Prognostic significance and biological implications of SM-like genes in mantle cell lymphoma.

BACKGROUND: SM-like (LSM) genes a family of RNA-binding proteins, are involved in mRNA regulation and can function as oncogenes by altering mRNA stability. However, their roles in B-cell progression and tumorigenesis remain poorly understood. METHODS: We analyzed gene expression profiles and overall survival data of 123 patients with mantle cell lymphoma (MCL). The LSM index was developed to assess its potential as a prognostic marker of MCL survival. RESULTS: Five of the eight LSM genes were identified as potential prognostic markers for survival in MCL, with particular emphasis on the LSM.index. The expression levels of these LSM genes demonstrated their potential utility as classifiers of MCL. The LSM.index-high group exhibited both poorer survival rates and lower RNA levels than did the overall transcript profile. Notably, LSM1 and LSM8 were overexpressed in the LSM.index-high group, with LSM1 showing 2.5-fold increase (p < 0.001) and LSM8 depicting 1.8-fold increase (p < 0.01) than those in the LSM.index-low group. Furthermore, elevated LSM gene expression was associated with increased cell division and RNA splicing pathway activity. CONCLUSIONS: The LSM.index demonstrates potential as a prognostic marker for survival in patients with MCL. Elevated expression of LSM genes, particularly LSM1 and LSM8, may be linked to poor survival outcomes through their involvement in cell division and RNA splicing pathways. These findings suggest that LSM genes may contribute to the aggressive behavior of MCL and represent potential targets for therapeutic interventions.

Dual modes of ZFC3H1 confer selectivity in nuclear RNA sorting.

The export and degradation pathways compete to sort nuclear RNAs, yet the default pathway remains unclear. Sorting of mature RNAs to degradation, facilitated by the exosome co-factor poly(A) exosome targeting (PAXT), is particularly challenging for their resemblance to mRNAs intended for translation. Here, we unveil that ZFC3H1, a core PAXT component, is co-transcriptionally loaded onto the first exon/intron of RNA precursors (pre-RNAs). Interestingly, this initial loading does not lead to pre-RNA degradation, as ZFC3H1 adopts a "closed" conformation, effectively blocking exosome recruitment. As processing progresses, RNA fate can be reshaped. Longer RNAs with more exons are allowed for nuclear export. By contrast, short RNAs with fewer exons preferentially recruit transient PAXT components ZC3H3 and RBM26/27 to the 3' end, triggering ZFC3H1 "opening" and subsequent exosomal degradation. Together, the decoupled loading and activation of ZFC3H1 pre-configures RNA fate for decay while still allowing a switch to nuclear export, depending on mature RNA features.

StructmRNA a BERT based model with dual level and conditional masking for mRNA representation.

In this study, we introduce StructmRNA, a new BERT-based model that was designed for the detailed analysis of mRNA sequences and structures. The success of DNABERT in understanding the intricate language of non-coding DNA with bidirectional encoder representations is extended to mRNA with StructmRNA. This new model uses a special dual-level masking technique that covers both sequence and structure, along with conditional masking. This enables StructmRNA to adeptly generate meaningful embeddings for mRNA sequences, even in the absence of explicit structural data, by capitalizing on the intricate sequence-structure correlations learned during extensive pre-training on vast datasets. Compared to well-known models like those in the Stanford OpenVaccine project, StructmRNA performs better in important tasks such as predicting RNA degradation. Thus, StructmRNA can inform better RNA-based treatments by predicting the secondary structures and biological functions of unseen mRNA sequences. The proficiency of this model is further confirmed by rigorous evaluations, revealing its unprecedented ability to generalize across various organisms and conditions, thereby marking a significant advance in the predictive analysis of mRNA for therapeutic design. With this work, we aim to set a new standard for mRNA analysis, contributing to the broader field of genomics and therapeutic development.

Functions and mechanisms of RNA tailing by nucleotidyl transferase proteins in plants.

