Mature mRNA Research Articles

Endonucleolytic processing plays a critical role in the maturation of ribosomal RNA in Methanococcus maripaludis

ABSTRACT Ribosomal RNA (rRNA) processing and maturation are fundamentally important for ribosome biogenesis, but the mechanisms in archaea, the third form of life, remains largely elusive. This study aimed to investigate the rRNA maturation process in Methanococcus maripaludis, a representative archaeon lacking known 3′-5′ exonucleases. Through cleavage site identification and enzymatic assays, the splicing endonuclease EndA was determined to process the bulge-helix-bulge (BHB) motifs in 16S and 23S rRNA precursors. After splicing, the circular processing intermediates were formed and this was confirmed by quantitative RT-PCR and Northern blot. Ribonuclease assay revealed a specific cleavage at a 10-nt A/U-rich motif at the mature 5′ end of pre-16S rRNA, which linearized circular pre-16S rRNA intermediate. Further 3′-RACE and ribonuclease assays determined that the endonuclease Nob1 cleaved the 3′ extension of pre-16S rRNA, and so generated the mature 3′ end. Circularized RT-PCR (cRT-PCR) and 5′-RACE identified two cleavage sites near helix 1 at the 5′ end of 23S rRNA, indicating that an RNA structure-based endonucleolytic processing linearized the circular pre-23S rRNA intermediate. In the maturation of pre-5S rRNA, multiple endonucleolytic processing sites were determined at the 10-nt A/U-rich motif in the leader and trailer sequence. This study demonstrates that endonucleolytic processing, particularly at the 10-nt A/U-rich motifs play an essential role in the pre-rRNA maturation of M. maripaludis, indicating diverse pathways of rRNA maturation in archaeal species.

Cell-type expression and activation by light of neuropsins in the developing and mature Xenopus retina.

Photosensitive opsins detect light and perform image- or nonimage-forming tasks. Opsins such as the "classical" visual opsins and melanopsin are well studied. However, the retinal expression and functions of a novel family of neuropsins are poorly understood. We explored the developmental time-course and cell-type specificity of neuropsin (opn5, 6a, 6b, and 8) expression in Xenopus laevis by in situ hybridization and immunohistochemistry. We compared the Xenopus results with publicly available single cell RNA sequencing (scRNA-seq) data from zebrafish, chicken, and mouse. Additionally, we analyzed light-activation of neuropsin-expressing cells through induction of c-fos mRNA. opn5 and opn8 expression begins at stage 37/38 when the retinal circuits begin to be activated. Once retinal circuits connect to the brain, opn5 mRNA is distributed across multiple retinal cell types, including bipolar (~70%-75%), amacrine (~10%), and retinal ganglion (~20%) cells, with opn8 present in amacrine (~70%) and retinal ganglion (~30%) cells. opn6a and opn6b mRNAs emerge in newborn-photoreceptors (stage 35), and are colocalized in rods and cones by stage 37/38. Interestingly, in the mature larval retina (stage 43/44), opn6a and opn6b mRNAs become preferentially localized to rods and cones, respectively, while newborn photoreceptors bordering the proliferative ciliary marginal zone express both genes. In zebrafish, opn6a and opn6b are also expressed in photoreceptors, while Müller glia and amacrine cells express opn8c. Most neuropsin-expressing retinal ganglion cells display c-fos expression in response to light, as do over half of the neuropsin-expressing interneurons. This study gave a better understanding of retinal neuropsin-expressing cells, their developmental onset, and light activation.

Advances in alternative splicing identification: deep learning and pantranscriptome.

In plants, alternative splicing is a crucial mechanism for regulating gene expression at the post-transcriptional level, which leads to diverse proteins by generating multiple mature mRNA isoforms and diversify the gene regulation. Due to the complexity and variability of this process, accurate identification of splicing events is a vital step in studying alternative splicing. This article presents the application of alternative splicing algorithms with or without reference genomes in plants, as well as the integration of advanced deep learning techniques for improved detection accuracy. In addition, we also discuss alternative splicing studies in the pan-genomic background and the usefulness of integrated strategies for fully profiling alternative splicing.

The human cellular protein NoL12 is a specific partner of the HIV-1 nucleocapsid protein NCp7.

