Transcription Termination Research Articles

Comprehensive transcriptomic analysis reveals turnip mosaic virus infection and its aphid vector Myzus persicae cause large changes in gene regulatory networks and co-transcription of alternative spliced mRNAs in Arabidopsis thaliana.

Virus infection and herbivory can alter the expression of stress-responsive genes in plants. This study employed high-throughput transcriptomic and alternative splicing analysis to understand the separate and combined impacts on host gene expression in Arabidopsis thaliana by Myzus persicae (green peach aphid), and turnip mosaic virus (TuMV). By investigating changes in transcript abundance, the data shows that aphids feeding on virus infected plants intensify the number of differentially expressed stress responsive genes compared to challenge by individual stressors. This study presents evidence that the combination of virus-vector-host interactions induces significant changes in hormone and secondary metabolite biosynthesis, as well as downstream factors involved in feedback loops within hormone signaling pathways. This study also shows that gene expressions are regulated through alternative pre-mRNA splicing and the use of alternative transcription start and termination sites. These combined data suggest that complex genetic changes occur as plants adapt to the combined challenges posed by aphids and the viruses they vector. This study also provides more advanced analyses that could be used in the future to dissect the genetic mechanisms mediating tripartite interactions and inform future breeding programs.

Mutational Analysis Supports Three-Hairpin Model of Attenuator for Transcription Regulation of ilvBNC Operon in Corynebacterium glutamicum

The ilvBNC operon in Corynebacterium glutamicum encodes key enzymes for the biosynthesis of branched-chain amino acids (L-isoleucine, L-leucine, and L-valine). This operon has been studied for quite a long time, and it is assumed that three hairpin mRNA structures can be formed in its regulatory region; however, their functionality and role in the attenuation mechanism of the ilvBNC operon are not completely clear. In the present work, we performed a mutational analysis of mRNA secondary structures in the regulatory region of the C. glutamicum ilvBNC operon, which allowed us to propose a model of the regulation of its transcription involving three mRNA hairpins that essentially act as a transcription terminator, an antiterminator, and an antiantiterminator. In this work, we proved the existence of a transcription terminator in this operon and experimentally confirmed the effectiveness of its influence on the expression of the ilvBNC operon, AHAS enzyme activity, and valine production. We demonstrated the unique functional features of this attenuator, which, due to the overlapping of the terminator and antiterminator hairpins, is capable of rapid low-energy transitions between them without the complete disruption of the hairpin structures.

RNA recognition by minimal ProQ from Neisseria meningitidis.

Neisseria meningitidis minimal ProQ is a global RNA binding protein belonging to the family of FinO-domain proteins. The N. meningitidis ProQ consists only of the FinO domain accompanied by short N- and C-terminal extensions. To better understand how this minimal FinO-domain protein recognizes RNAs, we compared its binding to seven different natural RNA ligands of this protein. Next, two of these RNAs, rpmG-3' and AniS, were subject to further mutational studies. The data showed that N. meningitidis ProQ binds the lower part of the intrinsic transcription terminator hairpin, and that the single-stranded sequences on the 5' and 3' side of terminator stem are required for tight binding. However, the specific lengths of 5' and 3' RNA sequences required for optimal binding differed between the two RNAs. Additionally, our data show that the 2'-OH and 3'-OH groups of the 3' terminal ribose contribute to RNA binding by N. meningitidis ProQ. In summary, the minimal ProQ protein from N. meningitidis has generally similar requirements for RNA binding as the isolated FinO domains of other proteins of this family, but differs from them in detailed RNA features that are optimal for specific RNA recognition.

Overexpression of the RNA-binding protein NrdA affects global gene expression and secondary metabolism in Aspergillus species.