The addition of non-templated nucleotides at the 3' terminus of RNA is a pervasive and evolutionarily conserved posttranscriptional modification in eukaryotes. Apart from canonical poly(A) polymerases (PAPs), which are responsible for catalyzing polyadenylation of messenger RNAs in the nucleus, a distinct group of non-canonical PAPs (ncPAPs), also known as nucleotidyl transferase proteins (NTPs), mediate the addition of uridine and adenosine or of more intricate combinations of nucleotides. Among these, HEN1 SUPPRESSOR 1 (HESO1) and UTP: RNA URIDYLYLTRANSFERASE (URT1) are the two most extensively studied NTPs responsible for the addition of uridine to the 3' ends of RNAs (RNA uridylation). Recent discoveries have improved our understanding of the functions and mechanisms of uridylation mediated by HESO1 and URT1 in RNA metabolism. Furthermore, more NTPs have been identified to function in the 3' tailing of RNA and not solely through uridylation. Accumulating evidence indicates that RNA tailing plays important roles in plant growth and development, stress responses, and disease resistance. In this review, we examined the latest developments in RNA tailing by NTPs, with a focus on RNA uridylation and metabolism in plants. We also discussed the essential aspects for future research in this field.

microRNAs as New Biomolecular Markers to Estimate Time since Death: A Systematic Review.

Estimating the post-mortem interval is still one of the most complex challenges in forensics. In fact, the main tools currently used are burdened by numerous limitations, which sometimes allow the time of death to be placed only within too large time intervals. In recent years, researchers have tried to identify new tools to try to narrow down the interval within which to place the time of death; among these, the analysis of microRNAs seems to be promising. An evidence-based systematic review of the literature has been conducted to evaluate the state of the art of knowledge, focusing on the potential correlation between miRNA degradation and PMI estimation. The research has been performed using the electronic databases PubMed, Scopus, and WOS. The results allowed us to highlight the usefulness of miRNAs both as markers for PMI estimation and for normalization, especially due to their stability. In fact, some miRNAs remain particularly stable for long periods and in different tissues, while others degrade faster. Furthermore, there are numerous factors capable of influencing the behavior of these molecules, among which the type of tissue, the cause of death, and the circadian rhythm appear to be the most relevant. Despite the promising results of the few articles present in the literature, because of the numerous limitations they are burdened by, further research is still necessary to achieve more solid and shareable results.

DeepKINET: a deep generative model for estimating single-cell RNA splicing and degradation rates.

Messenger RNA splicing and degradation are critical for gene expression regulation, the abnormality of which leads to diseases. Previous methods for estimating kinetic rates have limitations, assuming uniform rates across cells. DeepKINET is a deep generative model that estimates splicing and degradation rates at single-cell resolution from scRNA-seq data. DeepKINET outperforms existing methods on simulated and metabolic labeling datasets. Applied to forebrain and breast cancer data, it identifies RNA-binding proteins responsible for kinetic rate diversity. DeepKINET also analyzes the effects of splicing factor mutations on target genes in erythroid lineage cells. DeepKINET effectively reveals cellular heterogeneity in post-transcriptional regulation.

Diribonuclease activity eliminates toxic diribonucleotide accumulation.

RNA degradation is a central process required for transcriptional regulation. Eventually, this process degrades diribonucleotides into mononucleotides by specific diribonucleases. In Escherichia coli, oligoribonuclease (Orn) serves this function and is unique as the only essential exoribonuclease. Yet, related organisms, such as Pseudomonas aeruginosa, display a growth defect but are viable without Orn, contesting its essentiality. Here, we take advantage of P. aeruginosa orn mutants to screen for suppressors that restore colony morphology and identified yciV. Purified YciV (RNase AM) exhibits diribonuclease activity. While RNase AM is present in all γ-proteobacteria, phylogenetic analysis reveals differences that map to the active site. RNase AMPa expression in E. coli eliminates the necessity of orn. Together, these results show that diribonuclease activity prevents toxic diribonucleotide accumulation in γ-proteobacteria, suggesting that diribonucleotides may be utilized to monitor RNA degradation efficacy. Because higher eukaryotes encode Orn, these observations indicate a conserved mechanism for monitoring RNA degradation.

Cytosine methylation flags mitochondrial RNA for degradation.

