Mature mRNA Research Articles

Polypyrimidine tract binding protein 1 (PTBP1) contains a novel regulatory sequence, the rBH3, that binds the prosurvival protein MCL1

The maturation of RNA from its nascent transcription to ultimate utilization (e.g., translation, miR-mediated RNA silencing, etc.) involves an intricately coordinated series of biochemical reactions regulated by RNA-binding proteins (RBPs). Over the past several decades, there has been extensive effort to elucidate the biological factors that control specificity and selectivity of RNA target binding and downstream function. Polypyrimidine tract binding protein 1 (PTBP1) is an RBP that is involved in all steps of RNA maturation and serves as a key regulator of alternative splicing, and therefore, understanding its regulation is of critical biologic importance. While several mechanisms of RBP specificity have been proposed (e.g., cell-specific expression of RBPs and secondary structure of target RNA), recently, protein-protein interactions with individual domains of RBPs have been suggested to be important determinants of downstream function. Here, we demonstrate a novel binding interaction between the first RNA recognition motif 1 (RRM1) of PTBP1 and the prosurvival protein myeloid cell leukemia-1 (MCL1). Using both in silico and invitro analyses, we demonstrate that MCL1 binds a novel regulatory sequence on RRM1. NMR spectroscopy reveals that this interaction allosterically perturbs key residues in the RNA-binding interface of RRM1 and negatively impacts RRM1 association with target RNA. Furthermore, pulldown of MCL1 by endogenous PTBP1 verifies that these proteins interact in an endogenous cellular environment, establishing the biological relevance of this binding event. Overall, our findings suggest a novel mechanism of regulation of PTBP1 in which a protein-protein interaction with a single RRM can impact RNA association.

Deep intronic mutation in CRTAP results in unstable isoforms of the protein to induce type I collagen aggregation in a lethal type of osteogenesis imperfecta type VII

Genetic mutations are involved in Mendelian disorders. Unbuffered intronic mutations in gene variants can generate aberrant splice sites in mutant transcripts, resulting in mutant isoforms of proteins with modulated expression, stability, and function in diseased cells. Here, we identify a deep intronic variant, c.794_1403A>G, in CRTAP by genome sequencing of a male fetus with osteogenesis imperfecta (OI) type VII. The mutation introduces cryptic splice sites in intron-3 of CRTAP, resulting in two mature mutant transcripts with cryptic exons. While transcript-1 translates to a truncated isoform (277 amino acids) with thirteen C-terminal non-wild-type amino acids, transcript-2 translates to a wild-type protein sequence, except that this isoform contains an in-frame fusion of non-wild-type twenty-five amino acids in a tetratricopeptide repeat sequence. Both mutant isoforms of CRTAP are unstable due to the presence of a unique ‘GWxxI’ degron, which finally leads to loss of proline hydroxylation and aggregation of type I collagen. Although type I collagen aggregates undergo autophagy, the overall proteotoxicity resulted in death of the proband cells by senescence. In summary, we present a genetic disease pathomechanism by linking a novel deep intronic mutation in CRTAP to unstable mutant isoforms of the protein in lethal OI type VII.

Identification of a novel WASP splice-site mutation in a patient with thrombocytopenia

Background and aimWiskott-Aldrich syndrome (WAS) (MIM #301000) is a rare X-linked primary immunodeficiency due to mutations in Wiskott-Aldrich syndrome protein (WASP) gene, characterized by thrombocytopenia with small platelets, eczema, recurrent infections and an increased incidence of autoimmunity and malignancies. Aim of this report is to describe the functional characterization of a novel WASP mutation. MethodsHere we report on the case of a 2-month-old boy presenting with petechiae, thrombocytopenia (23000/mm3MPV 6.9 fL), slightly increased eosinophil count (620 cells/mm3) and reduced CD8+ (6%; 267 cells/mm3) and IgM levels (20.9 mg/dL). Targeted NGS revealed a novel 4 nucleotides deletion from position +3 to +6 of intron 8 (c.777+3_777+6delGACT) of WASP. To characterize the pathogenetic role of this deletion we performed a Polymerase Chain Reaction (PCR) analysis on cDNA obtained from white blood cells using primers annealing on exon 8 (FW) and 9 (Rev). Western Blot was used to evaluate protein expression. ResultsIn the mature transcript we observed the complete retention of intron 8, suggesting a splicing defect, due to the loss of a splice donor site at the 5ʹ-end of intron 8. By sequencing the PCR product, we identified a premature stop at codon 269. Real-time PCR confirmed that the expression levels of the transcript containing intron 8 in the patient were similar to those observed for the wild-type transcript in healthy controls, although no WAS protein was detectable in peripheral blood mononuclear cells (PBMC) from the patient. ConclusionsWe identified and characterized a novel splice site mutation in a patient presenting with petechiae and thrombocytopenia. The young age of the patient did not allow to define the phenotype (classical WAS vs X-linked thrombocytopenia) associated with the mutation.