The human immunodeficiency virus-1 (HIV-1) nucleocapsid protein (NCp7) is a nucleic acid chaperone protein with two highly conserved zinc fingers. To exert its key roles in the viral cycle, NCp7 interacts with several host proteins. Among them, the human NoL12 protein (hNoL12) was previously identified in genome wide screens as a potential partner of NCp7. hNoL12 is a highly conserved 25 kDa nucleolar RNA-binding protein implicated in the 5'end processing of ribosomal RNA in the nucleolus and thus in the assembly and maturation of ribosomes. In this work, we confirmed the NCp7/hNoL12 interaction in cells by Förster resonance energy transfer visualized by Fluorescence Lifetime Imaging Microscopy and co-immunoprecipitation. The interaction between NCp7 and hNoL12 was found to strongly depend on their both binding to RNA, as shown by the loss of interaction when the cell lysates were pretreated with RNase. Deletion mutants of hNoL12 were tested for their co-immunoprecipitation with NCp7, leading to the identification of the exonuclease domain of hNoL12 as the binding domain for NCp7. Finally, the interaction with hNoL12 was found to be specific of the mature NCp7 and to require NCp7 basic residues. IMPORTANCE HIV-1 mature nucleocapsid (NCp7) results from the maturation of the Gag precursor in the viral particle and is thus mostly abundant in the first phase of the infection which ends with the genomic viral DNA integration in the cell genome. Most if not all the nucleocapsid partners identified so far are not specific of the mature form. We described here the specific interaction in the nucleolus between NCp7 and the human nucleolar protein 12, a protein implicated in ribosomal RNA maturation and DNA damage response. This interaction takes place in the cell nucleolus, a subcellular compartment where NCp7 accumulates. The absence of binding between hNoL12 and Gag makes hNoL12 one of the few known specific cellular partners of NCp7.

Uncovering the effect of RNA polymerase steric interactions on gene expression noise: Analytical distributions of nascent and mature RNA numbers.

The telegraph model is the standard model of stochastic gene expression, which can be solved exactly to obtain the distribution of mature RNA numbers per cell. A modification of this model also leads to an analytical distribution of nascent RNA numbers. These solutions are routinely used for the analysis of single-cell data, including the inference of transcriptional parameters. However, these models neglect important mechanistic features of transcription elongation, such as the stochastic movement of RNA polymerases and their steric (excluded-volume) interactions. Here we construct a model of gene expression describing promoter switching between inactive and active states, binding of RNA polymerases in the active state, their stochastic movement including steric interactions along the gene, and their unbinding leading to a mature transcript that subsequently decays. We derive the steady-state distributions of the nascent and mature RNA numbers in two important limiting cases: constitutive expression and slow promoter switching. We show that RNA fluctuations are suppressed by steric interactions between RNA polymerases, and that this suppression can in some instances even lead to sub-Poissonian fluctuations; these effects are most pronounced for nascent RNA and less prominent for mature RNA, since the latter is not a direct sensor of transcription. We find a relationship between the parameters of our microscopic mechanistic model and those of the standard models that ensures excellent consistency in their prediction of the first and second RNA number moments over vast regions of parameter space, encompassing slow, intermediate, and rapid promoter switching, provided the RNA number distributions are Poissonian or super-Poissonian. Furthermore, we identify the limitations of inference from mature RNA data, specifically showing that it cannot differentiate between highly distinct RNA polymerase traffic patterns on a gene.

The bovine leukemia virus-derived long non-coding RNA AS1-S binds to bovine hnRNPM and alters the interaction between hnRNPM and host mRNAs.

