Mature mRNA Research Articles

The emerging roles of aberrant alternative splicing in glioma

Abstract Gliomas represent a heterogeneous group of uniformly fatal brain tumors. Low and high-grade gliomas have diverse molecular signatures. Despite successful advances in understanding glioma, several genetic, epigenetic, and post-transcriptional alterations leave various targeted therapies ineffective, leading to a poor prognosis for high-grade glioma. Recent advances have revealed the implication of dysregulated alternative splicing (AS) events in glioma development. AS is a process that produces, from a single genomic sequence, several mature messenger RNAs. Splicing of pre-messenger RNAs concerns at least 95% of transcripts and constitutes an important mechanism in gene expression regulation. Dysregulation of this process, through variations in spliceosome components, aberrant splicing factors and RNA-binding protein activity, disproportionate regulation of non-coding RNAs, and abnormal mRNA methylation, can contribute to the disruption of AS. Such disruptions are usually associated with the development of several cancers, including glioma. Consequently, AS constitutes a key regulatory mechanism that could serve as a target for future therapies. In this review, we explore how AS events, spliceosome components, and their regulatory mechanisms play a critical role in glioma development, highlighting their potential as targets for innovative therapeutic strategies against this challenging cancer.

An Investigation of the Conservatism of Sequences Defining trans-Splicing in the mod(mdg4) Locus across Drosophila and Silkworm Species.

Splicing, a key step in mRNA maturation, plays a crucial role in the regulation of eukaryotic gene expression. The formation of chimeric mRNAs from two transcripts during splicing is typically a prohibited process; however, in insects, trans-splicing is a primary mechanism for increasing protein diversity for several loci. The aim of this study is to investigate the evolutionary conservativeness of sequences responsible for trans-splicing in the mod(mdg4) locus among species from the Drosophilidae family (order Diptera) and the silkworm (Bombyx mori), which belongs to the order Lepidoptera. Using model transgenic lines, it was shown that sequences from distant Drosophila species retain the ability to support trans-splicing in D.melanogaster. In contrast, analogous sequences in Bombyx mori do not support trans-splicing. Thus, the RNA motifs and their binding hypothetical protein factors, defining trans-splicing, remain conserved among the Drosophilidae group, but have functionally diverged between Diptera and Lepidoptera.

Molecular assessment of splicing variants in a cohort of patients with inborn errors of immunity: methodological approach and interpretation remarks

BackgroundSplicing is the molecular mechanism to produce mature messenger RNA (mRNA) before its translation into protein. It is estimated that 50% of disease-causing mutations disrupt splicing, mostly of them affecting canonical positions. However, variants occurring in coding regions or deep-intronic variants can also affect splicing. In these cases, interpretation of the results may be challenging and molecular validation is required.MethodsThe study includes 23 patients with splicing variants out of a cohort of 187 patients diagnosed with inborn errors of immunity (IEI). Clinical features and immunophenotypes are shown. Reverse transcription-polymerase chain reaction (RT-PCR) is the molecular assay employed for pathogenicity validation.ResultsWe detected 23 patients of 20 pedigrees with splicing variants in IEI genes, which constitutes the 12.3% of our cohort. In total, 21 splicing variants were analyzed, 10 of which had previously been reported in the literature and 11 novel ones. Among the 23 patients, 16 showed variants at canonical splice sites. Molecular validation was required only in the cases of genes of uncertain significance (GUS), high homology pseudogenes or incompatible clinical phenotype. Seven patients showed variants outside canonical positions. All of them needed molecular validation, with the exception of two patients, whose variants had previously been well characterized in the medical literature.ConclusionThis study shows the proportion of splicing variants in a cohort of IEI patients, providing their clinical phenotypic characteristics and the methodology used to validate the splicing defects. Based on the results, an algorithm is proposed to clarify when a splicing variant should be validated by complementary methodology and when, by contrast, it can be directly considered disease causing.

