvement in fluorescence levels. Our findings contribute new tools for molecular biology research in the chloroplast and evidence fundamental gene regulation processes that could enable the development of more effective chloroplast engineering strategies. This work not only paves the way for more efficient genetic engineering of chloroplasts but also deepens our understanding of the regulatory mechanisms at play.

MYC Binding Near Transcriptional End Sites Regulates Basal Gene Expression, Read-Through Transcription and Intragenic Contacts.

The MYC oncoprotein regulates numerous genes involved in cellular processes such as cell cycle and mitochondrial and ribosomal structure and function. This requires heterodimerization with its partner, MAX, and binding to specific promoter and enhancer elements. Here, we show that MYC and MAX also bind near transcriptional end sites (TESs) of over one-sixth of all annotated genes. These interactions are dose-dependent, evolutionarily conserved, stabilize the normally short-lived MYC protein and regulate expression both in concert with and independent of MYC's binding elsewhere. MYC's TES binding occurs in association with other transcription factors, alters the chromatin landscape, increases nuclease susceptibility and can alter transcriptional read-through, particularly in response to certain stresses. MYC-bound TESs can directly contact promoters and may fine-tune gene expression in response to both physiologic and pathologic stimuli. Collectively, these findings support a previously unrecognized role for MYC in regulating transcription and its read-through via direct intragenic contacts between TESs and promoters.

Massively integrated coexpression analysis reveals transcriptional regulation, evolution and cellular implications of the yeast noncanonical translatome

BackgroundRecent studies uncovered pervasive transcription and translation of thousands of noncanonical open reading frames (nORFs) outside of annotated genes. The contribution of nORFs to cellular phenotypes is difficult to infer using conventional approaches because nORFs tend to be short, of recent de novo origins, and lowly expressed. Here we develop a dedicated coexpression analysis framework that accounts for low expression to investigate the transcriptional regulation, evolution, and potential cellular roles of nORFs in Saccharomyces cerevisiae.ResultsOur results reveal that nORFs tend to be preferentially coexpressed with genes involved in cellular transport or homeostasis but rarely with genes involved in RNA processing. Mechanistically, we discover that young de novo nORFs located downstream of conserved genes tend to leverage their neighbors’ promoters through transcription readthrough, resulting in high coexpression and high expression levels. Transcriptional piggybacking also influences the coexpression profiles of young de novo nORFs located upstream of genes, but to a lesser extent and without detectable impact on expression levels. Transcriptional piggybacking influences, but does not determine, the transcription profiles of de novo nORFs emerging nearby genes. About 40% of nORFs are not strongly coexpressed with any gene but are transcriptionally regulated nonetheless and tend to form entirely new transcription modules. We offer a web browser interface (https://carvunislab.csb.pitt.edu/shiny/coexpression/) to efficiently query, visualize, and download our coexpression inferences.ConclusionsOur results suggest that nORF transcription is highly regulated. Our coexpression dataset serves as an unprecedented resource for unraveling how nORFs integrate into cellular networks, contribute to cellular phenotypes, and evolve.

Epigenetic regulation of TERRA transcription and metacyclogenesis by base J in Leishmania major.

The hyper-modified DNA base J helps control termination of Pol II transcription at polycistronic transcription units (PTUs) in T. brucei and L. major , allowing epigenetic control of gene expression. The Telomere Repeat-containing RNA (TERRA) is synthesized in T. brucei by Pol I readthrough transcription of a telomeric PTU. While little is understood regarding TERRA synthesis and function, the hyper-modified DNA base J is highly enriched at telomeres in L. major promastigotes. We now show that TERRA is synthesized by Pol II in L. major and loss of base J leads to increased TERRA. For at least one site, the increased TERRA is by Pol II readthrough transcription from an adjacent PTU. Furthermore, Pol II readthrough defects and increased TERRA correlate with increased differentiation of promastigotes to the infectious metacyclic life stage and decreased cell viability. These results help explain the essential nature of base J in Leishmania and provide insight regarding epigenetic control of coding and non-coding RNA expression and parasite development during the life cycle of L. major .