Viruses utilize several strategies to cause latent infection and evade host immune responses. Long non-coding RNA (lncRNA), a class of non-protein-encoding RNA that regulates various cellular functions by interacting with RNA-binding proteins, plays important roles for viral latency in several viruses, such as herpesviruses and retroviruses, due to its lack of antigenicity. Bovine leukemia virus (BLV), which belongs to the family Retroviridae, encodes the BLV-derived lncRNA AS1-S, which is a major transcript expressed in latently infected cells. We herein identified bovine heterogeneous nuclear ribonucleoprotein M (hnRNPM), an RNA-binding protein located in the nucleus, as the binding partner of AS1-S using an RNA-protein pull-down assay. The pull-down assay using recombinant hnRNPM mutants showed that RNA recognition motifs (RRMs) 1 and 2, located in the N-terminal region of bovine hnRNPM, were responsible for the binding to AS1-S. Furthermore, RNA immunoprecipitation (RIP) assay results showed that the expression of AS1-S increased the number of mRNAs that co-immunoprecipitated with bovine hnRNPM in MDBK cells. These results suggested that AS1-S could alter the interaction between hnRNPM and host mRNAs, potentially interfering with cellular functions during the initial phase of mRNA maturation in the nucleus. Since most of the identified mRNAs that exhibited increased binding to hnRNPM were correlated with the KEGG term "Pathways in cancer," AS1-S might affect the proliferation and expansion of BLV-infected cells and contribute to tumor progression. IMPORTANCE BLV infects bovine B cells and causes malignant lymphoma, a disease that greatly affects the livestock industry. Due to its low incidence and long latent period, the molecular mechanisms underlying the progression of lymphoma remain enigmatic. Several non-coding RNAs (ncRNAs), such as miRNA and lncRNA, have recently been discovered in the BLV genome, and the relationship between BLV pathogenesis and these ncRNAs is attracting attention. However, most of the molecular functions of these transcripts remain unidentified. To the best of our knowledge, this is the first report describing a molecular function for the BLV-derived lncRNA AS1-S. The findings reported herein reveal a novel mechanism underlying BLV pathogenesis that could provide important insights for not only BLV research but also comparative studies of retroviruses.

Stochastic modeling of the mRNA life process: A generalized master equation

The mRNA life cycle is a complex biochemical process, involving transcription initiation, elongation, termination, splicing, and degradation. Each of these molecular events is multistep and can create a memory. The effect of this molecular memory on gene expression is not clear, although there are many related yet scattered experimental reports. To address this important issue, we develop a general theoretical framework formulated as a master equation in the sense of queue theory, which can reduce to multiple previously studied gene models in limiting cases. This framework allows us to interpret experimental observations, extract kinetic parameters from experimental data, and identify how the mRNA kinetics vary under regulatory influences. Notably, it allows us to evaluate the influences of elongation processes on mature RNA distribution; e.g., we find that the non-exponential elongation time can induce the bimodal mRNA expression and there is an optimal elongation noise intensity such that the mature RNA noise achieves the lowest level. In a word, our framework can not only provide insight into complex mRNA life processes but also bridge a dialogue between theoretical studies and experimental data.

RNA processing and degradation mechanisms shaping the mitochondrial transcriptome of budding yeasts.

Yeast mitochondrial genes are expressed as polycistronic transcription units that contain RNAs from different classes and show great evolutionary variability. The promoters are simple, and transcriptional control is rudimentary. Posttranscriptional mechanisms involving RNA maturation, stability, and degradation are thus the main force shaping the transcriptome and determining the expression levels of individual genes. Primary transcripts are fragmented by tRNA excision by RNase P and tRNase Z, additional processing events occur at the dodecamer site at the 3' end of protein-coding sequences. groups I and II introns are excised in a self-splicing reaction that is supported by protein splicing factors encoded by the nuclear genes, or by the introns themselves. The 3'-to-5' exoribonucleolytic complex called mtEXO is the main RNA degradation activity involved in RNA turnover and processing, supported by an auxiliary 5'-to-3' exoribonuclease Pet127p. tRNAs and, to a lesser extent, rRNAs undergo several different base modifications. This complex gene expression system relies on the coordinated action of mitochondrial and nuclear genes and undergoes rapid evolution, contributing to speciation events. Moving beyond the classical model yeast Saccharomyces cerevisiae to other budding yeasts should provide important insights into the coevolution of both genomes that constitute the eukaryotic genetic system.

Targeted splicing therapy: new strategies for colorectal cancer.