RNA-binding protein Nrd1 plays a role in RNA polymerase II transcription termination. In this study, we showed that the orthologous NrdA is important in global mRNA expression and secondary metabolism in Aspergillus species. We constructed an nrdA conditional expression strain using the Tet-On system in Aspergillus luchuenesis mut. kawachii. Downregulation of nrdA caused a severe growth defect, indicating that NrdA is essential for the proliferation of A. kawachii. Parallel RNA-sequencing and RNA immunoprecipitation-sequencing analysis identified potential NrdA-interacting transcripts, corresponding to 32% of the predicted protein-coding genes of A. kawachii. Subsequent gene ontology analysis suggested that overexpression of NrdA affects the production of secondary metabolites. To clarify this, we constructed Aspergillus nidulans, Aspergillus fumigatus, and Aspergillus oryzae strains overexpressing NrdA in the early developmental stage. Overexpression of NrdA reduced the production of sterigmatocystin and penicillin in A. nidulans, as well as that of helvolic acid and pyripyropene A in A. fumigatus. Moreover, it increased the production of kojic acid and reduced the production of penicillin in A. oryzae. These effects were accompanied by almost consistent changes in the mRNA levels of relevant genes. Collectively, these results suggest that NrdA is the essential RNA-binding protein, which plays a significant role in global gene expression and secondary metabolism in Aspergillus species.IMPORTANCENrd1, a component of the Nrd1-Nab3-Sen1 complex, is an essential RNA-binding protein involved in transcriptional termination in yeast. However, its role in filamentous fungi has not been studied. In this study, we characterized an orthologous NrdA in the Aspergillus species, identified potential NrdA-interacting mRNA, and investigated the effect of overexpression of NrdA on mRNA expression in Aspergillus luchuensis mut. kawachii. The results indicated that NrdA controls global gene expression involved in versatile metabolic pathways, including the secondary metabolic process, at least in the early developmental stage. We demonstrated that NrdA overexpression significantly affected the production of secondary metabolites in Aspergillus nidulans, Aspergillus oryzae, and Aspergillus fumigatus. Our findings are of importance to the fungal research community because the secondary metabolism is an industrially and clinically important aspect for the Aspergillus species.

Convergent and conserved roads lead to Rome: now archaea fill the gap of transcription termination.

Convergent and conserved roads lead to Rome: now archaea fill the gap of transcription termination.

Rapid and scalable personalized ASO screening in patient-derived organoids.

Personalized antisense oligonucleotides (ASOs) have achieved positive results in the treatment of rare genetic disease1. As clinical sequencing technologies continue to advance, the ability to identify patients with rare disease harbouring pathogenic genetic variants amenable to this therapeutic strategy will probably improve. Here we describe a scalable platform for generating patient-derived cellular models and demonstrate that these personalized models can be used for preclinical evaluation of patient-specific ASOs. We describe protocols for delivery of ASOs to patient-derived organoid models and confirm reversal of disease-associated phenotypes in cardiac organoids derived from a patient with Duchenne muscular dystrophy (DMD) with a structural deletion in the gene encoding dystrophin (DMD) that is amenable to treatment with existing ASO therapeutics. Furthermore, we designed novel patient-specific ASOs for two additional patients with DMD (siblings) with a deep intronic variant in the DMD gene that gives rise to a novel splice acceptor site, incorporation of a cryptic exon and premature transcript termination. We showed that treatment of patient-derived cardiac organoids with patient-specific ASOs results in restoration of DMD expression and reversal of disease-associated phenotypes. The approach outlined here provides the foundation for an expedited path towards the design and preclinical evaluation of personalized ASO therapeutics for a broad range of rare diseases.

The Psu protein of phage satellite P4 inhibits transcription termination factor ρ by forced hyper-oligomerization

Many bacteriophages modulate host transcription to favor expression of their own genomes. Phage satellite P4 polarity suppression protein, Psu, a building block of the viral capsid, inhibits hexameric transcription termination factor, ρ, by presently unknown mechanisms. Our cryogenic electron microscopy structures of ρ-Psu complexes show that Psu dimers clamp two inactive, open ρ rings and promote their expansion to higher-oligomeric states. ATPase, nucleotide binding and nucleic acid binding studies revealed that Psu hinders ρ ring closure and traps nucleotides in their binding pockets on ρ. Structure-guided mutagenesis in combination with growth, pull-down, and termination assays further delineated the functional ρ-Psu interfaces in vivo. Bioinformatic analyses revealed that Psu is associated with a wide variety of phage defense systems across Enterobacteriaceae, suggesting that Psu may regulate expression of anti-phage genes. Our findings show that modulation of the ρ oligomeric state via diverse strategies is a pervasive gene regulatory principle in bacteria.

The maize mTERF18 regulates transcriptional termination of the mitochondrial nad6 gene and is essential for kernel development.