HGG-40. UNRAVELING THE IMMUNOLOGIC VULNERABILITIES OF DIFFUSE HEMISPHERIC GLIOMA, H3 G34-MUTANT

Abstract BACKGROUND Insights into the pathogenesis of diffuse hemispheric glioma, H3 G34-mutant (DHG-G34), an incurable high-grade glioma, have opened new therapeutic possibilities. The DHG-G34 oncohistone mutation impairs the epigenetic regulator SETD2, which normally orchestrates DNA-mismatch-repair and negatively regulates Polycomb Repressive Complex 2 (PRC2). In DHG-G34, a mismatch-repair-deficiency phenotype is predicted, but an anti-tumor immune response is notably absent, which we hypothesize is linked to the aberrant transcriptional repression by PRC2. Here, we investigate DHG-G34 immunosuppression mechanisms in a real-world multi-omics cohort and cell line model. METHODS Clinical, molecular, and immunologic characteristics of a DHG-G34 cohort, sequenced at Caris Life Sciences, are contrasted against diffuse midline glioma (DMG) and pediatric low-grade glioma (LGG). KNS-42 DHG-G34 cell line was exposed to valemetostat (a EZH1/2 inhibitor) or DMSO in-vitro. Flow cytometry and RNA-sequencing were utilized to determine differential immunologic characteristics. RESULTS 29 DHG-G34, 51 DMG, and 52 LGG were identified. DHG-G34 median OS was 15.4 mo., like DMG (HR=1.1, p=0.686). Common DHG-G34 alterations were TP53 (93.1%), ATRX (69.0%), PDGFRA (31.0%). Tumor mutational burden (TMB) was high, >=10mt/MB, in 10% of DHG-G34, with 0 DMG or LGG events (p<0.01, q>0.05), but innate and adaptive immune fractions and antigen-presenting genes were decreased (fold-change: 0.19–0.83, q<0.05). In KNS-42, PRC2 inhibition upregulated MHC class 1 (p<0.001). Of 402 missense variants, 43 were upregulated following EHZ2 inhibition, and 4 were downregulated (q<0.05). Among immunostimulatory genes, P2RX7, which engages T-cells, was downregulated in patient samples (q<0.001) and upregulated in EZH2 inhibited KNS-42 (q<0.001). CONCLUSIONS DHG-G34 has a propensity for high TMB but remains immunologically cold. Our pilot experiment suggests inhibiting PRC2’s unique role in DHG-G34 may sensitize DHG-G34 to an immune response by inducing MHC class 1 expression and transcriptional read-through of mutated transcripts. Understanding whether treatment induces TMB in DHG-G34 may affect timing of an immunotherapeutic intervention.

Detection of novel PPP1R1B::STARD3 fusion transcript in acute myeloid leukemia: a case report

BackgroundAcute myeloid leukemia (AML) is the second most common type of leukemia in children. Although prognostic and diagnostic tests of AML patients have improved, there is still a great demand for new reliable clinical biomarkers for AML. Read-through fusion transcripts (RTFTs) are complex transcripts of adjacent genes whose molecular mechanisms are poorly understood. This is the first report of the presence of the PPP1R1B::STARD3 fusion transcript in an AML patient. Here, we investigated the presence of PPP1R1B::STARD3 RTFT in a case of AML using paired-end RNA sequencing (RNA-seq).Case presentationA Persian 12-year-old male was admitted to Dr. Sheikh Hospital of Mashhad, Iran, in September 2019 with the following symptoms, including fever, convulsions, hemorrhage, and bone pain. The patient was diagnosed with AML (non-M3-FAB subtype) based on cell morphologies and immunophenotypical features. Chromosomal analysis using the G-banding technique revealed t (9;22) (q34;q13).ConclusionsSingle-cell RNA sequencing (scRNA-seq) analysis suggested that the PPP1R1B promoter may be responsible for the PPP1R1B::STARD3 expression. Alterations in the level of lipid metabolites implicate cancer development, and this fusion can play a crucial role in the cholesterol movement in cancer cells. PPP1R1B::STARD3 may be considered a candidate for targeted therapies of the cholesterol metabolic and the PI3K/AKT signaling pathways involved in cancer development and progression.