Novel compound heterozygous variants in EMC1 associated with global developmental delay: a lesson from a non-silent synonymous exonic mutation.

The endoplasmic reticulum-membrane protein complex (EMC) as a molecular chaperone is required for the proper synthesis, folding and traffic of several transmembrane proteins. Variants in the subunit 1 of EMC (EMC1) have been implicated in neurodevelopmental disorders. Whole exome sequencing (WES) with Sanger sequencing validation was performed for a Chinese family, including the proband (a 4-year-old girl who displayed global developmental delay, severe hypotonia and visual impairment), her affected younger sister and her non-consanguineous parents. RT-PCR assay and Sanger sequencing were used to detect abnormal RNA splicing. Novel compound heterozygous variants in EMC1, including the maternally inherited chr1: 19566812_1956800delinsATTCTACTT[hg19];NM_015047.3:c.765_777delins ATTCTACTT;p.(Leu256fsTer10) and the paternally inherited chr1:19549890G> A[hg19];NM_015047.3:c.2376G>A;p.(Val792=) are identified in the proband and her affected sister. RT-PCR assay followed by Sanger sequencing reveals that the c.2376G>A variant leads to aberrant splicing, with retention of intron 19 (561bp) in the mature mRNA, which is presumed to introduce a premature translational termination codon (p.(Val792fsTer31)). Novel compound heterozygous variants in EMC1 have been identified in individuals with global developmental delay. Non-silent synonymous mutations should be kept in mind in genetic analysis.

Group II Introns: Highly Structured yet Dynamic.

RNA splicing, the removal of introns and ligation of exons, is a crucial process during mRNA maturation. Group II introns are large ribozymes that self-catalyze their splicing, as well as their transposition. They are living fossils of spliceosomal introns and eukaryotic retroelements. The yeast mitochondrial Sc.ai5γ is the first identified and best-studied self-splicing group II intron. A combination of biochemical, biophysical, and computational tools enables studying its catalytic properties, structure, and dynamics, while also serving to develop new therapeutic and biotechnological tools. We survey the history of group II intron studies paralleling the trends in RNA methodology with Sc.ai5γ in the spotlight.

Working a second job: Cell adhesion proteins that moonlight in the nucleus.

Cells are adept at sensing changes in their environment, transmitting signals internally to coordinate responses to external stimuli, and thereby influencing adaptive changes in cell states and behavior. Often, this response involves modulation of gene expression in the nucleus, which is seen largely as a physically separated process from the rest of the cell. Mechanosensing, whereby a cell senses physical stimuli, and integrates and converts these inputs into downstream responses including signaling cascades and gene regulatory changes, involves the participation of several macromolecular structures. Of note, the extracellular matrix (ECM) and its constituent macromolecules comprise an essential part of the cellular microenvironment, allowing cells to interact with each other, and providing both structural and biochemical stimuli sensed by adhesion transmembrane receptors. This highway of information between the ECM, cell adhesion proteins, and the cytoskeleton regulates cellular behavior, the disruption of which results in disease. Emerging evidence suggests a more direct role for some of these adhesion proteins in chromatin structure and gene regulation, RNA maturation and other non-canonical functions. While many of these discoveries were previously limited to observations of cytoplasmic-nuclear transport, recent advances in microscopy, and biochemical, proteomic and genomic technologies have begun to significantly enhance our understanding of the impact of nuclear localization of these proteins. This review will briefly cover known cell adhesion proteins that migrate to the nucleus, and their downstream functions. We will outline recent advances in this very exciting yet still emerging field, with impact ranging from basic biology to disease states like cancer.

Oncogenic roles of GPR176 in breast cancer: a potential marker of aggressiveness and a potential target of gene therapy.

