Nascent Transcripts Research Articles

A Systematic Identification of RNA-Binding Proteins (RBPs) Driving Aberrant Splicing in Cancer

Background: Alternative Splicing (AS) is a post-transcriptional process that allows a single RNA to produce different mRNA variants and, in some cases, multiple proteins. Various processes, many yet to be discovered, regulate AS. This study focuses on regulation by RNA-binding proteins (RBPs), which are not only crucial for splicing regulation but also linked to cancer prognosis and are emerging as therapeutic targets for cancer treatment. CLIP-seq experiments help identify where RBPs bind on nascent transcripts, potentially revealing changes in splicing status that suggest causal relationships. Selecting specific RBPs for CLIP-seq experiments is often driven by a priori hypotheses. Results: We developed an algorithm to detect RBPs likely related to splicing changes between conditions by integrating several CLIP-seq databases and a differential splicing detection algorithm. This work refines a previous study by improving splicing event prediction, testing different enrichment statistics, and performing additional validation experiments. The new method provides more accurate predictions and is included in the Bioconductor package EventPointer 3.14. We tested the algorithm in four experiments involving knockdowns of seven different RBPs. The algorithm accurately assessed the statistical significance of these RBPs using only splicing alterations. Additionally, we applied the algorithm to study sixteen cancer types from The Cancer Genome Atlas (TCGA) and three from TARGET. We identified relationships between RBPs and various cancer types, including alterations in CREBBP and MBNL2 in adenocarcinomas of the lung, liver, prostate, rectum, stomach, and colon. Some of these findings are validated in the literature, while others are novel. Conclusions: The developed algorithm enhances the ability to predict and understand RBP-related splicing changes, offering more accurate predictions and novel insights into cancer-related splicing alterations. This work highlights the potential of RBPs as therapeutic targets and contributes to the broader understanding of their roles in cancer biology.

The first chicken oocyte nucleus whole transcriptomic profile defines the spectrum of maternal mRNA and non-coding RNA genes transcribed by the lampbrush chromosomes.

Lampbrush chromosomes, with their unusually high rate of nascent RNA synthesis, provide a valuable model for studying mechanisms of global transcriptome up-regulation. Here, we obtained a whole-genomic profile of transcription along the entire length of all lampbrush chromosomes in the chicken karyotype. With nuclear RNA-seq, we obtained information about a wider set of transcripts, including long non-coding RNAs retained in the nucleus and stable intronic sequence RNAs. For a number of protein-coding genes, we visualized their nascent transcripts on the lateral loops of lampbrush chromosomes by RNA-FISH. The set of genes transcribed on the lampbrush chromosomes is required for basic cellular processes and is characterized by a broad expression pattern. We also present the first high-throughput transcriptome characterization of miRNAs and piRNAs in chicken oocytes at the lampbrush chromosome stage. Major targets of predicted piRNAs include CR1 and long terminal repeat (LTR) containing retrotransposable elements. Transcription of tandem repeat arrays was demonstrated by alignment against the whole telomere-to-telomere chromosome assemblies. We show that transcription of telomere-derived RNAs is initiated at adjacent LTR elements. We conclude that hypertranscription on the lateral loops of giant lampbrush chromosomes is required for synthesizing large amounts of transferred to the embryo maternal RNA for thousands of genes.

M6Am sequesters PCF11 to suppress premature termination and drive neuroblastoma differentiation

N6,2'-O-dimethyladenosine (m6Am) is an abundant mRNA modification that impacts multiple diseases, but its function remains controversial because the m6Am reader is unknown. Using quantitative proteomics, we identified transcriptional terminator premature cleavage factor II (PCF11) as a m6Am-specific reader in human cells. Direct quantification of mature versus nascent RNAs reveals that m6Am does not regulate mRNA stability but promotes nascent transcription. Mechanistically, m6Am functions by sequestering PCF11 away from proximal RNA polymerase II (RNA Pol II). This suppresses PCF11 from dissociating RNA Pol II near transcription start sites, thereby promoting full-length transcription of m6Am-modified RNAs. m6Am's unique relationship with PCF11 means m6Am function is enhanced when PCF11 is reduced, which occurs during all-trans-retinoic-acid (ATRA)-induced neuroblastoma-differentiation therapy. Here, m6Am promotes expression of ATF3, which represses neuroblastoma biomarker MYCN. Depleting m6Am suppresses MYCN repression in ATRA-treated neuroblastoma and maintains their tumor-stem-like properties. Collectively, we characterize m6Am as an anti-terminator RNA modification that suppresses premature termination and modulates neuroblastoma's therapeutic response.