Unraveling the immunologic vulnerabilities of diffuse hemispheric glioma, H3 G34-mutant.

2082 Background: Recent insights into the molecular basis of diffuse hemispheric glioma, H3 G34-mutant (DHG-G34), an incurable high-grade glioma, have opened new therapeutic possibilities. The oncohistone mutation in DHG-G34 impairs the epigenetic regulator SETD2, which normally orchestrates DNA-mismatch-repair and negatively regulates the transcriptional silencing activity of the Polycomb Repressive Complex 2 (PRC2). In DHG-G34, a mismatch-repair-deficiency phenotype is predicted, but an anti-tumor immune response is notably absent in clinical observations, which we posit is linked to the aberrant transcriptional repression by PRC2. Here, we investigate DHG-G34 immunosuppression mechanisms in a large real-world multi-omics cohort and human cell lines. Methods: Clinical, molecular, and immunologic characteristics of a DHG-G34 cohort, sequenced (DNA and RNA) at Caris Life Sciences (Phoenix, AZ), are contrasted against diffuse midline glioma (DMG) and pediatric low-grade glioma (LGG). A significant difference between genomic subgroups was defined as fold-change > 1.2. Statistical significance was determined using chi-square, Fishers-exact, and Mann Whitney U tests with corrections for multiple hypothesis testing (q < 0.05).Hazard ratio (HR) was calculated using the Cox proportional hazards model. KNS-42 DHG-G34 cell line was exposed in triplicate to 1 µM valemetostat (an EZH1/2 inhibitor) or DMSO in vitro. Flow cytometry and RNA-sequencing were utilized to determine differential immunologic characteristics. Results: 29 DHG-G34, 51 DMG, and 52 LGG were identified in Caris database. Median OS among DHG-G34 was 15.4 mo., similar to DMG (HR = 1.1, 95% CI: 0.70-1.73, p = 0.686). Most frequent alterations in DHG-G34 were TP53 (93.1%), ATRX (69.0%), PDGFRA (31.0%). Tumor mutational burden (TMB) was high, >=10mt/MB, in 10% of DHG-G34 and 0/51 in DMG and 0/52 LGG (p < 0.01, q > 0.05), but immune fractions including dendritic cells, T cells, macrophages, microglia, and antigen-presenting genes were decreased (fold-change: 0.19 – 0.83, q < 0.05). In KNS-42, PRC2 inhibition upregulated MHC class 1 (p < 0.001). Of 402 KNS-42 missense variants, transcription of 43 were upregulated following EHZ2 inhibition, and 4 were downregulated (q < 0.05). Among immunostimulatory genes, P2RX7, which engages T-cells and stimulates an inflammatory response, was downregulated in patient samples (q < 0.001) and upregulated in KNS-42 following EZH2 inhibition (q < 0.001). Conclusions: DHG-G34 has an increased propensity for high TMB but remains immunologically cold. Our pilot experiment suggests inhibiting PRC2’s unique role in DHG-G34 may sensitize DHG-G34 to an immune response by inducing MHC class 1 expression and transcriptional readthrough of mutated transcripts. Understanding whether treatment induces TMB in DHG-G34 may affect timing of an immunotherapeutic intervention.

Readon: a novel algorithm to identify read-through transcripts with long-read sequencing data.