Belonging to the G-protein coupled receptor 1 family, G protein-coupled receptor 176 (GPR176) is associated with the Gz/Gx G-protein subclass and is capable of decreasing cAMP production. GPR176 expression was detected by qRT-PCR, bioinformatics analysis, Western blot and immunohistochemistry, and compared with clinicopathological characteristics of breast cancer. GPR176-related genes and pathways were subjected to bioinformatic analysis. We also explored the effects of GPR176 on the phenotypes of breast cancer cells. Lower expression of GPR176 mRNA was seen in breast cancer than in normal tissues, but the opposite pattern was found for its protein (p < 0.05). GPR176 mRNA was associated with female sex, low T staging, non-Her-2+ subtypes, non-mutant p53 status in breast cancer (p < 0.05). GPR176 methylation was negatively correlated with its mRNA level and T staging in breast cancer, and was higher in breastcancer than normal tissues (p < 0.05). GPR176 protein expression was positively correlated with older age, small tumor size, and non-luminal-B subtype of breast cancers (p < 0.05). The differential genes of GPR176 were involved in receptor-ligand interaction, RNA maturation, and so forth (p < 0.05). GPR176-related genes were categorized into cell mobility, membrane structure, and so on (p < 0.05). GPR176 knockdown weakened the proliferation, glucose catabolism, anti-apoptosis, anti-pyroptosis, migration, invasion, and epithelial-mesenchymal transition of breast cancer cells. These results indicate that GPR176 might be involved in the tumorigenesis and subsequent progression of breast cancer by deteriorating aggressive phenotypes. It might be utilized as a potential biomarker to indicate the aggressive behaviors and poor prognosis of breast cancer and a potential target of genetic therapy.

Role of histone deacetylase 3 (HDAC3) in adipose tissue metabolism and immunophenotype: Selected Abstract - SITeCS Congress 2022

Introduction: Obesity is associated with comorbidities such as cardiovascular disease and type 2 diabetes. HDAC3 regulates adipose tissue physiology (WAT), and its genetic inactivation causes metabolic reprogramming of white adipocytes toward browning. The aim of this work is to evaluate the effect of HDAC3 silencing at different stages of differentiation and investigate the influence of adipocyte metabolism on the immunophenotype of WAT. Materials and Methods: Following HDAC3 silencing in mesenchymal stem cells and mature adipocytes, adipocyte function, RNA, DNA and protein levels, and proliferation at the end of differentiation were analyzed. Visceral WAT immunophenotype (vWAT) of Hdac3 KO mice in WAT (Hdac3fatKO) and controls (FL) was performed by FACS. Results: Silencing HDAC3 in precursors amplifies the expression of genes and proteins that regulate differentiation, oxidative metabolism, browning and mitochondrial activity. Following silencing, we found increased 1)phosphorylation of AKT (1.64 fold change, P&lt;0.0001), indicative of increased insulin signaling, and 2)proliferation, characteristic of the early phase of differentiation. Mitochondrial content was unchanged, but increased mitochondrial activity was observed in terms of maximal respiration (1.42 fold change, P=0.0151) and uncoupling of the electron transport chain (+11.6%, P&lt;0.0001). No difference was observed following HDAC3 silencing in mature adipocytes. We hypothesized that the enhancement of oxidative metabolism may cause cellular damage or senescence and, consequently, the immunophenotype of vWAT might be affected by HDAC3 ablation. Analysis reveals an increase of macrophages (2.48 fold change, P=0.0311) in the vWAT of Hdac3fatKO mice polarizing toward the M2 population. Coculture of adipocytes with macrophages from bone marrow indicates that HDAC3 silencing in adipocytes stimulates macrophage activation. Conclusions: HDAC3 is a key factor in the WAT phenotype, and its inactivation triggers mechanisms that support browning. Early epigenetic events mediated by HDAC3 silencing are crucial in directing adipocyte precursors toward the oxidative phenotype. Finally, results obtained from ex vivo and in vitro studies suggest that specific factors produced by KO adipocytes may be involved in determining the observed immunophenotype. [FONDAZIONE CARIPLO 2015-0641]

Influence of the 135 bp Intron on Stilbene Synthase VaSTS11 Transgene Expression in Cell Cultures of Grapevine and Different Plant Generations of Arabidopsis thaliana