Transcriptome-wide analysis reveal dynamic expression changes during endoplasmic reticulum stress in tomato

Endoplasmic reticulum (ER) stress is a cellular condition induced by environmental stressors. Transcriptomic approaches reveal the response of plants to ER stress and provide a broadened view. Tomato plants were treated with tunicamycin, and total RNA isolated from the tomato leaves was sequenced on an RNA-sequencing platform to be analyzed for differential expressions. A total of 856 differentially expressed genes (DEGs) were discovered in tunicamycin-treated tomato leaves. Upregulation of ER stress marker gene expressions was detected after 2h, while a prolonged ER stress downregulated most of the protein-coding genes. Pathways such as: response to stress, jasmonic acid mediated signaling, signal transduction regulation, plant hormone signal transduction, and protein processing in ER were enriched. Prolonged stress mostly resulted in reduced transcript levels, possibly related to a mechanism functioning to lower the load of nascent transcripts in the ER. Our findings emphasize the key role of JA-regulated signaling in tomato ER stress responses due to the differential expressions of the pathway components, along with other plant hormones and signaling-related or regulatory genes. ER stress being a common response to many stressors in plants, transcriptome data obtained here will provide for further studies of understanding plant stress tolerance or generating stress-resistant tomato plants.

PRC2-EZH1 contributes to circadian gene expression by orchestrating chromatin states and RNA polymerase II complex stability.

Circadian rhythmicity of gene expression is a conserved feature of cell physiology. This involves fine-tuning between transcriptional and post-transcriptional mechanisms and strongly depends on the metabolic state of the cell. Together these processes guarantee an adaptive plasticity of tissue-specific genetic programs. However, it is unclear how the epigenome and RNA Pol II rhythmicity are integrated. Here we show that the PcG protein EZH1 has a gateway bridging function in postmitotic skeletal muscle cells. On the one hand, the circadian clock master regulator BMAL1 directly controls oscillatory behavior and periodic assembly of core components of the PRC2-EZH1 complex. On the other hand, EZH1 is essential for circadian gene expression at alternate Zeitgeber times, through stabilization of RNA Polymerase II preinitiation complexes, thereby controlling nascent transcription. Collectively, our data show that PRC2-EZH1 regulates circadian transcription both negatively and positively by modulating chromatin states and basal transcription complex stability.

MONITTR allows real-time imaging of transcription and endogenous proteins in C. elegans.

Maximizing cell survival under stress requires rapid and transient adjustments of RNA and protein synthesis. However, capturing these dynamic changes at both single-cell level and across an organism has been challenging. Here, we developed a system named MONITTR (MS2-embedded mCherry-based monitoring of transcription) for real-time simultaneous measurement of nascent transcripts and endogenous protein levels in C. elegans. Utilizing this system, we monitored the transcriptional bursting of fasting-induced genes and found that the epidermis responds to fasting by modulating the proportion of actively transcribing nuclei and transcriptional kinetics of individual alleles. Additionally, our findings revealed the essential roles of the transcription factors NHR-49 and HLH-30 in governing the transcriptional kinetics of fasting-induced genes under fasting. Furthermore, we tracked transcriptional dynamics during heat-shock response and ER unfolded protein response and observed rapid changes in the level of nascent transcripts under stress conditions. Collectively, our study provides a foundation for quantitatively investigating how animals spatiotemporally modulate transcription in various physiological and pathological conditions.