There are many clustered transcriptionally active regions in the human genome, in which the transcription complex cannot immediately terminate transcription at the upstream gene termination site, but instead continues to transcribe intergenic regions and downstream genes, resulting in read-through transcripts. Several studies have demonstrated the regulatory roles of read-through transcripts in tumorigenesis and development. However, limited by the read length of next-generation sequencing, discovery of read-through transcripts has been slow. For long but also erroneous third-generation sequencing data, this study developed a novel minimizer sketch algorithm to accurately and quickly identify read-through transcripts. Readon initially splits the reference sequence into distinct active regions. It employs a sliding window approach within each region, calculates minimizers, and constructs the specialized structured arrays for query indexing. Following initial alignment anchor screening of candidate read-through transcripts, further confirmation steps are executed. Comparative assessments against existing software reveal Readon's superior performance on both simulated and validated real data. Additionally, two downstream tools are provided: one for predicting whether a read-through transcript is likely to undergo nonsense-mediated decay or encodes a protein, and another for visualizing splicing patterns. Readon is freely available on GitHub (https://github.com/Bulabula45/Readon).

Long-read sequencing for 29 immune cell subsets reveals disease-linked isoforms

Alternative splicing events are a major causal mechanism for complex traits, but they have been understudied due to the limitation of short-read sequencing. Here, we generate a full-length isoform annotation of human immune cells from an individual by long-read sequencing for 29 cell subsets. This contains a number of unannotated transcripts and isoforms such as a read-through transcript of TOMM40-APOE in the Alzheimer’s disease locus. We profile characteristics of isoforms and show that repetitive elements significantly explain the diversity of unannotated isoforms, providing insight into the human genome evolution. In addition, some of the isoforms are expressed in a cell-type specific manner, whose alternative 3’-UTRs usage contributes to their specificity. Further, we identify disease-associated isoforms by isoform switch analysis and by integration of several quantitative trait loci analyses with genome-wide association study data. Our findings will promote the elucidation of the mechanism of complex diseases via alternative splicing.

Expression of human RECQL5 in Saccharomyces cerevisiae causes transcription defects and transcription-associated genome instability

RECQL5 is a member of the conserved RecQ family of DNA helicases involved in the maintenance of genome stability that is specifically found in higher eukaryotes and associates with the elongating RNA polymerase II. To expand our understanding of its function we expressed human RECQL5 in the yeast Saccharomyces cerevisiae, which does not have a RECQL5 ortholog. We found that RECQL5 expression leads to cell growth inhibition, increased genotoxic sensitivity and transcription-associated hyperrecombination. Chromatin immunoprecipitation and transcriptomic analysis of yeast cells expressing human RECQL5 shows that this is recruited to transcribed genes and although it causes only a weak impact on gene expression, in particular at G + C-rich genes, it leads to a transcription termination defect detected as readthrough transcription. The data indicate that the interaction between RNAPII and RECQL5 is conserved from yeast to humans. Unexpectedly, however, the RECQL5-ID mutant, previously shown to have reduced the association with RNAPII in vitro, associates with the transcribing polymerase in cells. As a result, expression of RECQL5-ID leads to similar although weaker phenotypes than wild-type RECQL5 that could be transcription-mediated. Altogether, the data suggests that RECQL5 has the intrinsic ability to function in transcription-dependent and independent genome dynamics in S. cerevisiae.

Environment modulates protein heterogeneity through transcriptional and translational stop codon readthrough

Stop codon readthrough events give rise to longer proteins, which may alter the protein’s function, thereby generating short-lasting phenotypic variability from a single gene. In order to systematically assess the frequency and origin of stop codon readthrough events, we designed a library of reporters. We introduced premature stop codons into mScarlet, which enabled high-throughput quantification of protein synthesis termination errors in E. coli using fluorescent microscopy. We found that under stress conditions, stop codon readthrough may occur at rates as high as 80%, depending on the nucleotide context, suggesting that evolution frequently samples stop codon readthrough events. The analysis of selected reporters by mass spectrometry and RNA-seq showed that not only translation but also transcription errors contribute to stop codon readthrough. The RNA polymerase was more likely to misincorporate a nucleotide at premature stop codons. Proteome-wide detection of stop codon readthrough by mass spectrometry revealed that temperature regulated the expression of cryptic sequences generated by stop codon readthrough in E. coli. Overall, our findings suggest that the environment affects the accuracy of protein production, which increases protein heterogeneity when the organisms need to adapt to new conditions.