A Computational Recognition Analysis of Promising Prognostic Biomarkers in Breast, Colon and Lung Cancer Patients

Breast, colon, and lung carcinomas are classified as aggressive tumors with poor relapse-free survival (RFS), progression-free survival (PF), and poor hazard ratios (HRs) despite extensive therapy. Therefore, it is essential to identify a gene expression signature that correlates with RFS/PF and HR status in order to predict treatment efficiency. RNA-binding proteins (RBPs) play critical roles in RNA metabolism, including RNA transcription, maturation, and post-translational regulation. However, their involvement in cancer is not yet fully understood. In this study, we used computational bioinformatics to classify the functions and correlations of RBPs in solid cancers. We aimed to identify molecular biomarkers that could help predict disease prognosis and improve the therapeutic efficiency in treated patients. Intersection analysis summarized more than 1659 RBPs across three recently updated RNA databases. Bioinformatics analysis showed that 58 RBPs were common in breast, colon, and lung cancers, with HR values < 1 and >1 and a significant Q-value < 0.0001. RBP gene clusters were identified based on RFS/PF, HR, p-value, and fold induction. To define union RBPs, common genes were subjected to hierarchical clustering and were classified into two groups. Poor survival was associated with high genes expression, including CDKN2A, MEX3A, RPL39L, VARS, GSPT1, SNRPE, SSR1, and TIA1 in breast and colon cancer but not with lung cancer; and poor survival was associated with low genes expression, including PPARGC1B, EIF4E3, and SMAD9 in breast, colon, and lung cancer. This study highlights the significant contribution of PPARGC1B, EIF4E3, and SMAD9 out of 11 RBP genes as prognostic predictors in patients with breast, colon, and lung cancers and their potential application in personalized therapy.

The membrane-targeting-sequence motif is required for exhibition of recessive resurrection in Escherichia coli RNase E.

The essential homotetrameric endoribonuclease RNase E of Escherichia coli participates in global RNA turnover as well as stable RNA maturation. The protomer's N-terminal half (residues 1-529) bears the catalytic, allosteric, and tetramerization domains, including the active site residues D303 and D346. The C-terminal half (CTH, residues 530-1061) is dispensable for viability. We have previously described a phenomenon of recessive resurrection in RNase E that requires the CTH, wherein the wild-type homotetramer apparently displays nearly identical activity in vivo as a heterotetramer comprisingthree catalytically dead subunits (with D303A orD346A substitutions) and one wild-type subunit. Here, we show that recessive resurrection is exhibited even in dimeric RNase E with the CTH, and that it is largely dependent on the presence of a membrane-targeting-sequence motif (residues 565-582). A single F575E substitution also impaired recessive resurrection, whereas other CTH motifs (such as those for binding of RNA or of partner proteins) were dispensable. The phenomenon was independent of RNA 5'-monophosphate sensing by the enzyme. We propose that membrane-anchoring of RNase E renders it processive for endoribonucleolytic action, and that recessive resurrection and dominant negativity associated with mutant protomers are mutually exclusive manifestations of, respectively, processive and distributive catalytic mechanisms in a homo-oligomeric enzyme.

Mitochondrial mRNA and the small subunit rRNA in budding yeasts undergo 3'-end processing at conserved species-specific elements.

Respiration in eukaryotes depends on mitochondrial protein synthesis, which is performed by organelle-specific ribosomes translating organelle-encoded mRNAs. Although RNA maturation and stability are central events controlling mitochondrial gene expression, many of the molecular details in this pathway remain elusive. These include cis- and trans-regulatory factors that generate and protect the 3' ends. Here, we mapped the 3' ends of mitochondrial mRNAs of yeasts classified into multiple families of the subphylum Saccharomycotina. We found that the processing of mitochondrial 15S rRNA and mRNAs involves species-specific sequence elements, which we term 3'-end RNA processing elements (3'-RPEs). In Saccharomyces cerevisiae, the 3'-RPE has long been recognized as a conserved dodecamer sequence, which recent studies have shown specifically interacts with the nuclear genome-encoded pentatricopeptide repeat protein Rmd9. We also demonstrate that, analogous to Rmd9 in S. cerevisiae, two Rmd9 orthologs from the Debaryomycetaceae family interact with their respective 3'-RPEs found in mRNAs and 15S rRNA. Thus, Rmd9-dependent processing of mitochondrial RNA precursors may be a common mechanism among the families of the Saccharomycotina subphylum. Surprisingly, we observed that 3'-RPEs often occur upstream of stop codons in complex I subunit mRNAs from yeasts of the CUG-Ser1 clade. We examined two of these mature mRNAs and found that their stop codons are indeed removed. Thus, translation of these stop-codon-less transcripts would require a noncanonical termination mechanism. Our findings highlight Rmd9 as a key evolutionarily conserved factor in both mitochondrial mRNA metabolism and mitoribosome biogenesis in a variety of yeasts.