Modern plant biotechnology often faces the problem of obtaining a stable and powerful vector for gene overexpression. It is known that introns carry different regulatory elements whose effects on transgene expression have been poorly studied. To study the effect of an intron on transgene expression, the stilbene synthase 11 (VaSTS11) gene of grapevine Vitis amurensis Rupr. was selected and overexpressed in grapevine callus cell cultures and several plant generations of Arabidopsis thaliana as two forms, intronless VaSTS11c and intron-containing VaSTS11d. The STS genes play an important role in the biosynthesis of stilbenes, valuable plant secondary metabolites. VaSTS11d contained two exons and one intron, while VaSTS11c contained only two exons, which corresponded to the mature transcript. It has been shown that the intron-containing VaSTS11d was better expressed in several generations of transgenic A. thaliana than VaSTS11c and also exhibited a lower level of cytosine methylation. As a result, the content of stilbenes in the VaSTS11d-transgenic plants was much higher than in the VaSTS11c-transgenic plants. Similarly, the best efficiency in increasing the content of stilbenes was also observed in grapevine cell cultures overexpressing the intron-containing VaSTS11d transcript. Thus, the results indicate that an intron sequence with regulatory elements can have a strong positive effect on both transgene expression level and its biological functions in plants and plant cell cultures.

G-quadruplexes from non-coding RNAs.

Non-coding RNAs (ncRNAs) are significant regulators of gene expression in a wide range of biological processes, such as transcription, RNA maturation, or translation. ncRNAs interplay with proteins or other RNAs through not only classical sequence-based mechanisms but also unique higher-order structures such as RNA G-quadruplexes (rG4s). rG4s are predictably formed in guanine-rich sequences and are closely related to various human diseases, such as tumors, neurodegenerative diseases, and infections. This review focuses on the vital role of rG4s in ncRNAs, particularly lncRNAs and miRNAs. We outline the dynamic balance between rG4s and RNA stem-loop/hairpin structures and the interplay between ncRNAs and interactors, thereby modulating gene expression and disease progression. A complete understanding of the biological regulatory role and mechanism of rG4s in ncRNAs affirms the critical importance of folding into the appropriate three-dimensional structure in maintaining or modulating the functions of ncRNAs. It makes them novel therapeutic targets for adjusting potential-G4-containing-ncRNAs-associated diseases.

The HNRNPF/H RNA binding proteins and disease.

The members of the HNRNPF/H family of heterogeneous nuclear RNA proteins-HNRNPF, HNRNPH1, HNRNPH2, HNRNPH3, and GRSF1, are critical regulators of RNA maturation. Documented functions of these proteins include regulating splicing, particularly alternative splicing, 5' capping and 3' polyadenylation of RNAs, and RNA export. The assignment of these proteins to the HNRNPF/H protein family members relates to differences in the amino acid composition of their RNA recognition motifs, which differ from those of other RNA binding proteins (RBPs). HNRNPF/H proteins typically bind RNA sequences enriched with guanine (G) residues, including sequences that, in the presence of a cation, have the potential to form higher-order G-quadruplex structures. The need to further investigate members of the HNRNPF/H family of RBPs has intensified with the recent descriptions of their involvement in several disease states, including the pediatric tumor Ewing sarcoma and the hematological malignancy mantle cell lymphoma; newly described groups of developmental syndromes; and neuronal-related disorders, including addictive behavior. Here, to foster the study of the HNRNPF/H family of RBPs, we discuss features of the genes encoding these proteins, their structures and functions, and emerging contributions to disease. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Processing > Splicing Regulation/Alternative Splicing RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.

Principles, mechanisms, and biological implications of translation termination-reinitiation.

The gene expression pathway from DNA sequence to functional protein is not as straightforward as simple depictions of the central dogma might suggest. Each step is highly regulated, with complex and only partially understood molecular mechanisms at play. Translation is one step where the "one gene-one protein" paradigm breaks down, as often a single mature eukaryotic mRNA leads to more than one protein product. One way this occurs is through translation reinitiation, in which a ribosome starts making protein from one initiation site, translates until it terminates at a stop codon, but then escapes normal recycling steps and subsequently reinitiates at a different downstream site. This process is now recognized as both important and widespread, but we are only beginning to understand the interplay of factors involved in termination, recycling, and initiation that cause reinitiation events. There appear to be several ways to subvert recycling to achieve productive reinitiation, different types of stresses or signals that trigger this process, and the mechanism may depend in part on where the event occurs in the body of an mRNA. This perspective reviews the unique characteristics and mechanisms of reinitiation events, highlights the similarities and differences between three major scenarios of reinitiation, and raises outstanding questions that are promising avenues for future research.