Mitochondria are semi-autonomous organelle present in eukaryotic cells, containing their own genome and transcriptional machinery. However, their functions are intricately linked to proteins encoded by the nuclear genome. Mitochondrial transcription termination factors (mTERFs) are nucleic acid-binding proteins involved in RNA splicing and transcription termination within plant mitochondria and chloroplasts. Despite their recognized importance, the specific roles of mTERF proteins in maize remain largely unexplored. Here, we clone and functionally characterize maize mTERF18 gene. Our findings reveal that mTERF18 mutations lead to severely undifferentiated embryos, resulting in abortive phenotypes. Early kernel exhibits abnormal basal endosperm transfer layer and a significant reduction in both starch and protein accumulation in mterf18. We identify the mTERF18 gene through mapping-based cloning and validate this gene through allelic tests. mTERF18 is widely expressed across various maize tissues and encodes a highly conserved mitochondrial protein. Transcriptome data reveal that mTERF18 mutations disrupt transcriptional termination of the nad6 gene, leading to undetectable levels of Nad6 protein and reduced complex I assembly and activity. Furthermore, transmission electron microscopy observation of mterf18 endosperm uncover severe mitochondrial defects. Collectively, these findings highlight the critical role of mTERF18 in mitochondrial gene transcription termination and its pivotal impact on maize kernel development.

Deciphering the Coupling State-Dependent Transcription Termination in the Escherichia coli Galactose Operon.

The distance between the ribosome and the RNA polymerase active centers, known as the mRNA loop length, is crucial for transcription-translation coupling. Despite the existence of multiple expressomes with varying mRNA loop lengths, their invivo roles remain largely unexplored. This study examines the mechanisms governing transcription termination in the Escherichia coli galactose operon, revealing a crucial role in the transcription and translation coupling state. The operon utilizes both Rho-independent and Rho-dependent terminators. Our findings demonstrate that long-loop coupled transcription-translation complexes preferentially terminate at the upstream Rho-independent terminator, while short-loop complexes bypass it, terminating at the downstream Rho-dependent terminator. The efficiency of the Rho-independent terminator is enhanced by an extended U-track, suggesting a novel mechanism to overcome ribosome inhibition. These results uncover a new regulatory layer in transcription termination, challenging the traditional view of this process as random and highlighting a predetermined mechanism based on the coupling state. We propose that tandem terminators may function as regulatory checkpoints under fluctuating ribosome-RNAP coupling conditions, which can occur due to specific cellular states or factors affecting ribosome or RNAP binding efficiency. This suggests a previously overlooked mechanism that could refine transcription termination choices and expand our understanding of transcription regulation.

Identification of the arl1 locus controlling leaf rolling and its application in maize breeding

Increasing planting density is one of the most important strategies for generating higher maize yields. Moderate leaf rolling decreases mutual shading of leaves and increases the photosynthesis of the population and hence increases the tolerance for high-density planting. Few genes that control leaf rolling in maize have been identified, however, and their applicability for breeding programs remains unclear. Here we identified a maize abaxially rolled leaf1 (arl1) mutant with extreme abaxially rolled leaves and found that the size of the bulliform cells within the adaxial leaf blade surface increased in the arl1 mutant. Bulk segregation analysis mapping in an F2 population derived from a single cross between arl1 and inbred line Gui18421 with normal leaves identified the arl1 locus on chromosome 2. Sequential fine-mapping delimited the arl1 locus to a 233.56-kb genomic interval containing three candidate genes. Sequence alignment between arl1 and Gui18421 identified an 8-bp insertion in the coding region of Zm00001eb082500, which led to a frame shift causing premature transcription termination in arl1 mutant. Meanwhile, both deep sequencing and Sanger sequencing showed that Zm00001eb082520 was present in Gui18421 but was absent in arl1. A pair of near isogenic lines (NILs) carrying the Gui18421 allele (NILGui18421) and the arl1 allele (NILarl1) were developed, and the leaves of NILarl1 plants had greater light transmission and photosynthetic rate in the middle and lower canopy than did those of NILGui18421 plants under high-density planting. Furthermore, NILarl1 had a higher seed setting rate, more kernels per ear, and an increased kernel weight per ear than NILGui18421, and the grain yield of NILarl1 was not affected as the planting density increased, suggesting that the arl1 locus can be used for genetic improvement of high-density planting tolerance. Taken together, the identification of arl1 and evaluation of yield-related traits for NILGui18421 and NILarl1 provide an excellent target for future maize improvement.