Structural basis of Spliced Leader RNA recognition by the Trypanosoma brucei cap-binding complex

Kinetoplastids are a clade of eukaryotic protozoans that include human parasitic pathogens like trypanosomes and Leishmania species. In these organisms, protein-coding genes are transcribed as polycistronic pre-mRNAs, which need to be processed by the coupled action of trans-splicing and polyadenylation to yield monogenic mature mRNAs. During trans-splicing, a universal RNA sequence, the spliced leader RNA (SL RNA) mini-exon, is added to the 5’-end of each mRNA. The 5’-end of this mini-exon carries a hypermethylated cap structure and is bound by a trypanosomatid-specific cap-binding complex (CBC). The function of three of the kinetoplastid CBC subunits is unknown, but an essential role in cap-binding and trans-splicing has been suggested. Here, we report cryo-EM structures that reveal the molecular architecture of the Trypanosoma brucei CBC (TbCBC) complex. We find that TbCBC interacts with two distinct features of the SL RNA. The TbCBP20 subunit interacts with the m7G cap while TbCBP66 recognizes double-stranded portions of the SL RNA. Our findings pave the way for future research on mRNA maturation in kinetoplastids. Moreover, the observed structural similarities and differences between TbCBC and the mammalian cap-binding complex will be crucial for considering the potential of TbCBC as a target for anti-trypanosomatid drug development.

From Flies to Humans: Conserved Roles of CEBPZ, NOC2L, and NOC3L in rRNA Processing and Tumorigenesis.

NOC1, NOC2, and NOC3 are conserved nucleolar proteins essential for regulating ribosomal RNA (rRNA) maturation, a process critical for cellular homeostasis. NOC1, in Drosophila and yeast, enhances nucleolar activity to sustain rRNA processing, whereas its depletion leads to impaired polysome formation, reduced protein synthesis, and apoptosis. These genes have vertebrate homologs called CEBPZ, NOC2L, and NOC3l. In this study, we demonstrated that the RNA-regulatory functions of CEBPZ are conserved in vertebrates, and we showed that CEBPZ leads to the accumulation of 45S-pre rRNA, with consequent reduction in protein synthesis. Gene Ontology and bioinformatic analyses of CEBPZ, NOC2L, and NOC3L in tumors highlight a significant correlation between their reduction and the processes that regulate rRNA and 60S ribosomal maturation. Comparative analysis of their expression in tumor databases revealed that CEBPZ, NOC2L, and NOC3L exhibit contrasting expression patterns across tumor types. This dual role suggests that their overexpression promotes tumor growth, whereas reduced expression may exert tumor-suppressive effects, uncovering unexpected regulatory functions exerted by these proteins in cancer.

Comparative evaluation of ACetic - MEthanol high salt dissociation approach for single-cell transcriptomics of frozen human tissues.

Current dissociation methods for solid tissues in scRNA-seq studies do not guarantee intact single-cell isolation, especially for sensitive and complex human endocrine tissues. Most studies rely on enzymatic dissociation of fresh samples or nuclei isolation from frozen samples. Dissociating whole intact cells from fresh-frozen samples, commonly collected by biobanks, remains a challenge. Here, we utilized the acetic-methanol dissociation approach (ACME) to capture transcriptional profiles of individual cells from fresh-frozen tissue samples. This method combines acetic acid-based dissociation and methanol-based fixation. In our study, we optimized this approach for human endocrine tissue samples for the first time. We incorporated a high-salt washing buffer instead of the standard PBS to stabilize RNA and prevent RNases reactivation during rehydration. We have designated this optimized protocol as ACME HS (ACetic acid-MEthanol High Salt). This technique aims to preserve cell morphology and RNA integrity, minimizing transcriptome changes and providing a more accurate representation of mature mRNA. We compared the ability of enzymatic, ACME HS, and nuclei isolation methods to preserve major cell types, gene expression, and standard quality parameters across 41 tissue samples. Our results demonstrated that ACME HS effectively dissociates and fixes cells, preserving cell morphology and high RNA integrity. This makes ACME HS a valuable alternative for scRNA-seq protocols involving challenging tissues where obtaining a live cell suspension is difficult or disruptive.