Mitochondrial double-stranded RNA (dsRNA) can form spontaneously in mitochondria, blocking mitochondrial gene expression and triggering an immune response. A recent study by Kim, Tan, et al. identified a safeguard mechanism in which NOP2/Sun RNA methyltransferase 4 (NSUN4)-mediated RNA methylation (m5C) recruits the RNA degradation machinery to prevent dsRNA formation.

Potential Role of mRNA in Estimating Postmortem Interval: A Systematic Review.

Although the postmortem interval estimation still represents one of the main goals of forensic medicine, there are still several limitations that weigh on the methods most used for its determination: for this reason, even today, precisely estimating the postmortem interval remains one of the most important challenges in the forensic pathology field. To try to overcome these limitations, in recent years, numerous studies have been conducted on the potential use of the mRNA degradation time for reaching a more precise post mortem interval (PMI) estimation. An evidence-based systematic review of the literature has been conducted to evaluate the state of the art of the knowledge focusing on the potential correlation between mRNA degradation and PMI estimation. The research has been performed using the electronic databases PubMed and Scopus. The analysis conducted made it possible to confirm the potential applicability of mRNA for reaching a more precise PMI estimation. The analysis of the results highlighted the usefulness of some mRNAs, such as ß-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA, especially in short time frames, within a few hours or days of death. The matrices on which these analyses were conducted were also analyzed, resulting in less exposure to the external environment, including the heart, brain, and dental pulp. The major limitations were also reported, including the short time intervals analyzed in most of the articles, the lack of mathematical models, and the failure to report the error rate between the mRNA degradation time and PMI. Given the still small number of published articles, the lack of globally recognized standardized methods, and the numerous techniques used to evaluate the mRNA degradation times, numerous and larger studies are still necessary to reach more solid and shared evidence.

Membrane Localization of RNase Y Is Important for Global Gene Expression in Bacillus subtilis.

RNase Y is a key endoribonuclease that regulates global mRNA turnover and processing in Bacillus subtilis and likely many other bacteria. This enzyme is anchored to the cell membrane, creating a pseudo-compartmentalization that aligns with its role in initiating the decay of mRNAs primarily translated at the cell periphery. However, the reasons behind and the consequences of RNase Y's membrane attachment remain largely unknown. In our study, we examined a strain expressing wild-type levels of a cytoplasmic form of RNase Y from its chromosomal locus. This strain exhibits a slow-growth phenotype, similar to that of an RNase Y null mutant. Genome-wide data reveal a significant impact on the expression of hundreds of genes. While certain RNA substrates clearly depend on RNase Y's membrane attachment, others do not. We observed no correlation between mRNA stabilization in the mutant strains and the cellular location or function of the encoded proteins. Interestingly, the Y-complex, a specificity factor for RNase Y, also appears also recognize the cytoplasmic form of the enzyme, restoring wild-type levels of the corresponding transcripts. We propose that membrane attachment of RNase Y is crucial for its functional interaction with many coding and non-coding RNAs, limiting the cleavage of specific substrates, and potentially avoiding unfavorable competition with other ribonucleases like RNase J, which shares a similar evolutionarily conserved cleavage specificity.

A type II toxin-antitoxin system is responsible for the cell death at low temperature in Pseudomonas syringae Lz4W lacking RNase R.

RNase R (encoded by the rnr gene) is a highly processive 3' → 5' exoribonuclease essential for the growth of the psychrotrophic bacterium Pseudomonas syringae Lz4W at low temperature. The cell death of a rnr deletion mutant at low temperature has been previously attributed to processing defects in 16S rRNA, defective ribosomal assembly, and inefficient protein synthesis. We recently showed that RNase R is required to protect P. syringae Lz4W from DNA damage and oxidative stress, independent of its exoribonuclease activity. Here, we show that the processing defect in 16S rRNA does not cause cell death of the rnr mutant of P. syringae at low temperature. Our results demonstrate that the rnr mutant of P. syringae Lz4W, complemented with a RNase R deficient in exoribonuclease function (RNase RD284A), is defective in 16S rRNA processing but can grow at 4 °C. This suggested that the processing defect in ribosomal RNAs is not a cause of the cold sensitivity of the rnr mutant. We further show that the rnr mutant accumulates copies of the indigenous plasmid pLz4W that bears a type II toxin-antitoxin (TA) system (P. syringae antitoxin-P. syringae toxin). This phenotype was rescued by overexpressing antitoxin psA in the rnr mutant, suggesting that activation of the type II TA system leads to cold sensitivity of the rnr mutant of P. syringae Lz4W. Here, we report a previously unknown functional relationship between the cold sensitivity of the rnr mutant and a type II TA system in P. syringae Lz4W.