RNA splicing is the process of forming mature mRNA, which is an essential phase necessary for gene expression and controls many aspects of cell proliferation, survival, and differentiation. Abnormal gene-splicing events are closely related to the development of tumors, and the generation of oncogenic isoform in splicing can promote tumor progression. As a main process of tumor-specific splicing variants, alternative splicing (AS) can promote tumor progression by increasing the production of oncogenic splicing isoforms and/or reducing the production of normal splicing isoforms. This is the focus of current research on the regulation of aberrant tumor splicing. So far, AS has been found to be associated with various aspects of tumor biology, including cell proliferation and invasion, resistance to apoptosis, and sensitivity to different chemotherapeutic drugs. This article will review the abnormal splicing events in colorectal cancer (CRC), especially the tumor-associated splicing variants arising from AS, aiming to offer an insight into CRC-targeted splicing therapy.

Dissecting and improving gene regulatory network inference using single-cell transcriptome data.

Single-cell transcriptome data has been widely used to reconstruct gene regulatory networks (GRNs) controlling critical biological processes such as development and differentiation. Although a growing list of algorithms has been developed to infer GRNs using such data, achieving an inference accuracy consistently higher than random guessing has remained challenging. To address this, it is essential to delineate how the accuracy of regulatory inference is limited. Here, we systematically characterized factors limiting the accuracy of inferred GRNs and demonstrated that using pre-mRNA information can help improve regulatory inference compared to the typically used information (i.e., mature mRNA). Using kinetic modeling and simulated single-cell data sets, we showed that target genes' mature mRNA levels often fail to accurately report upstream regulatory activities because of gene-level and network-level factors, which can be improved by using pre-mRNA levels. We tested this finding on public single-cell RNA-seq data sets using intronic reads as proxies of pre-mRNA levels and can indeed achieve a higher inference accuracy compared to using exonic reads (corresponding to mature mRNAs). Using experimental data sets, we further validated findings from the simulated data sets and identified factors such as transcription factor activity dynamics influencing the accuracy of pre-mRNA-based inference. This work delineates the fundamental limitations of gene regulatory inference and helps improve GRN inference using single-cell RNA-seq data.

Formation of a stable RNase Y-RicT (YaaT) complex requires RicA (YmcA) and RicF (YlbF).

In Bacillus subtilis, the RicT (YaaT), RicA (YmcA), and RicF (YlbF) proteins, which form a stable ternary complex, are needed together with RNase Y (Rny) to cleave and thereby stabilize several key transcripts encoding enzymes of intermediary metabolism. We show here that RicT, but not RicA or RicF, forms a stable complex with Rny and that this association requires the presence of RicA and RicF. We propose that RicT is handed off from the ternary complex to Rny. We show further that the two iron-sulfur clusters carried by the ternary Ric complex are required for the formation of the stable RicT-Rny complex. We demonstrate that proteins of the degradosome-like network of B. subtilis, which also interact with Rny, are dispensable for processing of the gapA operon. Thus, Rny participates in distinct RNA-related processes, determined by its binding partners, and a RicT-Rny complex is likely the functional entity for gapA mRNA maturation. IMPORTANCE The action of nucleases on RNA is universal and essential for all forms of life and includes processing steps that lead to the mature and functional forms of certain transcripts. In Bacillus subtilis, it has been shown that key transcripts for energy-producing steps of glycolysis, for nitrogen assimilation, and for oxidative phosphorylation, all of them crucial processes of intermediary metabolism, are cleaved at specific locations, resulting in mRNA stabilization. The proteins required for these cleavages in B. subtilis [Rny (RNase Y), RicA (YmcA), RicF (YlbF), and RicT (YaaT)] are broadly conserved among the firmicutes, including several important pathogens, hinting that regulatory mechanisms they control may also be conserved. Several aspects of these regulatory events have been explored: phenotypes associated with the absence of these proteins have been described, the impact of these absences on the transcriptome has been documented, and there has been significant exploration of the biochemistry and structural biology of Rny and the Ric proteins. The present study further advances our understanding of the association of Ric proteins and Rny and shows that a complex of Rny with RicT is probably the entity that carries out mRNA maturation.