Single-nuclei RNA-sequencing fails to detect molecular dysregulation in the preeclamptic placenta.

Single-cell RNA-seq (scRNA-seq) revolutionized our understanding of tissue complexity in health and disease and revealed massive transcriptional dysregulation across placental cell classes in early-onset, but not late-onset preeclampsia (PE). However, the multinucleated syncytium is largely inaccessible to cell dissociation. Nuclei isolation and single-nuclei RNA-seq may be preferable in the placenta; not least considering compatibility with long-term tissue storage. Yet, nuclei contain a subsample of the cells' transcriptional profile. Mature transcripts critical to cellular function and disease may be missed. We analyzed placenta from pregnancies using single-cell and single-nuclei RNA-seq. The datasets comprise 45,836cells and 27,078 nuclei, from 10 to 7 early-onset preeclampsia (EPE) cases and 3 and 2 early idiopathic controls (ECT), respectively. We compared the methods' sensitivities, cell type detection, differential gene expression in PE, and performed histological validations. Mature syncytiotrophoblast were sampled ∼50x more efficiently after nuclei extraction. Yet, scRNA-seq was more sensitive in detection of genes, molecules and mature transcripts. In snRNA-seq, nuclei of all placental cell classes suffered ambient trophoblast contamination. Transcripts from extravillous trophoblast, stroma, vasculature and immune cells were profiled less comprehensively by single-nuclei RNA-seq (snRNA-seq), restricting cell-type detection. In EPE, we found dysregulation of angiogenic actors FLT1/PGF both in prefused syncytiotrophoblast after cell extraction, and mature syncytiotrophoblast after nuclei isolation. Disease-related stress and inflammation were undetected from nuclei. scRNA-seq has important advantages over snRNA-seq for comprehensive transcriptomics studies of the placenta, especially to understand cell-type resolved dysregulation in pathologies. Yet, to address the dilemma of an underrepresented syncytium, studies benefit from complementary nuclei extraction.

YY1 drives PARP1 expression essential for PARylation of NONO in mRNA maturation during neuroblastoma progression

BackgroundNeuroblastoma (NB), the most prevalent solid tumor in children, arises from sympathetic nervous system and accounts for 15% of pediatric cancer mortality. This malignancy exhibits substantial genetic and clinical heterogeneity, thus complicating treatment strategies. Poly(ADP-ribose) polymerase 1 (PARP1), a key enzyme catalyzing polyADP-ribosylation (PARylation), plays critical roles in various cellular processes, and contributes to tumorigenesis and aggressiveness. However, the functions and regulatory mechanisms of PARP1 in NB progression still remain to be determined.MethodsThe association of PARP1 expression with NB patients’ survival was analyzed by mining of R2 database. Western blotting, reverse transcription-polymerase chain reaction, MTT colorimetric, soft agar, and matrigel invasion assays were utilized to assess PARP1 expression and its effects on aggressiveness of NB cell lines. Chromatin immunoprecipitation (ChIP) sequencing and ChIP assays were employed to investigate the binding of Yin Yang 1 (YY1) to PARP1 promoter. Protein interactions were explored by BioGRID database analysis, molecular docking, and co-immunoprecipitation assay. RNA sequencing and crosslinking-immunoprecipitation high throughput sequencing datasets were used to identify precursor mRNA splicing targets of non-POU domain containing octamer binding protein (NONO).ResultsHigh PARP1 expression was associated with poor survival of NB patients. PARP1 over-expression enhanced the proliferation and invasion of NB cell lines, confirming its oncogenic roles. YY1 was identified as a key transcriptional regulator facilitating PARP1 expression. Additionally, PARP1 interacted with NONO to induce its PARylation, resulting in stabilization of NONO protein via preventing ubiquitin-mediated degradation. NONO facilitated the splicing and mRNA maturation of target genes a disintegrin and metalloproteinase domain 8 (ADAM8) and testis-expressed gene 14 (TEX14) in a PARylation-dependent manner. Rescue experiments indicated that YY1 facilitated PARP1-mediated PARylation of NONO and subsequent mRNA maturation of ADAM8 and TEX14 in NB cells. In clinical NB cases, high expression of YY1, PARP1, NONO, ADAM8, or TEX14 was associated with poor survival of patients.ConclusionsThese findings indicate that YY1 drives PARP1 expression essential for PARylation of NONO in mRNA maturation during NB progression.