Regulation of Oocyte mRNA Metabolism: A Key Determinant of Oocyte Developmental Competence.

The regulation of mRNA transcription and translation is uncoupled during oogenesis. The reason for this uncoupling is two-fold. Chromatin is only accessible to the transcriptional machinery during the growth phase as it condenses prior to resumption of meiosis to ensure faithful segregation of chromosomes during meiotic maturation. Thus, transcription rates are high during this time period in order to produce all of the transcripts needed for meiosis, fertilization, and embryo cleavage until the newly formed embryonic genome becomes transcriptionally active. To ensure appropriate timing of key developmental milestones including chromatin condensation, resumption of meiosis, segregation of chromosomes, and polar body extrusion, the translation of protein from transcripts synthesized during oocyte growth must be temporally regulated. This is achieved by the regulation of mRNA interaction with RNA binding proteins and shortening and lengthening of the poly(A) tail. This chapter details the essential factors that regulate the dynamic changes in mRNA synthesis, storage, translation, and degradation during oocyte growth and maturation.

Genome-wide quantification of RNA flow across subcellular compartments reveals determinants of the mammalian transcript life cycle.

Dissecting the regulatory mechanisms controlling mammalian transcripts from production to degradation requires quantitative measurements of mRNA flow across the cell. We developed subcellular TimeLapse-seq to measure the rates at which RNAs are released from chromatin, exported from the nucleus, loaded onto polysomes, and degraded within the nucleus and cytoplasm in human and mouse cells. These rates varied substantially, yet transcripts from genes with related functions or targeted by the same transcription factors and RNA-binding proteins flowed across subcellular compartments with similar kinetics. Verifying these associations uncovered a link between DDX3X and nuclear export. For hundreds of RNA metabolism genes, most transcripts with retained introns were degraded by the nuclear exosome, while the remaining molecules were exported with stable cytoplasmic lifespans. Transcripts residing on chromatin for longer had extended poly(A) tails, whereas the reverse was observed for cytoplasmic mRNAs. Finally, machine learning identified molecular features that predicted the diverse life cycles of mRNAs.

RNase R vs. PNPase: selecting the best-suited exoribonuclease for environmental adaptation.

3' → 5' exoribonucleases play a critical role in many aspects of RNA metabolism. RNase R, PNPase, and RNase II are the major contributors to RNA processing, maturation, and quality control in bacteria. Bacteria don't seem to have dedicated RNA degradation machineries to process different classes of RNAs. Under different environmental and physiological conditions, their roles can be redundant and sometimes overlapping. Here, I discuss why PNPase and RNase R may have switched their physiological roles in some bacterial species to adapt to environmental conditions, despite being biochemically distinct exoribonucleases.

Peripheral Blood CD8+ T-Lymphocyte Immune Response in Benign and Subpopulations of Breast Cancer Patients.

Peripheral blood CD8+ T lymphocytes play a crucial role in cell-mediated immunity and tumor-related immune responses in breast cancer. In this study, label-free quantification analysis and gene set enrichment analysis (GSEA) of CD8+ T lymphocytes in the peripheral blood of benign patients and patients with different breast cancer (BC) subtypes, i.e., luminal A, luminal B, and triple-negative breast cancer (TNBC), were performed using nano-UHPLC and Orbitrap mass spectrometry. Differential protein expression in CD8+ T lymphocytes revealed significant downregulation (log2 FC ≥ 0.38 or ≤-0.38, adj. p < 0.05), particularly in proteins involved in cytotoxicity, cytolysis, and proteolysis, such as granzymes (GZMs) and perforin 1 (PRF1). This downregulation was observed in the benign group (GZMH, GZMM, and PRF1) and luminal B (GZMA, GZMH) subtypes, whereas granzyme K (GZMK) was upregulated in TNBC in comparison to healthy controls. The RNA degradation pathway was significantly downregulated (p < 0.05, normalized enrichment score (NES) from -1.47 to -1.80) across all BC subtypes, suggesting a potential mechanism for regulating gene expression during T cell activation. Also, the Sm-like proteins (LSM2, LSM3, and LSM5) were significantly downregulated in the RNA degradation pathway. Proteomic analysis of CD8+ T lymphocytes in peripheral blood across different breast cancer subtypes provides a comprehensive view of the molecular mechanisms of the systemic immune response that can significantly contribute to advancements in the diagnosis, treatment, and prognosis of this disease.