Abstract 3722: Exploring the role of eIF4E in cancer cells with targeted protein degradation

Abstract Initiation of translation is considered the main rate-limiting step of protein synthesis and requires the recognition of 5’ m7G-cap on mature mRNAs and the formation of eukaryotic translation initiation factor 4F (eIF4F) multi-protein mRNA cap-binding complex. Formation of this complex requires the interaction of eIF4E and the scaffold protein eIF4G, and RNA helicase eIF4A. This eIF4F complex along with eIF3 mediate the recruitment of the 40S ribosomal particle to the 5′ cap of mRNA. Activation of eIF4E is a regulatory hub of many major oncogenic pathways, thus, targeting eIF4E has emerged as a potential therapeutic strategy in cancer. Here we have used a targeted protein degradation approach coupled with genetic rescue to explore the molecular and cellular dependency of cancer cells on eIF4E. Stable H1299 human NSCLC clones expressing FKBP12F36V-tagged eIF4E but lacking endogenous eIF4E were established. Treatment of multiple N- or C-tagged-eIF4E clones with dTAGv-1, an FKBP12F36V selective heterobifunctional molecule that recruits VHL, induced rapid degradation of eIF4E to undetectable levels by 6hr exposure. This also resulted in reduced expression of MCL1, a previously reported biomarker of eIF4E activity. Longer exposures to dTAGv-1 resulted in a cytostasis that was not associated with cell death. A diastereomer negative control of dTAGv-1 that cannot recruit VHL did not elicit loss of eIF4E or the downstream events associated with its loss. Global analysis of protein synthesis initiation by RIBOseq and proteome profiling following dTAGv-1 treatment out to 32hr exposure demonstrated surprisingly few alterations in protein expression despite the significant effect on cancer cell growth. We also expressed wild-type or eIF4E mutants predicted to disrupt key functions and determined their ability to rescue molecular or cellular phenotype associate with eIF4E-loss following dTAGv-1 treatment. Expression of wild-type eIF4E completely rescued cell growth and MCL1 expression. A W56A mutant predicted to disrupt mRNA-cap binding was unable to rescue eIF4E loss. In contrast, expression of W73F or S290A mutants (predicted to disrupt eIF4G binding or exhibit reduced eIF4E activity, respectively) were able to rescue the loss of eIF4E. In summary, our rescue experiments show that mRNA-cap binding by eIF4E is required and that eIF4E:eIF4G interaction in cells may be more complex than predicted. This may also explain the challenges associated with developing selective and cellularly potent inhibitors of the eIF4E:eIF4G interaction and that targeting mRNA-cap binding may be a more effective strategy. We predicted that removing eIF4E would impact on the global synthesis of many proteins. However, our data demonstrate that targeting eIF4E leads to limited effects on protein synthesis that remain sufficient to inhibit cancer cell growth. Further experiments are underway to understand which proteins drive this dependency on eIF4E. Citation Format: Swee Y. Sharp, Marianna Martella, Christopher I. Milton, George Ward, Caroline Richardson, Andrew Woodhead, Paul A. Clarke. Exploring the role of eIF4E in cancer cells with targeted protein degradation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3722.

Abstract 2051: MDN1, a novel potential early biomarker for endocrine resistant breast cancer