The Vsr-like protein FASTKD4 regulates the stability and polyadenylation of the MT-ND3 mRNA.

Expression of the compact mitochondrialgenome is regulated by nuclear encoded, mitochondrially localized RNA-binding proteins (RBPs). RBPs regulate the lifecycles of mitochondrial RNAs from transcription to degradation by mediating RNA processing, maturation, stability and translation. The Fas-activated serine/threonine kinase (FASTK) family of RBPs has been shown to regulate and fine-tune discrete aspects of mitochondrial gene expression. Although the roles of specific targets of FASTK proteins have been elucidated, the molecular mechanisms of FASTK proteins in mitochondrial RNA metabolism remain unclear. Therefore, we resolved the structure of FASTKD4 at atomic level that includes the RAP domain and the two FAST motifs, creating a positively charged cavity resembling that of the very short patch repair endonuclease. Our biochemical studies show that FASTKD4 binds the canonical poly(A) tail of MT-ND3 enabling its maturation and translation. The in vitro role of FASTKD4 is consistent with its loss in cells that results in decreased MT-ND3 polyadenylation, which destabilizes thismessenger RNA in mitochondria.

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.

RNA Metabolism and the Role of Small RNAs in Regulating Multiple Aspects of RNA Metabolism.

RNA metabolism is focused on RNA molecules and encompasses all the crucial processes an RNA molecule may or will undergo throughout its life cycle. It is an essential cellular process that allows all cells to function effectively. The transcriptomic landscape of a cell is shaped by the processes such as RNA biosynthesis, maturation (RNA processing, folding, and modification), intra- and inter-cellular transport, transcriptional and post-transcriptional regulation, modification, catabolic decay, and retrograde signaling, all of which are interconnected and are essential for cellular RNA homeostasis. In eukaryotes, sRNAs, typically 20-31 nucleotides in length, are a class of ncRNAs found to function as nodes in various gene regulatory networks. sRNAs are known to play significant roles in regulating RNA population at the transcriptional, post-transcriptional, and translational levels. Along with sRNAs, such as miRNAs, siRNAs, and piRNAs, new categories of ncRNAs, i.e., lncRNAs and circRNAs, also contribute to RNA metabolism regulation in eukaryotes. In plants, various genetic screens have demonstrated that sRNA biogenesis mutants, as well as RNA metabolism pathway mutants, exhibit similar growth and development defects, misregulated primary and secondary metabolism, as well as impaired stress response. In addition, sRNAs are both the "products" and the "regulators" in broad RNA metabolism networks; gene regulatory networks involving sRNAs form autoregulatory loops that affect the expression of both sRNA and the respective target. This review examines the interconnected aspects of RNA metabolism with sRNA regulatory pathways in plants. It also explores the potential conservation of these pathways across different kingdoms, particularly in plants and animals. Additionally, the review highlights how cellular RNA homeostasis directly impacts adaptive responses to environmental changes as well as different developmental aspects in plants.

FUS and METTL3 collaborate to regulate RNA maturation, preventing unfolded protein response and promoting gastric cancer progression