Transcriptional inhibition after irradiation occurs preferentially at highly expressed genes in a manner dependent on cell cycle progression

In response to DNA double-strand damage, ongoing transcription is inhibited to facilitate accurate DNA repair while transcriptional recovery occurs after DNA repair is complete. However, the mechanisms at play and the identity of the transcripts being regulated in this manner are unclear. In contrast to the situation following UV damage, we found that transcriptional recovery after ionizing radiation (IR) occurs in a manner independent of the HIRA histone chaperone. Sequencing of the nascent transcripts identified a programmed transcriptional response, where certain transcripts and pathways are rapidly downregulated after IR, while other transcripts and pathways are upregulated. Specifically, most of the loss of nascent transcripts occurring after IR is due to inhibition of transcriptional initiation of the highly transcribed histone genes and the rDNA. To identify factors responsible for transcriptional inhibition after IR in an unbiased manner, we performed a whole genome gRNA library CRISPR/Cas9 screen. Many of the top hits on our screen were factors required for protein neddylation. However, at short times after inhibition of neddylation, transcriptional inhibition still occurred after IR, even though neddylation was effectively inhibited. Persistent inhibition of neddylation blocked transcriptional inhibition after IR, and it also leads to cell cycle arrest. Indeed, we uncovered that many inhibitors and conditions that lead to cell cycle arrest in G1 or G2 phase also prevent transcriptional inhibition after IR. As such, it appears that transcriptional inhibition after IR occurs preferentially at highly expressed genes in cycling cells.

Transcriptional inhibition after irradiation occurs preferentially at highly expressed genes in a manner dependent on cell cycle progression.

In response to DNA double-strand damage, ongoing transcription is inhibited to facilitate accurate DNA repair while transcriptional recovery occurs after DNA repair is complete. However, the mechanisms at play and the identity of the transcripts being regulated in this manner are unclear. In contrast to the situation following UV damage, we found that transcriptional recovery after ionizing radiation (IR) occurs in a manner independent of the HIRA histone chaperone. Sequencing of the nascent transcripts identified a programmed transcriptional response, where certain transcripts and pathways are rapidly downregulated after IR, while other transcripts and pathways are upregulated. Specifically, most of the loss of nascent transcripts occurring after IR is due to inhibition of transcriptional initiation of the highly transcribed histone genes and the rDNA. To identify factors responsible for transcriptional inhibition after IR in an unbiased manner, we performed a whole genome gRNA library CRISPR/Cas9 screen. Many of the top hits on our screen were factors required for protein neddylation. However, at short times after inhibition of neddylation, transcriptional inhibition still occurred after IR, even though neddylation was effectively inhibited. Persistent inhibition of neddylation blocked transcriptional inhibition after IR, and it also leads to cell cycle arrest. Indeed, we uncovered that many inhibitors and conditions that lead to cell cycle arrest in G1 or G2 phase also prevent transcriptional inhibition after IR. As such, it appears that transcriptional inhibition after IR occurs preferentially at highly expressed genes in cycling cells.

Pathological R-loops in bacteria from engineered expression of endogenous antisense RNAs whose synthesis is ordinarily terminated by Rho.

In many bacteria, the essential factors Rho and NusG mediate termination of synthesis of nascent transcripts (including antisense RNAs) that are not being simultaneously translated. It has been proposed that in Rho's absence toxic RNA-DNA hybrids (R-loops) may be generated from nascent untranslated transcripts, and genome-wide mapping studies in Escherichia coli have identified putative loci of R-loop formation from more than 100 endogenous antisense transcripts that are synthesized only in a Rho-deficient strain. Here we provide evidence that engineered expression in wild-type E. coli of several such individual antisense regions on a plasmid or the chromosome generates R-loops that, in an RNase H-modulated manner, serve to disrupt genome integrity. Rho inhibition was associated with increased prevalence of antisense R-loops also in Xanthomonas oryzae pv. oryzae and Caulobacter crescentus. Our results confirm the essential role of Rho in several bacterial genera for prevention of toxic R-loops from pervasive yet cryptic endogenous antisense transcripts. Engineered antisense R-looped regions may be useful for studies on both site-specific impediments to bacterial chromosomal replication and the mechanisms of their resolution.

Cockayne syndrome B protein is implicated in transcription and associated chromatin dynamics in homeostatic and genotoxic conditions.

