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Illustration of alternative splicing and polyadenylation patterns. A, Schematic of alternative splicing patterns including cassette exon, alternative 5′ or 3′ splice site selection, mutually exclusive exons, and intron retention. Constitutive exons are depicted in gray and alternatively splicing exons are depicted in purple or yellow. Thick lines represent alternatives to the canonical (thin line) splicing patterns. B, Examples of alternative polyadenylation patterns include tandem 3′UTR, skipped alternative terminal exon, and composite alternative terminal exon. While the proximal and distal PAS are in proximity to each other within tandem 3′UTRs, proximal PAS may be located within upstream sequences and its inclusion may be subjected to regulation like alternative splicing. Thick lines represent alternatives to the canonical (thin line) splicing patterns. Red hexagon represents translation stop codon. Purple and yellow regions indicate 3′UTRs
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The latest advances in next-generation sequencing studies and transcriptomic profiling over the past decade have highlighted a surprising frequency of genes regulated by RNA processing mechanisms in the immune system. In particular, two control steps in mRNA maturation, namely alternative splicing and alternative polyadenylation, are now recognized...
Citations
... To assess age-related changes in RNA processing in different tissues, we used APA as a readout. APA is a crucial RNA processing mechanism linked to alternative splicing 36,37 . For example, a previous study demonstrated that ret-1, encoding C. elegans Reticulon, exhibits two different splicing forms in muscle and intestine, and the splicing difference is reduced with aging 38 . ...
Organismal aging involves functional declines in both somatic and reproductive tissues. Multiple strategies have been discovered to extend lifespan across species. However, how age-related molecular changes differ among various tissues and how those lifespan-extending strategies slow tissue aging in distinct manners remain unclear. Here we generated the transcriptomic Cell Atlas of Worm Aging (CAWA, http://mengwanglab.org/atlas) of wild-type and long-lived strains. We discovered cell-specific, age-related molecular and functional signatures across all somatic and germ cell types. We developed transcriptomic aging clocks for different tissues and quantitatively determined how three different pro-longevity strategies slow tissue aging distinctively. Furthermore, through genome-wide profiling of alternative polyadenylation (APA) events in different tissues, we discovered cell-type-specific APA changes during aging and revealed how these changes are differentially affected by the pro-longevity strategies. Together, this study offers fundamental molecular insights into both somatic and reproductive aging and provides a valuable resource for in-depth understanding of the diversity of pro-longevity mechanisms.
... While iFLAS provides new insights into isoform identification and functional analysis from the perspective of full-length transcriptome, the integration of multiple tools may reduce its efficiency. Additionally, we did not consider the combined correlation between AS and APA in the PA site identification process, as well as the correlation between AS and allele-specific expression in the ASAS identification process, despite their correlation being extensively discussed (Park et al., 2018;Blake & Lynch, 2021). Since iFLAS is still in development, we are dedicated to optimizing its analytical processes, improving its operational efficiency, and enhancing the comprehensiveness of its functional modules. ...
The advent of full‐length transcriptome sequencing technologies has accelerated the discovery of novel splicing isoforms. However, existing alternative splicing (AS) tools are either tailored for short‐read RNA‐Seq data or designed for human and animal studies. The disparities in AS patterns between plants and animals still pose a challenge to the reliable identification and functional exploration of novel isoforms in plants.
Here, we developed integrated full‐length alternative splicing analysis (iFLAS), a plant‐optimized AS toolkit that introduced a semi‐supervised machine learning method known as positive‐unlabeled (PU) learning to accurately identify novel isoforms. iFLAS also enables the investigation of AS functions from various perspectives, such as differential AS, poly(A) tail length, and allele‐specific AS (ASAS) analyses.
By applying iFLAS to three full‐length transcriptome sequencing datasets, we systematically identified and functionally characterized maize (Zea mays) AS patterns. We found intron retention not only introduces premature termination codons, resulting in lower expression levels of isoforms, but may also regulate the length of 3′UTR and poly(A) tail, thereby affecting the functional differentiation of isoforms. Moreover, we observed distinct ASAS patterns in two genes within heterosis offspring, highlighting their potential value in breeding.
These results underscore the broad applicability of iFLAS in plant full‐length transcriptome‐based AS research.
... In addition to direct involvement in the APA pathway, RBPs can recruit or compete with polyadenylation-associated proteins to regulate the CPA process in target mRNAs [52]. HnRNP and SR family proteins can also regulate the polyadenylation process in mRNAs [53] (Figure 4). . RBPs form complexes with snRNP and polypeptides that together regulate the process of selective splicing. ...
... In addition to direct involvement in the APA pathway, RBPs can recruit or compete with polyadenylation-associated proteins to regulate the CPA process in target mRNAs [52]. HnRNP and SR family proteins can also regulate the polyadenylation process in mRNAs [53] (Figure 4). ...