Functionally important components of the transcription elongation complex involved in Rho-dependent termination.

Bacterial transcription terminator, Rho is an RNA-dependent ATPase that terminates transcription. Several structures of pre-termination complexes of the Rho-transcription elongation complex (EC) revealed a static picture of components of the EC that come close to the nascent RNA-bound Rho, where many of the residues of EC reside≤10Å from the Rho residues. However, the in vitro-formed Rho-EC complexes do not reveal the in vivo Rho-EC dynamic interaction patterns during the termination process. Here we report synthetic defect analyses of various combinations of the mutations in RNAP β, β' and ω-subunits, NusA, NusG, and Rho proteins to delineate the functional network of this process. Several mutations in the β-flap and β'-Zn-finger and -Clamp helices domains of RNAP are synthetically defective in the presence of Rho mutants indicating functional involvement of these domains. Mutations in the NusA RNA-binding domains were synthetically defective with the Rho mutants suggesting its involvement. Our genetic analyses also revealed functional antagonisms between the ω-subunit of RNAP and the NusG-CTD during termination. We concluded that the regions surrounding the RNA exit channel, the RNA-binding domains of NusA, the RNAP ω-subunit, and NusG-CTD constitute a functional network with Rho just before the onset of in vivo Rho-dependent termination.

Genomic insights into the probiotic potential and genes linked to gallic acid metabolism in Pediococcus pentosaceus MBBL6 isolated from healthy cow milk.

Pediococcus pentosaceus is well known for its probiotic properties, including roles in improving health, antimicrobial production, and enhancing fermented food quality. This study aimed to comprehensively analyze the whole genome of P. pentosaceus MBBL6, isolated from healthy cow milk, to assess its probiotic and antimicrobial potentials. P. pentosaceus MBBL6, isolated from a healthy cow milk at BSMRAU dairy farm, Gazipur, Bangladesh, underwent comprehensive genomic analysis, including whole genome sequencing, assembly, annotation, phylogenetic comparison, and assessment of metabolic pathways and secondary metabolites. Antimicrobial efficacy was evaluated through in-vitro and in-vivo studies, alongside in-silico exploration for potential mastitis therapy. We predicted 1,906 genes and 204 SEED sub-systems involved in carbohydrate metabolism and vitamin B complex biosynthesis, with a focus on lactose metabolism in MMBL6. Notably, 43 putative carbohydrate-active enzyme genes, including lysozymes, suggest the ability of MBBL6 for carbohydrate biotransformation and antimicrobial activity. The genome also revealed primary metabolic pathways for arginine and gallic acid metabolism and secondary metabolite gene clusters, including T3PKS and RiPP-like regions. Importantly, two bacteriocin biosynthesis gene clusters namely bovicin_255_variant and penocin_A, were identified in MBBL6. The safety assessment of MBBL6 genome revealed no virulence genes and a low pathogenicity score (0.196 out of 1.0). Several genes related to survival in gastrointestinal tract and colonization were also identified. Furthermore, MBBL6 exhibited susceptibility to a wide range of antibiotics in-vitro, and effectively suppressed mastitis pathogens in an in-vivo mouse mastitis model trial. The observed bacteriocin, particularly bovicin, demonstrated the ability to disrupt the function of an essential protein, Rho factor of mastitis pathogens by blocking transcription termination process. Taken together, our in-depth genomic analysis underscores the metabolic versatility, safety profile, and antimicrobial potential of P. pentosaceus MBBL6, suggesting its promise for applications in therapeutics, bioremediation, and biopreservation.

Defining gene ends: RNA polymerase II CTD threonine 4 phosphorylation marks transcription termination regions genome-wide.