The integrity of the actively transcribed genome against helix-distorting DNA lesions relies on a multilayered cellular response that enhances Transcription-Coupled Nucleotide Excision Repair (TC-NER). When defective, TC-NER is causatively associated with Cockayne-Syndrome (CS), a rare severe human progeroid disorder. Although the presence of unresolved transcription-blocking lesions is considered a driver of the aging process, the molecular features of the transcription-driven response to genotoxic stress in CS-B cells remain largely unknown. Here, an in-depth view of the transcriptional and associated chromatin dynamics that occur in CS-B cells illuminates the role of CSB therein. By employing high-throughput genome-wide approaches, we observed that absence of a functional CSB protein results in a delay in transcription progression, more positioned +1 nucleosomes, and less dynamic chromatin structure, compared to normal cells. We found that early after exposure to UV, CS-B cells released RNA polymerase II (RNAPII) from promoter-proximal pause sites into elongation. However, the magnitude of this response and the progression of RNAPII were reduced compared to normal counterparts. Notably, we detected increased post-UV retainment of unprocessed nascent RNA transcripts and chromatin-associated elongating RNAPII molecules. Contrary to the prevailing models, we found that transcription initiation is operational in CS-B fibroblasts early after UV and that chromatin accessibility showed a marginal increase. Our study provides robust evidence for the role of CSB in shaping the transcription and chromatin landscape both in homeostasis and in response to genotoxic insults, which is independent of its known role in TC-NER, and which may underlie major aspects of the CS phenotype.

8530 Characterizing the Genomic Effects of Low-Dose Estrogen in Breast Cancer Cells

Abstract Disclosure: H. Kim: None. T.S. Nandu: None. W.L. Kraus: None. Estrogens are steroid hormones that play a key role in a wide range of physiological and pathological processes. The predominant nuclear receptor for estrogens, estrogen receptor-alpha (ERɑ), functions primarily as a nuclear transcription factor (TF) through ligand-dependent activation by 17β-estradiol (E2), the major naturally-occurring estrogen. The physiological levels of E2 are regulated by production, sequestration, and degradation, which can limit the amount of bioavailable E2. However, many studies examining the genomic effects of E2-regulated signaling in cell-based assays used supraphysiological levels of hormone. While this has allowed us to understand the extremes of E2 signaling, it limits our ability to examine cellular functions which operate at lower hormone concentrations. The genomic actions of ligand-bound ERɑ are mediated through transcriptional enhancer formation and function. Alterations in protein and DNA components dictate different enhancer molecular subtypes that specify distinct regulatory mechanisms and downstream cellular phenotypes. How the amount of available hormone affects these genomic processes is unknown. In order to understand the genomic effects of physiological low-dose levels of E2, we have treated MCF-7 ERɑ-positive human breast cancer cells with a range of E2 concentrations and examined the global regulation of gene expression using a multi-omics approach (RNA-seq, ERɑ ChIP-seq, H3K27ac ChIP-seq, PRO-seq, ATAC-seq). Interestingly, we identified gene sets that are preferentially expressed maximally at picomolar or nanomolar concentrations. We are using these genes sets to explore the mechanisms of estrogen-regulated gene expression at physiologically relevant of E2 concentrations. Analysis of nascent RNA transcription using PRO-seq indicates regulation at the transcriptional level. Preliminary ERɑ chromatin binding assays show that while many loci require high levels of E2 for maximum ERɑ binding, others show maximum ERɑ binding at lower doses of E2. Further analyses show distinct enhancer features, such as chromatin accessibility and H3K27ac enrichment, may play a role in determining the response to E2 dosage. We are now determining the molecular mechanisms of dosage sensitivity using a multipronged approach integrating genomics, molecular biology, and cell-based assays. With these studies, we hope to understand a long overlooked physiological aspect of E2 with a comprehensive and global analysis of signal-dependent genomic responses. Presentation: 6/3/2024