RNA-binding proteins (RBPs), being pivotal elements in both physiological and pathological processes, possess the ability to directly impact RNA, thereby exerting a profound influence on cellular life. Furthermore, the dysregulation of RBPs not only induces alterations in the expression levels of genes associated with cancer but also impairs the occurrence of post-transcriptional regulatory mechanisms. Consequently, these circumstances can give rise to aberrations in cellular processes, ultimately resulting in alterations within the proteome. An aberrant proteome can disrupt the equilibrium between oncogenes and tumor suppressor genes, promoting cancer progression. Given their significant role in modulating gene expression and post-transcriptional regulation, directing therapeutic interventions towards RBPs represents a viable strategy for combating drug resistance in cancer treatment. RBPs possess significant potential as diagnostic and prognostic markers for diverse cancer types. Gaining comprehensive insights into the structure and functionality of RBPs, along with delving deeper into the molecular mechanisms underlying RBPs in tumor drug resistance, can enhance cancer treatment strategies and augment the prognostic outcomes for individuals afflicted with cancer.
... Alternative splicing (AS) contributes to phenotypic heterogeneity An introduction to AS: Most human protein-coding genes undergo AS, a key transcriptional and posttranscriptional process that leads to the formation of multiple transcript variants or splicing variants (SVs) that exert diverse effects via multiple mechanisms, including nonsense-mediated mRNA decay (NMD) (Fig. 7A). These splicing events are functionally important for innate and adaptive immune responses [305,306] due to their capacity to generate tissue-and cell type-specific or stimulusresponsive SVs [307][308][309], which have diverse or even opposing functions [310,311]. Abnormal SVs preferentially produced in various diseases have been proposed as biomarkers for diagnosis and treatment, and studies of such SVs have revealed precision therapy approaches to correct disease-specific defects caused by mis-splicing [312,313]. ...
... Although splicing factors and the processing cascades necessary for spliceosome function are well known [307][308][309], most of the abnormal chemokine SVs detected at the transcriptional level can be translated into distinct protein isoforms. As confusion mounts over the role of RNA isoforms in functional diversity and phenotypic plasticity [314,315], most chemokine transcript variants have not been studied, and their contribution to immune disorders and malignancy remains unknown [305,306,315]. A few notable examples of chemokine SVs with altered ligand-binding or signaling properties have been reported [316][317][318][319][320][321][322]. ...
Over the past thirty years, the importance of chemokines and their seven-transmembrane G protein-coupled receptors (GPCRs) has been increasingly recognized. Chemokine interactions with receptors trigger signaling pathway activity to form a network fundamental to diverse immune processes, including host homeostasis and responses to disease. Genetic and nongenetic regulation of both the expression and structure of chemokines and receptors conveys chemokine functional heterogeneity. Imbalances and defects in the system contribute to the pathogenesis of a variety of diseases, including cancer, immune and inflammatory diseases, and metabolic and neurological disorders, which render the system a focus of studies aiming to discover therapies and important biomarkers. The integrated view of chemokine biology underpinning divergence and plasticity has provided insights into immune dysfunction in disease states, including, among others, coronavirus disease 2019 (COVID-19). In this review, by reporting the latest advances in chemokine biology and results from analyses of a plethora of sequencing-based datasets, we outline recent advances in the understanding of the genetic variations and nongenetic heterogeneity of chemokines and receptors and provide an updated view of their contribution to the pathophysiological network, focusing on chemokine-mediated inflammation and cancer. Clarification of the molecular basis of dynamic chemokine-receptor interactions will help advance the understanding of chemokine biology to achieve precision medicine application in the clinic.
... Since coding genes of eukaryotes are composed of exons and introns, their initial transcript precursors undergo AS to remove introns, even some exons in some cases, to generate mature mRNAs [34]. Representative alternative splicing RBPs include serine/arginine-rich (SR) family proteins and hnRNP proteins [35]. Classical SRs bind with RNAs through their RRM domains. ...
... A number of RBPs have been identified to regulate APA [41]. Except mentioned RBPs, other RBPs such as hnRNPs and SR proteins can also regulate splicing and polyadenylation of mRNAs [35]. ...
Therapy resistance remains a huge challenge for current breast cancer treatments. Exploring molecular mechanisms of therapy resistance might provide therapeutic targets for patients with advanced breast cancer and improve their prognosis. RNA-binding proteins (RBPs) play an important role in regulating therapy resistance. Here we summarize the functions of RBPs, highlight their tremendously important roles in regulating therapy sensitivity and resistance and we also reveal current therapeutic approaches reversing abnormal functions of RBPs in breast cancer.