Defining the beginning of a eukaryotic protein-coding gene is relatively simple. It corresponds to the first ribonucleotide incorporated by RNA polymerase II (Pol II) into the nascent RNA molecule. This nucleotide is protected by capping and maintained in the mature messenger RNA (mRNA). However, in higher eukaryotes, the end of mRNA is separated from the sites of transcription termination by hundreds to thousands of base pairs. Currently used genomic annotations only take account of the end of the mature transcript - the sites where pre-mRNA cleavage occurs, while the regions in which transcription terminates are unannotated. Here, we describe the evidence for a marker of transcription termination, which could be widely applicable in genomic studies. Pol II termination regions can be determined genome-wide by detecting Pol II phosphorylated on threonine 4 of its C-terminal domain (Pol II CTD-T4ph). Pol II in this state pauses before leaving the DNA template. Up to date this potent mark has been underused because the evidence for its place and role in termination is scattered acrossmultiple publications. We summarize the observations regarding Pol II CTD-T4ph in termination regions and present bioinformatic analyses that further support Pol II CTD-T4ph as a global termination mark in animals.

Proteomic characterization of regenerated cartilage following knee joint distraction; a human case-study

ABSTRACT Purpose Knee joint distraction is a surgical procedure with cartilage-regenerating properties. The composition of joint distraction-regenerated cartilage in human patients is poorly documented. In this case-study, provided a unique opportunity to biomolecularly characterize the regenerated tissue from a patient who underwent bilateral distraction and later knee replacements. Methods Knee joint distraction was conducted using an external fixation frame and total knee arthroplasty was performed several years later. Radiographic imaging was performed to assess the status of the knee joint prior, during and after clinical interventions. Following total knee replacement, cartilage biopsies were collected and processed for tissue sectioning and histochemical staining. Tandem mass-spectrometry proteomics analysis was used to characterize and compare the proteomic composition. Results Both knee joints showed joint-space improvement pre- and post-knee joint distraction. Regenerated cartilage was white with an irregular surface, while native (lateral) cartilage had a yellow appearance and smooth surface. Histochemical staining showed higher Safranin-O positivity in native cartilage compared to regenerated cartilage, and differences in collagen structure. Proteomic analysis did not reveal major differences in cartilage extracellular matrix protein abundance. Bioinformatic analyses revealed enrichment in ribosomal proteins (regenerated cartilage) and RNA Polymerase II Transcription Termination (native cartilage). Conclusion Histologically, knee joint distraction-regenerated cartilage showed less glycosaminoglycans and disorganized collagen compared to native cartilage. However, mass-spectrometry has no major differences in extracellular matrix protein abundance, with proteomic clues suggesting protein translation regulation as a potential mechanism for regeneration.

Mutagenesis studies suggest a mechanism for influenza polymerase stalling during polyadenylation.

Influenza polymerase (FluPol) carries out both viral transcription and replication using the same viral genome segment as a template to yield distinct end products. However, it remains largely unclear how FluPol synthesizes transcripts containing poly (A) tails during transcription termination, while producing fully complementary products during replication termination. In this study, through structural analysis combined with cell-based and biochemical assays, we identified that the PB1 Leu675/Asn676 and PB2 Arg38 residues of FluPol are critical for transcription termination and polyadenylation. During transcription termination, these three residues adopt the PB1 Leu675/Asn676down and PB2 Arg38out conformations, with their side chains positioned against the G12 and G14 residues of the RNA template at the 5' end. These steric hindrances block template translocation and facilitate FluPol 'stuttering' at U17, which is required for viral messenger RNA polyadenylation. Importantly, both structural analysis and mutational studies suggest that this specific conformation of these residues is unique to the transcription termination state. Overall, our findings provide novel insights into the mechanisms by which FluPol generates distinct 3' end products during transcription and replication termination.

Control of iron acquisition by multiple small RNAs unravels a new role for transcriptional terminator loops in gene regulation.

Small RNAs (sRNAs) controlling gene expression by imperfect base-pairing with mRNA(s) are widespread in bacteria. They regulate multiple genes, including genes involved in iron homeostasis, through a wide variety of mechanisms. We previously showed that OmrA and OmrB sRNAs repress the synthesis of the Escherichia coli FepA receptor for iron-enterobactin complexes. We now report that five additional sRNAs, namely RprA, RybB, ArrS, RseX and SdsR, responding to different environmental cues, also repress fepA, independently of one another. While RprA follows the canonical mechanism of pairing with the translation initiation region, repression by ArrS or RseX requires a secondary structure far upstream within the long fepA 5' untranslated region. We also demonstrate a dual action of SdsR, whose 5'-part pairs with the fepA translation initiation region while its 3'-end behaves like ArrS or RseX. Strikingly, mutation analysis shows a key role for the loops of these sRNAs' intrinsic terminators in the regulation. Furthermore, regulation depends on both the Hfq chaperone and the RNase E endonuclease. Overall, our data strongly suggest that FepA levels must be tightly controlled under a variety of conditions and highlight the diversity of mechanisms that underly the regulation of gene expression by sRNAs in bacteria.