Assessing the effect of sample storage time on viral detection using a rapid and cost-effective CTAB-based extraction method.

BACKGROUND: Cassava leaf samples degrade quickly during storage and transportation from distant areas. Proper sampling and efficient, low-cost storage methods are critical for obtaining sufficient quality DNA and RNA for plant virus epidemiology and improving disease control understanding. This is useful when samples are collected from remote areas far from a laboratory or in developing countries where money and materials for virus diagnostics are scarce. RESULTS: The effect of sample storage duration on nucleic acid (N.A.) quality on virus detection was investigated in this study. A simple, rapid, and cost-effective CTAB-based approach (M3) for single N.A. extraction was optimized and tested alongside two existing CTAB-based methods (M1 and M2) for N.A. extraction from fresh and herbarium cassava leaves stored for; 1, 8, 26, and 56 months. The amount and quality of DNA and RNA were determined using Nanodrop 2000 c U.V.-vis Spectrophotometer and agarose gel electrophoreses. The sample degradation rate was estimated using a simple mathematical model in Matlab computational software. The results show no significant difference in mean DNA concentration between M1 and M2 but a significant difference between M3 and the other two methods at p < 0.005. The mean DNA concentration extracted using M3 was higher for 1 and 8 months of leave storage. M3 and M2 produced high concentrations at 26 and 56 months of leave storage. Using a developed scale for quality score, M3 and M2 produced high-quality DNA from fresh samples. All methods produced poor-quality DNA and RNA at 8 and 26 months of leave storage and no visual bands at the age of 56 months. Statistically, there was a significant difference in the mean DNA quality between M1 and M2, but there was no significant difference between M3 and the other two methods at p < 0.005. However, Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) were readily detected by RT-PCR from RNA isolated using M3. The quality of DNA declined per storage time at 0.0493 and 0.0521/month, while RNA was 0.0678 and 0.0744/month. Compared to the existing two methods, modified CTAB extracted enough high-quality N.A. in one-third the time of the existing two methods. CONCLUSION: Our method provides cost-effective, quick, and simple processing of fresh and dry samples, which will quicken and guide the decision on when and what type of sample to process for plant disease management and surveillance actions.

Quantifying mRNA in Highly Degraded Fixed Tissues by Nanostring Technology: A Comparative Study.

Archive tissues are the most available source of human tissues useful for molecular analysis in translational research. The main issues for those specimens are the modification and degradation of biomolecules, namely proteins, DNA, and RNA. In the last decade, several high-throughput analytical methods have been applied to archive tissues. Although histological tissues are fixed in neutral-buffered formalin nowadays, in the recent past, Bouin's solution was also used in tissue processing. The present study aims to investigate the feasibility of nCounter Nanostring hybridization in quantifying mRNA in highly degraded samples, such as Bouin's fixed and paraffin-embedded (BFPE) tissues, in comparison to the standard formalin-fixed and paraffin-embedded (FFPE) tissues as a source of RNA. A total of 16 paraffin-embedded tissue blocks from eight patients were analyzed (8 were FFPE and 8 were BEPE). Nanostring technology was applied to 300 ng of each RNA sample, whereas 360 ng of the same templates were retrotranscribed and submitted to qPCR and ddPCR. Our results show that the Nanostring technology outperforms the reference methods (ddPCR and qPCR) in detecting target mRNA in FFPE and BFPE samples. However, even Nanostring technology does not escape the limitation imposed by the degradation of the RNA templates, which could lead to misleading conclusions on the gene expression level.