Abstract P3018: SCARNA20 Promotes M2 Macrophage Polarization Under Endotoxemia And Reduces Inflammation

Background: Macrophage polarization plays a key role in the inflammatory process. M1 pro-inflammatory and M2 anti-inflammatory phenotypes can down-regulate each other and may contribute to the pathogenesis of various diseases. Small Cajal Body Associated RNAs (SCARNAs) play a key role in alternative splicing and maturation of mRNAs. Our recent studies found that a combination of Procainamide (P, DNMT1 inhibitor) and Tubastatin A (T, HDAC6 inhibitor) reduces inflammation upon lipopolysaccharide (LPS) stimuli. Hypothesis: We hypothesize that the P+T treatment increases the expression of SCARNA20 hence reducing the proinflammatory response and promoting M2 macrophage polarization after LPS challenge. Methods and Results: To elucidate SCARNAs of interest during the inflammatory response, we treated macrophages with LPS (1μg/mL) and P (400nM/mL) + T (2.5μM/mL) for 1hr and performed RNA sequencing. Based on the sequencing results and qRT-PCR validation, SCARNA20 was the most significantly differentially expressed SCARNA between conditions (Fig. A) . The expression of TNFα was significantly reduced with the P+T treatment (Fig. B) . SCARNA20 overexpression (OE) showed an increase in the expression of the M2 marker CD163 and a reduction in the expression of the M1 marker CD80 (Fig. C) . Additionally, TNFα expression was significantly reduced in the SCARNA20 OE samples after the LPS challenge without treatment with P+T (Fig. D) . Finally, SCARNA20 knockdown macrophages (Fig. E) treated with P+T did not suppress TNFα expression (Fig. F) . Conclusions: We report for the first time that SCARNA20 is an important regulator of M2 macrophage polarization and pro-inflammatory response.

Abstract P1111: Dysfunctional SCARNAs And Failed Pseudouridylation Result In Irregulation Of Cardiac Genes In Noonan Syndrome Patient-derived Induced Cardiomyocytes

Background: Small Cajal body associated RNAs (scaRNAs) can control alternative splicing and the maturation of mRNAs by regulating the pseudouridylation (PsU) of small nuclear RNAs (snRNAs), which are involved in the formation of the spliceosome complex, via RNA-dependent mechanism. Failure of snRNA PsU results in dysregulated mRNA splicing and maturation which causes congenital heart defects. In this study, we aim to find the importance of snRNAs PsU in Noonan syndrome (NS) patients-derived induced pluripotent stem cell-derived cardiomyocytes (NS-iCM) when compared to normal skin fibroblast iCM (SF-iCM). Hypothesis: We hypothesize that snRNAs PsU by scaRNAs will have a significant role in the development of cardiomyocytes in NS patients. Methods and Results: We generated iCMs from SF-iPSCs and NS patient-derived NS-iPSCs. The qRT-PCR analysis showed a significantly lower expression of scaRNA6, scaRNA20, and scaRNA28 in NS-iCMs when compared with control SF-iCMs (Fig. A). The PsU of snRNA U12 was quantified by RNA Immunoprecipitation (RIP) using the Pseudouridine (Ψ)-specific antibody and mouse IgGas a negative control. The data showed that snRNA U12 was significantly reduced in the NS-iCM when compared with negative IgG (Fig. B). Furthermore, the PsU modification was quantified by using the 1-cyclohexyl- (2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate (CMCT) method. Our data showed that relative Ψ units were significantly reduced in the NS-iCMs when compared with normal SF-iCMs (Fig. C). Conclusion: Our results indicate that snRNAs PsU by scaRNAs were dysfunctional in NS-iCMCs, which may result in cardiac defects in NS patients.

Decoding the prognostic significance of integrator complex subunit 9 (INTS9) in glioma: links to TP53 mutations, E2F signaling, and inflammatory microenvironments