9273 Characterizing the Genomic Effects of Low-Dose Estrogen in Breast Cancer Cells

Abstract Disclosure: H. Kim: None. T.S. Nandu: None. W.L. Kraus: None. Estrogens are steroid hormones that play a key role in a wide range of physiological and pathological processes. The predominant nuclear receptor for estrogens, estrogen receptor-alpha (ERɑ), functions primarily as a nuclear transcription factor (TF) through ligand-dependent activation by 17β-estradiol (E2), the major naturally-occurring estrogen. The physiological levels of E2 are regulated by production, sequestration, and degradation, which can limit the amount of bioavailable E2. However, many studies examining the genomic effects of E2-regulated signaling in cell-based assays used supraphysiological levels of hormone. While this has allowed us to understand the extremes of E2 signaling, it limits our ability to examine cellular functions which operate at lower hormone concentrations. The genomic actions of ligand-bound ERɑ are mediated through transcriptional enhancer formation and function. Alterations in protein and DNA components dictate different enhancer molecular subtypes that specify distinct regulatory mechanisms and downstream cellular phenotypes. How the amount of available hormone affects these genomic processes is unknown. In order to understand the genomic effects of physiological low-dose levels of E2, we have treated MCF-7 ERɑ-positive human breast cancer cells with a range of E2 concentrations and examined the global regulation of gene expression using a multi-omics approach (RNA-seq, ERɑ ChIP-seq, H3K27ac ChIP-seq, PRO-seq, ATAC-seq). Interestingly, we identified gene sets that are preferentially expressed maximally at picomolar or nanomolar concentrations. We are using these genes sets to explore the mechanisms of estrogen-regulated gene expression at physiologically relevant of E2 concentrations. Analysis of nascent RNA transcription using PRO-seq indicates regulation at the transcriptional level. Preliminary ERɑ chromatin binding assays show that while many loci require high levels of E2 for maximum ERɑ binding, others show maximum ERɑ binding at lower doses of E2. Further analyses show distinct enhancer features, such as chromatin accessibility and H3K27ac enrichment, may play a role in determining the response to E2 dosage. We are now determining the molecular mechanisms of dosage sensitivity using a multipronged approach integrating genomics, molecular biology, and cell-based assays. With these studies, we hope to understand a long overlooked physiological aspect of E2 with a comprehensive and global analysis of signal-dependent genomic responses. Presentation: 6/3/2024

The Escherichia coli ribB riboswitch senses flavin mononucleotide within a defined transcriptional window.

Riboswitches are metabolite-binding RNA regulators that modulate gene expression at the levels of transcription and translation. One of the hallmarks of riboswitch regulation is that they undergo structural changes upon metabolite binding. While a lot of effort has been put to characterize how the metabolite is recognized by the riboswitch, there is still relatively little information regarding how ligand sensing is performed within a transcriptional context. Here, we study the ligand-dependent cotranscriptional folding of the FMN-sensing ribB riboswitch of Escherichia coli. Using RNase H assays to study nascent ribB riboswitch transcripts, DNA probes targeting the P1 and sequestering stems indicate that FMN binding leads to the protection of these regions from RNase H cleavage, consistent with the riboswitch inhibiting translation initiation when bound to FMN. Our results show that ligand sensing is strongly affected by the position of elongating RNA polymerase, which is defining an FMN binding transcriptional window that is bordered in its 3' extremity by a transcriptional pause site. Also, using successively overlapping DNA probes targeting a subdomain of the riboswitch, our data suggest the presence of a previously unsuspected helical region involving the 3' strand of the P1 stem. Our results show that this helical region is conserved across bacterial species, thus suggesting that this predicted structure, the anti*-P1 stem, is involved in the FMN-free conformation of the ribB riboswitch. Overall, our study further demonstrates that intricate folding strategies may be used by riboswitches to perform metabolite sensing during the transcriptional process.

The zinc-finger transcription factor Sfp1 imprints specific classes of mRNAs and links their synthesis to cytoplasmic decay

To function effectively as an integrated system, the transcriptional and post-transcriptional machineries must communicate through mechanisms that are still poorly understood. Here, we focus on the zinc-finger Sfp1, known to regulate transcription of proliferation-related genes. We show that Sfp1 can regulate transcription either by binding to promoters, like most known transcription activators, or by binding to the transcribed regions (gene bodies), probably via RNA polymerase II (Pol II). We further studied the first mode of Sfp1 activity and found that, following promoter binding, Sfp1 binds to gene bodies and affects Pol II configuration, manifested by dissociation or conformational change of its Rpb4 subunit and increased backtracking. Surprisingly, Sfp1 binds to a subset of mRNAs co-transcriptionally and stabilizes them. The interaction between Sfp1 and its client mRNAs is controlled by their respective promoters and coincides with Sfp1’s dissociation from chromatin. Intriguingly, Sfp1 dissociation from the chromatin correlates with the extent of the backtracked Pol II. We propose that, following promoter recruitment, Sfp1 accompanies Pol II and regulates backtracking. The backtracked Pol II is more compatible with Sfp1’s relocation to the nascent transcripts, whereupon Sfp1 accompanies these mRNAs to the cytoplasm and regulates their stability. Thus, Sfp1’s co-transcriptional binding imprints the mRNA fate, serving as a paradigm for the cross-talk between the synthesis and decay of specific mRNAs, and a paradigm for the dual-role of some zinc-finger proteins. The interplay between Sfp1’s two modes of transcription regulation remains to be examined.