FUS-mediated alternative splicing and METTL3-regulated RNA methylation play crucial roles in RNA processing. The purpose of this study was to investigate the interactive roles of FUS and METTL3 in gastric cancer (GC) progression. RNA sequencing data were obtained from the TCGA-STAD dataset. Differentially expressed genes (DEGs) were analyzed across groups stratified by the medians of FUS, METTL3, and NEAT1, respectively. Endoplasmic reticulum (ER) stress markers PERK, IRE1, pIRE1, Bip, and CHOP, as well as related apoptosis stress markers PARP, cleaved-PARP, (Cleaved) Caspase 7, and (Cleaved) Caspase 3, were assessed through western blotting. Alternative splicing and N6-methyladenosine (m(6)A) methylation of specific genes were detected with MeRIP-PCR. Finally, in vivo experiments were conducted using nude mice bearing sh-FUS-transfected HGC27 xenograft tumors. FUS and METTL3 expression levels were elevated in GC tissues. A significant overlap of DEGs was observed between the FUS- and METTL3-stratified groups. These overlapping DEGs were predominantly enriched in mRNA processing and protein processing in the ER. ER stress and apoptosis were induced by sh-FUS or sh-METTL3, which was further enhanced by ER stress inducer tunicamycin in both MKN45 and HGC27 cells. Similarly, DEGs for NEAT1 high- and low-expressed groups were enriched in protein processing in the ER and spliceosome. To a lesser extent, ER stress was also induced by sh-NEAT1 and enhanced by tunicamycin in HGC27 cells. Furthermore, sh-FUS or sh-METTL3 influenced alternative splicing and methylation of specific mRNAs, including FUS, NEAT1, PCNA, MCM2, and BIRC5. Tumor progression was inhibited by sh-FUS in mice, and ER stress and apoptosis were induced, which were further enhanced by tunicamycin. FUS and METTL3 collaborate to facilitate RNA maturation. Inhibiting FUS or METTL3 promoted ER stress and apoptosis and inhibited progression in GC.Graphical abstractAberrant levels of FUS and METTL3 can evoke endoplasmic reticulum (ER) stress and apoptosis by generating splicing and methylation variants of mRNAs in gastric cancer, supporting the therapeutic potential of inducing ER stress

SLAM-seq reveals independent contributions of RNA processing and stability to gene expression in African trypanosomes.

Gene expression is a multi-step process that converts DNA-encoded information into proteins, involving RNA transcription, maturation, degradation, and translation. While transcriptional control is a major regulator of protein levels, the role of post-transcriptional processes such as RNA processing and degradation is less well understood due to the challenge of measuring their contributions individually. To address this challenge, we investigated the control of gene expression in Trypanosoma brucei, a unicellular parasite assumed to lack transcriptional control. Instead, mRNA levels in T. brucei are controlled by post-transcriptional processes, which enabled us to disentangle the contribution of both processes to total mRNA levels. In this study, we developed an efficient metabolic RNA labeling approach and combined ultra-short metabolic labeling with transient transcriptome sequencing (TT-seq) to confirm the long-standing assumption that RNA polymerase II transcription is unregulated in T. brucei. In addition, we established thiol (SH)-linked alkylation for metabolic sequencing of RNA (SLAM-seq) to globally quantify RNA processing rates and half-lives. Our data, combined with scRNA-seq data, indicate that RNA processing and stability independently affect total mRNA levels and contribute to the variability seen between individual cells in African trypanosomes.

Intron RPS25Ai, a Novel DNA Element, Has Global Effects on Synthetic Pathway Engineering by Empowering Protein Synthesis.

Classical genetic components in synthetic biology encompass essential elements of promoters, transcription factors, protein-coding genes, and terminators while both academic and industrial needs require novel engineering tools. Our study explores the potential of introns as versatile, novel biological DNA elements. Using intron RPS25Ai from Saccharomyces cerevisiae, the expression of mCherry was enhanced by 18.4-fold, demonstrating spatiotemporal regulatory patterns at both transcriptional and translational levels. A molecular mechanism study shows that this distinctive fine-tuning control relies on correct splicing events and extends to post-transcriptional processes. Intron RPS25Ai was applied to a heterologous metabolic pathway in engineered yeast, increasing β-carotene production by 4.29-fold. RPS25Ai functioned as a multilevel regulatory genetic element, enabling the increase in the expression of crtYB both at the pre-mRNA (99%) and mature RNA level (64%), with a splicing efficiency of 82%. Furthermore, the intron-engineered strain achieved a genome-scale regulation, upregulating 67% of "intron-containing" genes, with an average expression increase of 27%, compared with the upregulation of only 37% of "no-intron" genes. In addition, RPS25Ai induced a comprehensive rearrangement of ribosomal components, with the expression of 89% of ribosomal genes being upregulated, further empowering protein synthesis in the β-carotene-producing yeast cell factory.

Arabidopsis RNA-binding proteins interact with viral structural proteins and modify turnip yellows virus accumulation.