IntroductionGliomas, a type of brain neoplasm, are prevalent and often fatal. Molecular diagnostics have improved understanding, but treatment options are limited. This study investigates the role of INTS9 in processing small nuclear RNA (snRNA), which is crucial to generating mature messenger RNA (mRNA). We aim to employ advanced bioinformatics analyses with large-scale databases and conduct functional experiments to elucidate its potential role in glioma therapeutics.Materials and methodsWe collected genomic, proteomic, and Whole-Exon-Sequencing data from The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) for bioinformatic analyses. Then, we validated INTS9 protein expression through immunohistochemistry and assessed its correlation with P53 and KI67 protein expression. Gene Set Enrichment Analysis (GSEA) was performed to identify altered signaling pathways, and functional experiments were conducted on three cell lines treated with siINTS9. Then, we also investigate the impacts of tumor heterogeneity on INTS9 expression by integrating single-cell sequencing, 12-cell state prediction, and CIBERSORT analyses. Finally, we also observed longitudinal changes in INTS9 using the Glioma Longitudinal Analysis (GLASS) dataset.ResultsOur findings showed increased INTS9 levels in tumor tissue compared to non-neoplastic components, correlating with high tumor grading and proliferation index. TP53 mutation was the most notable factor associated with upregulated INTS9, along with other potential contributors, such as combined chromosome 7 gain/10 loss, TERT promoter mutation, and increased Tumor Mutational Burden (TMB). In GSEA analyses, we also linked INTS9 with enhanced cell proliferation and inflammation signaling. Downregulating INTS9 impacted cellular proliferation and cell cycle regulation during the function validation. In the context of the 12 cell states, INTS9 correlated with tumor-stem and tumor-proliferative-stem cells. CIBERSORT analyses revealed increased INTS9 associated with increased macrophage M0 and M2 but depletion of monocytes. Longitudinally, we also noticed that the INTS9 expression declined during recurrence in IDH wildtype.ConclusionThis study assessed the role of INTS9 protein in glioma development and its potential as a therapeutic target. Results indicated elevated INTS9 levels were linked to increased proliferation capacity, higher tumor grading, and poorer prognosis, potentially resulting from TP53 mutations. This research highlights the potential of INTS9 as a promising target for glioma treatment.

The induction of p53 correlates with defects in the production, but not the levels, of the small ribosomal subunit and stalled large ribosomal subunit biogenesis.

Ribosome biogenesis is one of the biggest consumers of cellular energy. More than 20 genetic diseases (ribosomopathies) and multiple cancers arise from defects in the production of the 40S (SSU) and 60S (LSU) ribosomal subunits. Defects in the production of either the SSU or LSU result in p53 induction through the accumulation of the 5S RNP, an LSU assembly intermediate. While the mechanism is understood for the LSU, it is still unclear how SSU production defects induce p53 through the 5S RNP since the production of the two subunits is believed to be uncoupled. Here, we examined the response to SSU production defects to understand how this leads to the activation of p53 via the 5S RNP. We found that p53 activation occurs rapidly after SSU production is blocked, prior to changes in mature ribosomal RNA (rRNA) levels but correlated with early, middle and late SSU pre-rRNA processing defects. Furthermore, both nucleolar/nuclear LSU maturation, in particular late stages in 5.8S rRNA processing, and pre-LSU export were affected by SSU production defects. We have therefore uncovered a novel connection between the SSU and LSU production pathways in human cells, which explains how p53 is induced in response to SSU production defects.

In vitro oogenesis from murine premeiotic germ cells using a new three-dimensional culture system

A faithful reconstitution of the complete process of oogenesis in vitro is helpful for understanding the molecular mechanisms, genetics, and epigenetic changes related to gametogenesis; it can also be useful for clinical drug screening, disease research, and regenerative medicine. To this end, given the consensus that murine female germ cells initiate meiosis at E13.5, substantial works have reported the successful generation of fertile oocytes using E12.5 female gonads as starting materials. Nevertheless, our data demonstrated that murine germ cells at E12.5 have heterogeneously initiated a meiotic transcriptional program based on a measurement of pre‐mRNAs (unspliced) and mature mRNAs (spliced) at a single-cell level. Therefore, to establish a platform that faithfully recapitulates the entire process in vitro (from premeiotic murine germ cells to fully developed oocytes), we here report a novel three-dimensional organoid culture (3-DOC) system, which successfully induced fully developed oocytes from E11.5 premeiotic female germ cells (oogonia). Compared with 2D culture and other 3D culture methods, this new culture system is more cost-effective and can create high-quality oocytes similar to in vivo oocytes. In summary, our new culture platform provides an experimental model for future research in regenerative medicine and reproductive biology.

When Size Really Matters: The Eccentricities of Dystrophin Transcription and the Hazards of Quantifying mRNA from Very Long Genes.