The zinc-finger transcription factor Sfp1 imprints specific classes of mRNAs and links their synthesis to cytoplasmic decay.

To function effectively as an integrated system, the transcriptional and post-transcriptional machineries must communicate through mechanisms that are still poorly understood. Here, we focus on the zinc-finger Sfp1, known to regulate transcription of proliferation-related genes. We show that Sfp1 can regulate transcription either by binding to promoters, like most known transcription activators, or by binding to the transcribed regions (gene bodies), probably via RNA polymerase II (Pol II). We further studied the first mode of Sfp1 activity and found that, following promoter binding, Sfp1 binds to gene bodies and affects Pol II configuration, manifested by dissociation or conformational change of its Rpb4 subunit and increased backtracking. Surprisingly, Sfp1 binds to a subset of mRNAs co-transcriptionally and stabilizes them. The interaction between Sfp1 and its client mRNAs is controlled by their respective promoters and coincides with Sfp1's dissociation from chromatin. Intriguingly, Sfp1 dissociation from the chromatin correlates with the extent of the backtracked Pol II. We propose that, following promoter recruitment, Sfp1 accompanies Pol II and regulates backtracking. The backtracked Pol II is more compatible with Sfp1's relocation to the nascent transcripts, whereupon Sfp1 accompanies these mRNAs to the cytoplasm and regulates their stability. Thus, Sfp1's co-transcriptional binding imprints the mRNA fate, serving as a paradigm for the cross-talk between the synthesis and decay of specific mRNAs, and a paradigm for the dual-role of some zinc-finger proteins. The interplay between Sfp1's two modes of transcription regulation remains to be examined.

Histone H3.3 lysine 9 and 27 control repressive chromatin at cryptic enhancers and bivalent promoters

Histone modifications are associated with distinct transcriptional states, but it is unclear whether they instruct gene expression. To investigate this, we mutate histone H3.3 K9 and K27 residues in mouse embryonic stem cells (mESCs). Here, we find that H3.3K9 is essential for controlling specific distal intergenic regions and for proper H3K27me3 deposition at promoters. The H3.3K9A mutation resulted in decreased H3K9me3 at regions encompassing endogenous retroviruses and induced a gain of H3K27ac and nascent transcription. These changes in the chromatin environment unleash cryptic enhancers, resulting in the activation of distinctive transcriptional programs and culminating in protein expression normally restricted to specialized immune cell types. The H3.3K27A mutant disrupts the deposition and spreading of the repressive H3K27me3 mark, particularly impacting bivalent genes with higher basal levels of H3.3 at promoters. Therefore, H3.3K9 and K27 crucially orchestrate repressive chromatin states at cis-regulatory elements and bivalent promoters, respectively, and instruct proper transcription in mESCs.

Cotranscriptional Folding of a 5′ Stem-loop in the Escherichia coli tbpA Riboswitch at Single-nucleotide Resolution

Transcription elongation is one of the most important processes in the cell. During RNA polymerase elongation, the folding of nascent transcripts plays crucial roles in the genetic decision. Bacterial riboswitches are prime examples of RNA regulators that control gene expression by altering their structure upon metabolite sensing. It was previously revealed that the thiamin pyrophosphate-sensing tbpA riboswitch in Escherichia coli cotranscriptionally adopts three main structures leading to metabolite sensing. Here, using single-molecule FRET, we characterize the transition in which the first nascent structure, a 5′ stem-loop, is unfolded during transcription elongation to form the ligand-binding competent structure. Our results suggest that the structural transition occurs in a relatively abrupt manner, i.e., within a 1–2 nucleotide window. Furthermore, a highly dynamic structural exchange is observed, indicating that riboswitch transcripts perform rapid sampling of nascent co-occurring structures. We also observe that the presence of the RNAP stabilizes the 5′ stem-loop along the elongation process, consistent with RNAP interacting with the 5′ stem-loop. Our study emphasizes the role of early folding stem-loop structures in the cotranscriptional formation of complex RNA molecules involved in genetic regulation.