As obligate intracellular parasites, viruses depend on host proteins and pathways for their multiplication. Among these host factors, specific nuclear proteins are involved in the life cycle of some cytoplasmic replicating RNA viruses, although their role in the viral cycle remains largely unknown. The polerovirus turnip yellows virus (TuYV) encodes a major coat protein (CP) and a 74 kDa protein known as the readthrough (RT) protein. The icosahedral viral capsid is composed of the CP and a minor component RT*, arising from a C-terminal cleavage of the full-length RT. In this study, we identified Arabidopsis (Arabidopsis thaliana) ALY family proteins as interacting partners of TuYV structural proteins using yeast 2-hybrid assays and co-immunoprecipitations in planta. ALY proteins are adaptor proteins of the THO-TREX-1 complex essential to the nuclear export of mature messenger RNAs (mRNAs). Although all 4 ALY proteins colocalized with the CP and the RT protein in the nucleus upon co-expression in agro-infiltrated Nicotiana benthamiana leaves, only the CP remained nuclear and colocalized with ALY proteins in TuYV-infected cells, suggesting that the CP is an essential partner of ALY proteins. Importantly, TuYV-infected A. thaliana 4xaly knock-out mutants showed a significant increase in viral accumulation, indicating that TuYV infection is affected by an unknown ALY-mediated antiviral defense mechanism or impairs the cellular mRNA export pathway to favor viral RNA translation. This finding underpins the crucial role played by nuclear factors in the life cycle of cytoplasmic RNA viruses.

DRBD18 acts as a transcript-specific RNA editing auxiliary factor in Trypanosoma brucei.

Uridine insertion/deletion (U-indel) RNA editing of mitochondrial transcripts is a posttranscriptional modification in kinetoplastid organisms, resulting in the generation of mature mRNAs from cryptic precursors. This RNA editing process involves a multi-protein complex holoenzyme and multiple accessory factors. Recent investigations have highlighted the pivotal involvement of accessory RNA binding proteins (RBPs) in modulating RNA editing in T. brucei, often in a transcript-specific manner. DRBD18 is a multifunctional RBP that reportedly impacts the stability, processing, export and translation of nuclear-encoded mRNAs. However, mass spectrometry studies report DRBD18-RESC interactions, prompting us to investigate its role in mitochondrial U-indel RNA editing. In this study, we demonstrate the specific and RNase-sensitive interaction of DRBD18 with multiple RESC factors. Depletion of DRBD18 through RNA interference in procyclic form T. brucei leads to a significant reduction in the levels of edited A6 and COIII mitochondrial transcripts, while its overexpression causes a notable increase in the abundance of these edited mRNAs. RNA immunoprecipitation/qRT-PCR analysis indicates a direct role for DRBD18 in A6 and COIII mRNA editing. We also examined the impact of arginine methylation of DRBD18 in the editing process, revealing that the hypomethylated form of DRBD18, rather than the arginine-methylated version, is essential for promoting these editing events. In conclusion, our findings demonstrate that DRBD18 directly affects the editing of A6 and COIII mRNAs, with its function being modulated by its arginine methylation status, marking the first report of a mitochondrial function for this protein and identifying it as a newly characterized RNA editing auxiliary factor.

Orthrus: Towards Evolutionary and Functional RNA Foundation Models.

In the face of rapidly accumulating genomic data, our ability to accurately pre-dict key mature RNA properties that underlie transcript function and regulation remains limited. Pre-trained genomic foundation models offer an avenue to adapt learned RNA representations to biological prediction tasks. However, existing genomic foundation models are trained using strategies borrowed from textual or visual domains that do not leverage biological domain knowledge. Here, we intro-duce Orthrus, a Mamba-based mature RNA foundation model pre-trained using a novel self-supervised contrastive learning objective with biological augmentations. Orthrus is trained by maximizing embedding similarity between curated pairs of RNA transcripts, where pairs are formed from splice isoforms of 10 model organ-isms and transcripts from orthologous genes in 400+ mammalian species from the Zoonomia Project. This training objective results in a latent representation that clusters RNA sequences with functional and evolutionary similarities. We find that the generalized mature RNA isoform representations learned by Orthrus significantly outperform existing genomic foundation models on five mRNA prop-erty prediction tasks, and requires only a fraction of fine-tuning data to do so. Finally, we show that Orthrus is capable of capturing divergent biological function of individual transcript isoforms.