At 2.3 megabases in length, the dystrophin gene is enormous: transcription of a single mRNA requires approximately 16 h. Principally expressed in skeletal muscle, the dystrophin protein product protects the muscle sarcolemma against contraction-induced injury, and dystrophin deficiency results in the fatal muscle-wasting disease, Duchenne muscular dystrophy. This gene is thus of key clinical interest, and therapeutic strategies aimed at eliciting dystrophin restoration require quantitative analysis of its expression. Approaches for quantifying dystrophin at the protein level are well-established, however study at the mRNA level warrants closer scrutiny: measured expression values differ in a sequence-dependent fashion, with significant consequences for data interpretation. In this manuscript, we discuss these nuances of expression and present evidence to support a transcriptional model whereby the long transcription time is coupled to a short mature mRNA half-life, with dystrophin transcripts being predominantly nascent as a consequence. We explore the effects of such a model on cellular transcriptional dynamics and then discuss key implications for the study of dystrophin gene expression, focusing on both conventional (qPCR) and next-gen (RNAseq) approaches.

Martynoside rescues 5-fluorouracil-impaired ribosome biogenesis by stabilizing RPL27A

Martynoside (MAR), a bioactive component in several well-known tonic traditional Chinese herbs, exhibits pro-hematopoietic activity during 5-fluorouracil (5-FU) treatment. However, the molecular target and the mechanism of MAR are poorly understood. Here, by adopting the mRNA display with a library of even-distribution (md-LED) method, we systematically examined MAR-protein interactions in vitro and identified the ribosomal protein L27a (RPL27A) as a key cellular target of MAR. Structural and mutational analysis confirmed the specific interaction between MAR and the exon 4,5-encoded region of RPL27A. MAR attenuated 5-FU-induced cytotoxicity in bone marrow nucleated cells, increased RPL27A protein stability, and reduced the ubiquitination of RPL27A at lys92 (K92) and lys94 (K94). Disruption of MAR binding at key residues of RPL27A completely abolished the MAR-induced stabilization. Furthermore, by integrating label-free quantitative ubiquitination proteomics, transcriptomics, and ribosome function assays, we revealed that MAR restored RPL27A protein levels and thus rescued ribosome biogenesis impaired by 5-FU. Specifically, MAR increased mature ribosomal RNA (rRNA) abundance, prevented ribosomal protein degradation, facilitated ribosome assembly, and maintained nucleolar integrity. Collectively, our findings characterize the target of a component of Chinese medicine, reveal the importance of ribosome biogenesis in hematopoiesis, and open up a new direction for improving hematopoiesis by targeting RPL27A.

The DEAD-box RNA helicase Dbp5 is a key protein that couples multiple steps in gene expression.

Cell viability largely depends on the surveillance of mRNA export and translation. Upon pre-mRNA processing and nuclear quality control, mature mRNAs are exported into the cytoplasm via Mex67-Mtr2 attachment. At the cytoplasmic site of the nuclear pore complex, the export receptor is displaced by the action of the DEAD-box RNA helicase Dbp5. Subsequent quality control of the open reading frame requires translation. Our studies suggest an involvement of Dbp5 in cytoplasmic no-go-and non-stop decay. Most importantly, we have also identified a key function for Dbp5 in translation termination, which identifies this helicase as a master regulator of mRNA expression.

Analogs of the Catechol Derivative Dynasore Inhibit HIV-1 Ribonuclease H, SARS-CoV-2 nsp14 Exoribonuclease, and Virus Replication.

Viral replication often depends on RNA maturation and degradation processes catalyzed by viral ribonucleases, which are therefore candidate targets for antiviral drugs. Here, we synthesized and studied the antiviral properties of a novel nitrocatechol compound (1c) and other analogs that are structurally related to the catechol derivative dynasore. Interestingly, compound 1c strongly inhibited two DEDD box viral ribonucleases, HIV-1 RNase H and SARS-CoV-2 nsp14 3'-to-5' exoribonuclease (ExoN). While 1c inhibited SARS-CoV-2 ExoN activity, it did not interfere with the mRNA methyltransferase activity of nsp14. In silico molecular docking placed compound 1c in the catalytic pocket of the ExoN domain of nsp14. Finally, 1c inhibited SARS-CoV-2 replication but had no toxicity to human lung adenocarcinoma cells. Given its simple chemical synthesis from easily available starting materials, these results suggest that 1c might be a lead compound for the design of new antiviral compounds that target coronavirus nsp14 ExoN and other viral ribonucleases.