Transcription termination and antitermination are critical for the fitness and function of the integrative and conjugative element Tn916.

Premature expression of genes in mobile genetic elements can be detrimental to their bacterial hosts. Tn916, the founding member of a large family of integrative and conjugative elements (ICEs; aka conjugative transposons), confers tetracycline-resistance and is found in several Gram-positive bacterial species. We identified a transcription terminator near one end of Tn916 that functions as an insulator that prevents expression of element genes when Tn916 is integrated downstream from an active host promoter. The terminator blocked expression of Tn916 genes needed for unwinding and rolling circle replication of the element DNA, and loss of the terminator caused a fitness defect for the host cells. Further, we identified an element-encoded antiterminator (named canT for conjugation-associated antitermination) that is essential for transcription of Tn916 genes after excision of the element from the host chromosome. We found that the antiterminator is orientation-specific, functions with heterologous promoters and terminators, is processive and is most likely a cis-acting RNA. Insulating gene expression in conjugative elements that are integrated in the chromosome is likely a key feature of the interplay between mobile genetic elements and their hosts and appears to be critical for the function and evolution of the large family of Tn916-like elements.

Influenza A virus transcription generates capped cRNAs that activate RIG-I.

During influenza A virus (IAV) infection, host pathogen receptor retinoic acid-inducible gene I (RIG-I) detects the partially complementary, 5'-triphosphorylated ends of the viral genome segments and non-canonical replication products. However, it has also been suggested that innate immune responses may be triggered by viral transcription. In this study, we investigated whether an immunostimulatory RNA is produced during IAV transcription. We show that the IAV RNA polymerase can read though the polyadenylation signal during transcription termination, generating a capped complementary RNA (ccRNA), which contains the 5' cap of an IAV mRNA and the 3' terminus of a cRNA instead of a poly(A) tail. ccRNAs are detectable in vitro and in both ribonucleoprotein reconstitution assays and IAV infections. Mutations that disrupt polyadenylation enhance ccRNA synthesis and increase RIG-I-dependent innate immune activation. Notably, while ccRNA itself is not immunostimulatory, it forms a RIG-I agonist by hybridizing with a complementary negative-sense viral RNA. These findings thus identify a novel non-canonical IAV RNA species and suggest an alternative mechanism for RIG-I activation during IAV infection.

Progress on molecular mechanisms of bacterial transcription termination.

Transcription is the process by which genetic information is copied from DNA to RNA, and it can be divided into three stages: transcription initiation, elongation, and termination. Transcription termination is the last step of gene transcription and is crucial for accurate gene expression. Two prevailing modes of transcription termination exist in bacteria: Rho-dependent termination and intrinsic termination (Rho-independent termination). Transcription termination is positively and negatively regulated by bacterial or bacteriophage proteins. In this review, we summarize the research progress of bacterial transcription and its regulation, with the aim of providing a theoretical foundation for further studies and understanding of transcription termination process.

RNA 3′end tailing safeguards cells against products of pervasive transcription termination

Premature transcription termination yields a wealth of unadenylated (pA−) RNA. Although this can be targeted for degradation by the Nuclear EXosome Targeting (NEXT) complex, possible backup pathways remain poorly understood. Here, we find increased levels of 3′ end uridylated and adenylated RNAs upon NEXT inactivation. U-tailed RNAs are mostly short and modified by the cytoplasmic tailing enzymes, TUT4/7, following their PHAX-dependent nuclear export and prior to their degradation by the cytoplasmic exosome or the exoribonuclease DIS3L2. Longer RNAs are instead adenylated redundantly by enzymes TENT2, PAPOLA and PAPOLG. These transcripts are either degraded via the nuclear Poly(A) tail eXosome Targeting (PAXT) connection or exported and removed by the cytoplasmic exosome in a translation-dependent manner. Failure to do so decreases global translation and induces cell death. We conclude that post-transcriptional 3′ end modification and removal of excess pA− RNA is achieved by tailing enzymes and export factors shared with productive RNA pathways.