... For example, as shown in Figure 2G, we found that rs12068974 was located on HNRNPU, and may potentially affect APA events on CD55 in breast cancer. HNRNPU is a critical regulator of APA decisions (41). The alteration of HNRNPU may lead to distinct APA events on target genes. ...
Alternative polyadenylation (APA) is a widespread posttranscriptional regulation process. APA generates diverse mRNA isoforms with different 3’ UTR lengths, affecting mRNA expression, miRNA binding regulation and alternative splicing events. Previous studies have demonstrated the important roles of APA in tumorigenesis and cancer progression through diverse aspects. Thus, a comprehensive functional landscape of diverse APA events would aid in a better understanding of the underlying mechanisms related to APA in human cancers. Here, we built CAFuncAPA (https://relab.xidian.edu.cn/CAFuncAPA/) to systematically annotate the functions of 15478 APA events in human pan-cancers. Specifically, we first identified APA events associated with cancer survival and tumor progression. We annotated the potential downstream effects of APA on genes/isoforms expression, regulation of miRNAs, RNA binding proteins (RBPs) and alternative splicing events. Moreover, we also identified up-regulators of APA events, including the effects of genetic variants on poly(A) sites and RBPs, as well as the effect of methylation phenotypes on APA events. These findings suggested that CAFuncAPA can be a helpful resource for a better understanding of APA regulators and potential functions in cancer biology.
... Sequencing of the human genome and transcriptome has revealed that over 90% of multi-exon genes are alternatively spliced in a tissue-specific manner [45,46] . AS is regulated by RNAbinding protein (RBP) activity [47,48] , many RBPs are known to control AS in immune cells [47,48] , and there is increasing evidence that Akt controls the function of these proteins [15,49] . ...
... Sequencing of the human genome and transcriptome has revealed that over 90% of multi-exon genes are alternatively spliced in a tissue-specific manner [45,46] . AS is regulated by RNAbinding protein (RBP) activity [47,48] , many RBPs are known to control AS in immune cells [47,48] , and there is increasing evidence that Akt controls the function of these proteins [15,49] . ...
The activation and differentiation of CD4+ T cells is a complex process that is controlled by many factors. A critical component of the signaling pathway triggered following T-cell receptor (TCR) engagement is the serine threonine kinase Akt. Akt is involved in the control of many cellular processes including proliferation, metabolism, and differentiation of specific TH-cell subsets. Recent work has shown that, depending on the nature or strength of the TCR activation, Akt may activate different sets of substrates which then lead to differential cellular outcomes. Akt plays an important role in controlling the strength of the TCR signal and several recent studies have identified novel mechanisms including control of the expression of negative regulators of TCR signaling, and the influence on regulatory T cells (Treg) and TH17 differentiation. Many of these functions are mediated via control of the FoxO family of transcription factors, that play an important role in metabolism and Th cell differentiation. A theme that is emerging is that Akt does not function in the same way in all T-cell types. We highlight differences between CD4 and CD8 T cells as well as between Treg, TH17, and TFH cells. While Akt activity has been implicated in the control of alternative splicing in tumor cells, recent studies are emerging that indicate that similar functions may exist in CD4 T cells. In this mini review, we highlight some of the recent advances in these areas of Akt function that demonstrate the varied role that Akt plays in the function of CD4 T cells.
... Some of the best characterized examples of how alternative splicing can switch protein activity and cellular function come from the apoptosis signaling pathway (Blake and Lynch, 2021). Apoptosis is an immunologically silent form of cell death in which cells undergo shrinkage, phosphatidylserine exposure on the extracellular membrane, DNA fragmentation, and cellular blebbing (Elmore, 2007). ...
... Like FAS, the gene encoding Bcl-x is alternatively spliced to produce two isoforms: Bcl-xS, which promotes the activity of Bax and apoptosis, and a longer isoform, Bcl-xL that inhibits Bax activity and thus is anti-apoptotic (Billen et al., 2008;Stevens and Oltean, 2019). Bax and Bim also have reported alternatively spliced isoforms; however, the functional relevance of these is less well characterized (Blake and Lynch, 2021). Finally, once released from the mitochondria, cytosolic cytochrome c activates the enzymatic activity of the initiator caspase-9, through formation of the apoptosome, which then also cleaves the aforementioned effector caspases to promote apoptosis (McIlwain et al., 2013;Singh et al., 2019). ...