Abstract Midasin (MDN1) is a chaperone protein required for maturation and nuclear export of pre-ribosomal RNA. Recent investigation regarding the mutational status of MDN1 in breast cancer (BC) revealed an increased tumor mutational burden in BC patients. Interestingly, our previously published work demonstrated significantly high MDN1 protein levels in AI-resistant mammospheres. Therefore, we investigated the overall status of MDN1 in the TCGA database in breast cancer tumors and identified MDN1 expression in breast cancer cells that were sensitive or resistant to AIs. The purpose of this study was to evaluate whether there is an association between MDN1 expression or mutation status to examine if MDN1 functions as an early high-risk indicator for breast cancers resistant to AIs or endocrine therapy. Using the cBioportal for Cancer Genomics, in silico analysis was conducted and BC patients with altered MDN1 status demonstrated several clinical attributes that were significantly associated with higher mutation counts (Kruskal Wallis test, p value = 1.41 × 10-10 and q value = 4.11 × 10-9); MSI sensor score, (p value = 2.74 × 10-5 and q value = 2.27 × 10-4); and Fraction Altered Genome score (p value = 2.37 × 10-3 and q value = 0.011). Next, aneuploidy in breast cancer significantly evolved and was positively correlated with higher MDN1 expression (Spearman: 0.64, p value = 3.92 × 10-122). Compared to the other BC subtypes, MDN1 was significantly elevated in basal breast cancer (Chi square test p value &amp;lt; 10.0 × 10-10, q value = 4.11 × 10-9) and significantly higher in deceased versus living BC patients. The DepMap breast cancer cell line database analysis also revealed significantly increased MDN1 expression in both ER+/HER2- cell lines (p value = 7.93 × 10-3; 12 cell lines) and in ER-/HER2- cell lines (p value = 2.88 × 10-2; 31 cell lines). Primary and metastatic BC cell lines were compared and MDN1 expression was significantly higher in metastatic BC cells (p value = 8.04 × 10-5; 33 metastatic cell lines). Likewise, AI-resistant cell lines exhibited altered MDN1 expression compared to their sensitive counterparts. Additionally, there was a statistically significant association between higher ER copy number and increased MDN1 expression in BC cell lines (p value = 1.44 × 10-3; 61 cell lines). Further in-depth analysis of proteins that were coexpressed in MDN1-altered tumors revealed a panel of genes (MSH2, MSH6, RBM15, FOXM1,CCNE2, and CHEK1) that could represent a signature of early markers of AI-resistant BC since there was a positive correlation between MDN1 expression and associated pathways. In conclusion, increased expression of MDN1 in BC tumors or cells may serve as preliminary marker to predict resistance to endocrine therapy due to its significant association with genes such as MSH2, MSH6 and FOXM1 that are strongly established as indicators of endocrine resistance. This work was supported by NIH grant # SC1GM126617 and U54MD007582. Citation Format: Afia Ohemeng, Akash Gupta, Bipika Banjara, Mounika M. Pamukuntla, Manasa Kotina, A Michael Davidson, Syreeta L. Tilghman. MDN1, a novel potential early biomarker for endocrine resistant breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2051.

464 Alternative RNA Splicing Changes Underlie Meningioma DNA Methylation Groups

INTRODUCTION: Meningiomas are the most common primary intracranial tumor. They are associated with genetic mutations found across broad epigenetic or transcriptomic groups. Alternative RNA splicing (AS) is a critical step in gene expression that involves the differential inclusion of sequences into the mature mRNA. Dysregulation of AS is a hallmark of human tumors, but its contributions to meningioma tumorigenesis are incompletely understood. In particular, whether meningioma DNA-methylation groups are associated with specific downstream changes in AS is unknown. METHODS: AS events from 184 meningiomas were characterized using rMATS, a computational tool that quantifies percent splicing event inclusion (PSI) from short-read RNA-sequencing. Differential gene expression was quantified using DESeq2. RESULTS: Our analysis revealed 135 differentially spliced events in Immune-enriched vs. Hypermitotic meningiomas, 126 events in Immune-enriched vs. Merlin-intact meningiomas, and 185 events in Merlin-intact vs. Hypermitotic meningiomas. Hierarchical clustering of samples based on differential splicing events separated Hypermitotic from Merlin-intact and Immune-enriched meningiomas. The proto-oncogenes Misshapen-like Kinase 1 (MINK1) and Nuclear Auto-antigenic Sperm Protein (NASP) were differentially spliced across WHO grades, DNA methylation groups, and in primary vs. recurrent meningiomas and correlated with recurrence free survival. Differential gene expression analysis revealed alterations in the expression of several splicing factors across methylation groups, including those involved in tumorigenesis (MEX3A, SNRPE, SRSF5). CONCLUSIONS: AS promotes differential isoform expression across clinically important DNA-methylation groups of meningiomas. Our analyses capture a splicing signature that correlates with meningioma aggressiveness, identifies prognostic splicing events in known human proto-oncogenes (MINK1, NASP), and uncovers their potential upstream regulators. Validation of how these events impact meningioma tumorigenesis may allow for the development of novel diagnostic tests and therapies targeting AS.

Long non‐coding RNA‐regulated alternative splicing modulates key cancer pathway in lung adenocarcinoma

Long non‐coding RNA‐regulated alternative splicing modulates key cancer pathway in lung adenocarcinoma

3'-End Processing of Eukaryotic mRNA: Machinery, Regulation, and Impact on Gene Expression.