T7 RNA polymerase catalyzed transcription of the epimerizable DNA lesion, Fapy•dG and 8-oxo-2′-deoxyguanosine

Fapy•dG (N6-(2-deoxy-α,β-D-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine) and 8-OxodGuo (8-oxo-7,8-dihydro-2′-deoxyguanosine) are major products of 2’-deoxyguanosine oxidation. Fapy•dG is unusual in that it exists as a dynamic mixture of anomers. Much less is known about the effects of Fapy•dG than 8-OxodGuo on transcriptional bypass. The data presented here indicate that T7 RNA polymerase (T7 RNAP) bypass of Fapy•dG is more complex than that of 8-OxodGuo. Primer dependent transcriptional bypass of Fapy•dG by T7 RNAP is hindered compared to dG. T7 RNAP incorporates cytidine opposite Fapy•dG in a miniscaffold at least 13-fold more rapidly than A, G or U. Fitting of reaction data indicate that Fapy•dG anomers are kinetically distinguishable. Extension of a nascent transcript past Fapy•dG is weakly dependent on the nucleotide opposite the lesion. The rate constants describing extension past fast or slow reacting base pairs vary less than 2-fold as a function of the nucleotide opposite the lesion. Promoter dependent T7 RNAP bypass of Fapy•dG and 8-OxodGuo was carried out side-by-side. 8-OxodGuo bypass results in >55% A opposite it. When the shuttle vector contains a Fapy•dG:dA base pair, as high as 20% point mutations and 9% single-nucleotide deletions are produced upon Fapy•dG bypass. Error-prone bypass of a Fapy•dG:dC base pair accounts for ∼9% of the transcripts. Transcriptional bypass mutation frequencies of Fapy•dG and 8-OxodGuo measured in RNA products are comparable to or greater than replication errors, suggesting that these lesions could contribute to mutations significantly through transcription.

Calmodulin Triggers Activity-Dependent rRNA Biogenesis via Interaction with DDX21.

Protein synthesis in response to neuronal activity, known as activity-dependent translation, is critical for synaptic plasticity and memory formation. However, the signaling cascades that couple neuronal activity to the translational events remain elusive. In this study, we identified the role of calmodulin (CaM), a conserved Ca2+-binding protein, in ribosomal RNA (rRNA) biogenesis in neurons. We found the CaM-regulated rRNA synthesis is Ca2+-dependent and necessary for nascent protein synthesis and axon growth in hippocampal neurons. Mechanistically, CaM interacts with nucleolar DEAD (Asp-Glu-Ala-Asp) box RNA helicase (DDX21) in a Ca2+-dependent manner to regulate nascent rRNA transcription within nucleoli. We further found CaM alters the conformation of DDX21 to liberate the DDX21-sequestered RPA194, the catalytic subunit of RNA polymerase I, to facilitate transcription of ribosomal DNA. Using high-throughput screening, we identified the small molecules batefenterol and indacaterol that attenuate the CaM-DDX21 interaction and suppress nascent rRNA synthesis and axon growth in hippocampal neurons. These results unveiled the previously unrecognized role of CaM as a messenger to link the activity-induced Ca2+ influx to the nucleolar events essential for protein synthesis. We thus identified the ability of CaM to transmit information to the nucleoli of neurons in response to stimulation.

Rho-dependent transcriptional switches regulate the bacterial response to cold shock

Binding of the bacterial Rho helicase to nascent transcripts triggers Rho-dependent transcription termination (RDTT) in response to cellular signals that modulate mRNA structure and accessibility of Rho utilization (Rut) sites. Despite the impact of temperature on RNA structure, RDTT was never linked to the bacterial response to temperature shifts. We show that Rho is a central player in the cold-shock response (CSR), challenging the current view that CSR is primarily a posttranscriptional program. We identify Rut sites in 5'-untranslated regions of key CSR genes/operons (cspA, cspB, cspG, and nsrR-rnr-yjfHI) that trigger premature RDTT at 37°C but not at 15°C. High concentrations of RNA chaperone CspA or nucleotide changes in the cspA mRNA leader reduce RDTT efficiency, revealing how RNA restructuring directs Rho to activate CSR genes during the cold shock and to silence them during cold acclimation. These findings establish a paradigm for how RNA thermosensors can modulate gene expression.