Protein phosphatase PP1 regulation of RNA polymerase II transcription termination and allelic exclusion of VSG genes in trypanosomes.

The genomes of Leishmania and trypanosomes are organized into polycistronic transcription units flanked by a modified DNA base J involved in promoting RNA polymerase II (Pol II) termination. We recently characterized a Leishmania complex containing a J-binding protein, PP1 protein phosphatase 1, and PP1 regulatory protein (PNUTS) that controls transcription termination potentially via dephosphorylation of Pol II by PP1. While T. brucei contains eight PP1 isoforms, none purified with the PNUTS complex, complicating the analysis of PP1 function in termination. We now demonstrate that the PP1-binding motif of TbPNUTS is required for function in termination in vivo and that TbPP1-1 modulates Pol II termination in T. brucei and dephosphorylation of the large subunit of Pol II. PP1-1 knock-down results in increased cellular levels of phosphorylated RPB1 accompanied by readthrough transcription and aberrant transcription of the chromosome by Pol II, including Pol I transcribed loci that are typically silent, such as telomeric VSG expression sites involved in antigenic variation. These results provide important insights into the mechanism underlying Pol II transcription termination in primitive eukaryotes that rely on polycistronic transcription and maintain allelic exclusion of VSG genes.

Multiple regulatory inputs including cell envelope stress orchestrate expression of the Escherichia coli rpoN operon.

The rpoN operon, an important regulatory hub in Enterobacteriaceae, includes rpoN encoding sigma factor σ54, hpf involved in ribosome hibernation, rapZ regulating glucosamine-6-phosphate levels, and two genes encoding proteins of the nitrogen-related phosphotransferase system. Little is known about regulatory mechanisms controlling the abundance of these proteins. This study employs transposon mutagenesis and chemical screens to dissect the complex expression of the rpoN operon. We find that envelope stress conditions trigger read-through transcription into the rpoN operon from a promoter located upstream of the preceding lptA-lptB locus. This promoter is controlled by the envelope stress sigma factor E and response regulator PhoP is required for its full response to a subset of stress signals. σE also stimulates ptsN-rapZ-npr expression using an element downstream of rpoN, presumably by interfering with mRNA processing by RNase E. Additionally, we identify a novel promoter in the 3' end of rpoN that directs transcription of the distal genes in response to ethanol. Finally, we show that translation of hpf and ptsN is individually regulated by the RNA chaperone Hfq, perhaps involving small RNAs. Collectively, our work demonstrates that the rpoN operon is subject to complex regulation, integrating signals related to envelope stress and carbon source quality.

Kinetic pathway of HIV-1 TAR cotranscriptional folding.

The Trans-Activator Receptor (TAR) RNA, located at the 5'-end untranslated region (5' UTR) of the human immunodeficiency virus type 1 (HIV-1), is pivotal in the virus's life cycle. As the initial functional domain, it folds during the transcription of viral mRNA. Although TAR's role in recruiting the Tat protein for trans-activation is established, the detailed kinetic mechanisms at play during early transcription, especially at points of temporary transcriptional pausing, remain elusive. Moreover, the precise physical processes of transcriptional pause and subsequent escape are not fully elucidated. This study focuses on the folding kinetics of TAR and the biological implications by integrating computer simulations of RNA folding during transcription with nuclear magnetic resonance (NMR) spectroscopy data. The findings reveal insights into the folding mechanism of a non-native intermediate that triggers transcriptional pause, along with different folding pathways leading to transcriptional pause and readthrough. The profiling of the cotranscriptional folding pathway and identification of kinetic structural intermediates reveal a novel mechanism for viral transcriptional regulation, which could pave the way for new antiviral drug designs targeting kinetic cotranscriptional folding pathways in viral RNAs.