Formation of the 3' end of a eukaryotic mRNA is a key step in the production of a mature transcript. This process is mediated by a number of protein factors that cleave the pre-mRNA, add a poly(A) tail, and regulate transcription by protein dephosphorylation. Cleavage and polyadenylation specificity factor (CPSF) in humans, or cleavage and polyadenylation factor (CPF) in yeast, coordinates these enzymatic activities with each other, with RNA recognition, and with transcription. The site of pre-mRNA cleavage can strongly influence the translation, stability, and localization of the mRNA. Hence, cleavage site selection is highly regulated. The length of the poly(A) tail is also controlled to ensure that every transcript has a similar tail when it is exported from the nucleus. In this review, we summarize new mechanistic insights into mRNA 3'-end processing obtained through structural studies and biochemical reconstitution and outline outstanding questions in the field.

Regulation of mature mRNA levels by RNA processing efficiency.

Transcription and co-transcriptional processes, including pre-mRNA splicing and mRNA cleavage and polyadenylation, regulate the production of mature mRNAs. The carboxyl terminal domain (CTD) of RNA polymerase (pol) II, which comprises 52 repeats of the Tyr1Ser2Pro3Thr4Ser5Pro6Ser7 peptide, is involved in the coordination of transcription with co-transcriptional processes. The pol II CTD is dynamically modified by protein phosphorylation, which regulates recruitment of transcription and co-transcriptional factors. We have investigated whether mature mRNA levels from intron-containing protein-coding genes are related to pol II CTD phosphorylation, RNA stability, and pre-mRNA splicing and mRNA cleavage and polyadenylation efficiency. We find that genes that produce a low level of mature mRNAs are associated with relatively high phosphorylation of the pol II CTD Thr4 residue, poor RNA processing, increased chromatin association of transcripts, and shorter RNA half-life. While these poorly-processed transcripts are degraded by the nuclear RNA exosome, our results indicate that in addition to RNA half-life, chromatin association due to a low RNA processing efficiency also plays an important role in the regulation of mature mRNA levels.

Profiling the Binding Activities of Peptides and Inhibitors to the U2 Auxiliary Factor Homology Motif (UHM) Domains.

RNA splicing is a biological process to generate mature mRNA (mRNA) by removing introns and annexing exons in the nascent RNA transcript and is executed by a multiprotein complex called spliceosome. To aid RNA splicing, a class of splicing factors use an atypical RNA recognition domain (UHM) to bind with U2AF ligand motifs (ULMs) in proteins to form modules that recognize splice sites and splicing regulatory elements on mRNA. Mutations of UHM containing splicing factors have been found frequently in myeloid neoplasms. To profile the selectivity of UHMs for inhibitor development, we established binding assays to measure the binding activities between UHM domains and ULM peptides and a set of small-molecule inhibitors. Additionally, we computationally analyzed the targeting potential of the UHM domains by small-molecule inhibitors. Our study provided the binding assessment of UHM domains to diverse ligands that may guide development of selective UHM domain inhibitors in the future.

Roles of alternative splicing in infectious diseases: from hosts, pathogens to their interactions.

Alternative splicing (AS) is an evolutionarily conserved mechanism that removes introns and ligates exons to generate mature messenger RNAs (mRNAs), extremely improving the richness of transcriptome and proteome. Both mammal hosts and pathogens require AS to maintain their life activities, and inherent physiological heterogeneity between mammals and pathogens makes them adopt different ways to perform AS. Mammals and fungi conduct a two-step transesterification reaction by spliceosomes to splice each individual mRNA (named cis -splicing). Parasites also use spliceosomes to splice, but this splicing can occur among different mRNAs (named trans -splicing). Bacteria and viruses directly hijack the host's splicing machinery to accomplish this process. Infection-related changes are reflected in the spliceosome behaviors and the characteristics of various splicing regulators (abundance, modification, distribution, movement speed, and conformation), which further radiate to alterations in the global splicing profiles. Genes with splicing changes are enriched in immune-, growth-, or metabolism-related pathways, highlighting approaches through which hosts crosstalk with pathogens. Based on these infection-specific regulators or AS events, several targeted agents have been developed to fight against pathogens. Here, we summarized recent findings in the field of infection-related splicing, including splicing mechanisms of pathogens and hosts, splicing regulation and aberrant AS events, as well as emerging targeted drugs. We aimed to systemically decode host-pathogen interactions from a perspective of splicing. We further discussed the current strategies of drug development, detection methods, analysis algorithms, and database construction, facilitating the annotation of infection-related splicing and the integration of AS with disease phenotype.