The role of the 5' sensing function of ribonuclease E in cyanobacteria.

RNA degradation is critical for synchronising gene expression with changing conditions in prokaryotic and eukaryotic organisms. In bacteria, the preference of the central ribonucleases RNase E, RNase J and RNase Y for 5'-monophosphorylated RNAs is considered important for RNA degradation. For RNase E, the underlying mechanism is termed 5' sensing, contrasting to the alternative 'direct entry' mode, which is independent of monophosphorylated 5' ends. Cyanobacteria, such as Synechocystis sp. PCC 6803 (Synechocystis), encode RNase E and RNase J homologues. Here, we constructed a Synechocystis strain lacking the 5' sensing function of RNase E and mapped on a transcriptome-wide level 283 5'-sensing-dependent cleavage sites. These included so far unknown targets such as mRNAs encoding proteins related to energy metabolism and carbon fixation. The 5' sensing function of cyanobacterial RNase E is important for the maturation of rRNA and several tRNAs, including tRNAGluUUC. This tRNA activates glutamate for tetrapyrrole biosynthesis in plant chloroplasts and in most prokaryotes. Furthermore, we found that increased RNase activities lead to a higher copy number of the major Synechocystis plasmids pSYSA and pSYSM. These results provide a first step towards understanding the importance of the different target mechanisms of RNase E outside Escherichia coli.

The putative RNA helicase DDX1 associates with the nuclear RNA exosome and modulates RNA/DNA hybrids (R-loops).

The RNA exosome is a ribonuclease complex that mediates both RNA processing and degradation. This complex is evolutionarily conserved, ubiquitously expressed, and required for fundamental cellular functions, including rRNA processing. The RNA exosome plays roles in regulating gene expression and protecting the genome, including modulating the accumulation of RNA-DNA hybrids (R-loops). The function of the RNA exosome is facilitated by cofactors, such as the RNA helicase MTR4, which binds/remodels RNAs. Recently, missense mutations in RNA exosome subunit genes have been linked to neurological diseases. One possibility to explain why missense mutations in genes encoding RNA exosome subunits lead to neurological diseases is that the complex may interact with cell- or tissue-specific cofactors that are impacted by these changes. To begin addressing this question, we performed immunoprecipitation of the RNA exosome subunit, EXOSC3, in a neuronal cell line (N2A), followed by proteomic analyses to identify novel interactors. We identified the putative RNA helicase, DDX1, as an interactor. DDX1 plays roles in double-strand break repair, rRNA processing, and R-loop modulation. To explore the functional connections between EXOSC3 and DDX1, we examined the interaction following double-strand breaks and analyzed changes in R-loops in N2A cells depleted for EXOSC3 or DDX1 by DNA/RNA immunoprecipitation followed by sequencing. We find that EXOSC3 interaction with DDX1 is decreased in the presence of DNA damage and that loss of EXOSC3 or DDX1 alters R-loops. These results suggest EXOSC3 and DDX1 interact during events of cellular homeostasis and potentially suppress unscrupulous expression of genes promoting neuronal projection.

Structural basis for competitive binding of productive and degradative co-transcriptional effectors to the nuclear cap-binding complex.

The nuclear cap-binding complex (CBC) coordinates co-transcriptional maturation, transport, or degradation of nascent RNA polymerase II (Pol II) transcripts. CBC with its partner ARS2 forms mutually exclusive complexes with diverse "effectors" that promote either productive or destructive outcomes. Combining AlphaFold predictions with structural and biochemical validation, we show how effectors NCBP3, NELF-E, ARS2, PHAX, and ZC3H18 form competing binary complexes with CBC and how PHAX, NCBP3, ZC3H18, and other effectors compete for binding to ARS2. In ternary CBC-ARS2 complexes with PHAX, NCBP3, or ZC3H18, ARS2 is responsible for the initial effector recruitment but inhibits their direct binding to the CBC. We show that in vivo ZC3H18 binding to both CBC and ARS2 is required for nuclear RNA degradation. We propose that recruitment of PHAX to CBC-ARS2 can lead, with appropriate cues, to competitive displacement of ARS2 and ZC3H18 from the CBC, thus promoting a productive rather than a degradative RNA fate.