Alternative splicing occurs in the vast majority of human genes, giving rise to distinct mRNA and protein isoforms. We, and others, have previously identified hundreds of genes that change their isoform expression upon T cell activation via alternative splicing; however, how these changes link activation input with functional output remains largely unknown. Here we investigate how costimulation of T cells through the CD28 receptor impacts alternative splicing in T cells activated through the T cell receptor (CD3) and find that while CD28 signaling alone has minimal impact on splicing, it enhances the extent of change for up to 20% of TCR-induced alternative splicing events. Interestingly, a set of CD28-enhanced splicing events occur within genes encoding key components of the apoptotic signaling pathway; namely caspase-9, Bax and Bim. Using both CRISPR-edited cells and antisense oligos to force expression of specific isoforms, we show for all three of these genes that the isoform induced by CD3/CD28 costimulation promotes resistance to apoptosis, and that changes in all three genes together function combinatorially to further promote cell viability. Finally, we show that the JNK signaling pathway, induced downstream of CD3/CD28 costimulation, is required for each of these splicing events, further highlighting their co-regulation. Together these findings demonstrate that alternative splicing is a key mechanism by which costimulation of CD28 promotes viability of activated T cells.
... (2) Costimulation induces hundreds of AS and APA changes in CD8 + T cells, (3) RNA binding protein Tardbp controls AS and APA events in CD8 + T cells, and (4) Tardbp is necessary for optimal CD8 + T effector function after costimulation. AS and APA changes have been previously shown during T cell activation 40 . However, few largescale studies have conclusively demonstrated the necessity of RNA binding proteins regulating AS and APA for optimal T cell function after activation in vivo. ...
... Given that we found splicing sites in TCR signaling genes specifically controlled by Tardbp, this suggests an essential role for Tardbp in controlling TCR signaling gene isoforms in the process of T cell activation (Fig. 5). Further, prior work has shown a role for additional RBPs during T cell activation 40 . As we found 18 other RBPs to be upregulated after costimulation and found binding sites for many RBPs near splicing sites changed after T cell costimulation, it would be of interest to test the role of these RBPs in T cell function and contributions to the actions of costimulation. ...
Boosting T cell activation through costimulation directs defense against cancer and viral infections. Despite multiple studies targeting costimulation in clinical trials, the increased potency and reprogramming of T cells endowed by costimulation is poorly understood. Canonical dogma states that transcription mediates T cell activation. Here, we show that the spliceosome, controlling post-transcriptional alternative splicing and alternative polyadenylation, is the most enriched pathway in T cells after CD134/CD137 costimulation. Costimulation of CD8+ T cells significantly increases expression of 29 RNA-binding proteins while RNA-seq uncovers over 1000 differential alternative splicing and polyadenylation events. Using in vivo mouse and in vitro human models, we demonstrate that RNA-binding protein Tardbp is required for effector cytokine production, CD8+ T cell clonal expansion, and isoform regulation after costimulation. The prospect of immune response optimization through reprogramming of mRNA isoform production offered herein opens new avenues for experimentally and therapeutically tuning the activities of T cells. The provision of costimulatory signals to T cells are known to be vital in the direction and induction of a T cell driven response. Here the authors show an optimal T cell response requires a costimulatory induced modification of the transcript landscape and achieves this via modulation of the RNA binding proteins.
... Additional consideration should be given to the fact that PFS-mediated alternative splicing is central to T-cell activation. Thus, there is a possibility that CAR-T cell activation (via the CAR CD28-costimulatory domain) may be altered following binding/sequestration of nuclear PFS by VL30 mRNA [91][92][93]. ...
For more than a decade, genetically engineered autologous T-cells have been successfully employed as immunotherapy drugs for patients with incurable blood cancers. The active components in some of these game-changing medicines are autologous T-cells that express viral vector-delivered chimeric antigen receptors (CARs), which specifically target proteins that are preferentially expressed on cancer cells. Some of these therapeutic CAR expressing T-cells (CAR-Ts) are engineered via transduction with -retroviral vectors (-RVVs) produced in a stable producer cell line that was derived from murine PG13 packaging cells (ATCC CRL-10686). Earlier studies reported on the copackaging of murine virus-like 30S RNA (VL30) genomes with -retroviral vectors generated in murine stable packaging cells. In an earlier study, VL30 mRNA was found to enhance the metastatic potential of human melanoma cells. These findings raise biosafety concerns regarding the possibility that therapeutic CAR-Ts have been inadvertently contaminated with potentially oncogenic VL30 retrotransposons. In this study, we demonstrated the presence of infectious VL30 particles in PG13 cell-conditioned media and observed the ability of these particles to deliver transcriptionally active VL30 genomes to human cells. Notably, VL30 genomes packaged by HIV-1-based vector particles transduced naïve human cells in culture. Furthermore, we detected the transfer and expression of VL30 genomes in clinical-grade CAR-T cells generated by transduction with PG13 cell-derived -retroviral vectors. Our findings raise biosafety concerns regarding the use of murine packaging cell lines in ongoing clinical applications.
