Long-read sequencing reveals the landscape of aberrant alternative splicing and novel therapeutic target in colorectal cancer.

BACKGROUND: Alternative splicing complexity plays a vital role in carcinogenesis and cancer progression. Improved understanding of novel splicing events and the underlying regulatory mechanisms may contribute new insights into developing new therapeutic strategies for colorectal cancer (CRC). METHODS: Here, we combined long-read sequencing technology with short-read RNA-seq methods to investigate the transcriptome complexity in CRC. By using experiment assays, we explored the function of newly identified splicing isoform TIMP1 Δ4-5. Moreover, a CRISPR/dCasRx-based strategy to induce the TIMP1 exon 4-5 exclusion was introduced to inhibit neoplasm growth. RESULTS: A total of 90,703 transcripts were identified, of which > 62% were novel compared with current transcriptome annotations. These novel transcripts were more likely to be sample specific, expressed at relatively lower levels with more exons, and oncogenes displayed a characteristic to generate more transcripts in CRC. Clinical outcome data analysis showed that 1472 differentially expressed alternative splicing events (DEAS) were tightly associated with CRC patients' prognosis, and many novel isoforms were likely to be important determinants for patient survival. Among these, newly identified splicing isoform TIMP1 Δ4-5 was significantly downregulated in CRC. Further in vitro and in vivo assays demonstrated that ectopic expression of TIMP1 Δ4-5 significantly suppresses tumor cell growth and metastasis. Serine/arginine-rich splicing factor 1 (SRSF1) acts as a onco-splicing regulator through sustaining the inclusion of TIMP1 exon 4-5. Furthermore, CRISPR/dCasRx-based strategies designed to induce TIMP1 exon 4-5 exclusion have the potential to restrain the CRC growth. CONCLUSIONS: This data provides a rich resource for deeper studies of gastrointestinal malignancies. Newly identified splicing isoform TIMP1 Δ4-5 plays an important role in mediating CRC progression and may be a potential therapy target in CRC.

Global Shift in Alternative Splicing and Therapeutic Susceptibilities in Leukemia Driven by METTL3 Overexpression.

SUMMARY: Mutations in splicing factors are commonly observed in chronic lymphocytic leukemia (CLL); however, other mechanisms can also contribute to the dysregulation of alternative splicing. One example is the overexpression of the m6A RNA methyltransferase METTL3, that by depositing the epitranscriptomic mark in spliceosome transcripts leads to aberrant splicing, but at the same time creates vulnerability to METTL3 inhibitors. See related article by Wu et al., p. 228 (8) .

Alternative mRNA Splicing and Promising Therapies in Cancer.

Cancer is among the leading causes of mortality worldwide. While considerable attention has been given to genetic and epigenetic sources of cancer-specific cellular activities, the role of alternative mRNA splicing has only recently received attention as a major contributor to cancer initiation and progression. The distribution of alternate mRNA splicing variants in cancer cells is different from their non-cancer counterparts, and cancer cells are more sensitive than non-cancer cells to drugs that target components of the splicing regulatory network. While many of the alternatively spliced mRNAs in cancer cells may represent "noise" from splicing dysregulation, certain recurring splicing variants have been shown to contribute to tumor progression. Some pathogenic splicing disruption events result from mutations in cis-acting splicing regulatory sequences in disease-associated genes, while others may result from shifts in balance among naturally occurring alternate splicing variants among mRNAs that participate in cell cycle progression and the regulation of apoptosis. This review provides examples of cancer-related alternate splicing events resulting from each step of mRNA processing and the promising therapies that may be used to address them.

Intron retention is a stress response in sensor genes and is restored by Japanese herbal medicines: A basis for future clinical applications.

Intron retention (IR) is a regulatory mechanism that can retard protein production by acting at the level of mRNA processing. We recently demonstrated that IR occurs at the pre-symptomatic state during the aging process of a mouse model of aging, providing a promising biomarker for that state, and can be restored to the normal state by juzentaihoto (JTT), a Japanese herbal medicine (Kampo) (Okada et al. 2021). Here we characterized the genes that accumulate retained introns, examined the biological significance of increased IR in these genes for the host, and determined whether drugs other than JTT can have this effect. By analyzing RNA-sequencing data generated from the hippocampus of the 19-week-old SAMP8 mouse, a model for studying age-related depression and Alzheimer's disease, we showed that genes with increased IR are generally involved in multiple metabolic pathways and have pivotal roles in sensing homeostasis. We thus propose that IR is a stress response and works to fine-tune the expression of many downstream target genes, leading to lower levels of their translation under stress conditions. Interestingly, Kampo medicines, as well as other organic compounds, restored splicing of a specific set of retained introns in these sensor genes in accordance with the physiological recovery conditions of the host, which corresponds with the recovery of transcripts represented by differentially expressed genes. Thus, analysis of IR genes may have broad applicability in evaluating the pre-symptomatic state based on the extent of IR of selective sensor genes, opening a promising early diagnosis of any diseases and a strategy for evaluating efficacies of several drugs based on the extent of IR restoration of these sensor genes.

Alternative RNA splicing in tumour heterogeneity, plasticity and therapy.

Alternative splicing is a process by which a single gene is able to encode multiple different protein isoforms. It is regulated by the inclusion or exclusion of introns and exons that are joined in different patterns prior to protein translation, thus enabling transcriptomic and proteomic diversity. It is now widely accepted that alternative splicing is dysregulated across nearly all cancer types. This widespread dysregulation means that nearly all cellular processes are affected - these include processes synonymous with the hallmarks of cancer - evasion of apoptosis, tissue invasion and metastasis, altered cellular metabolism, genome instability and drug resistance. Emerging evidence indicates that the dysregulation of alternative splicing also promotes a permissive environment for increased tumour heterogeneity and cellular plasticity. These are fundamental regulators of a patient's response to therapy. In this Review, we introduce the mechanisms of alternative splicing and the role of aberrant splicing in cancer, with particular focus on newfound evidence of alternative splicing promoting tumour heterogeneity, cellular plasticity and altered metabolism. We discuss recent in vivo models generated to study alternative splicing and the importance of these for understanding complex tumourigenic processes. Finally, we review the effects of alternative splicing on immune evasion, cell death and genome instability, and how targeting these might enhance therapeutic efficacy.

Detection of HAC1 mRNA Splicing by RT-PCR in Saccharomyces cerevisiae.

HAC1 mRNA remains translationally repressed in the cytoplasm of the budding yeast Saccharomyces cerevisiae. Under conditions of cellular stress, a dual kinase RNase IRE1 (Inositol Requiring Enzyme-1) cleaves out an intervening sequence from the HAC1 mRNA. Cleaved mRNAs are then ligated by tRNA ligase, thus generating a spliced mRNA that translates an active transcription factor. This unconventional splicing of HAC1 mRNA in the cytoplasm is a molecular marker for various cellular stresses including oxidative stress and endoplasmic reticulum (ER) stress. This article describes a PCR-based protocol to detect the HAC1 mRNA splicing.

Prostate cancer: Alternatively spliced mRNA transcripts in tumor progression and their uses as therapeutic targets.

Prostate cancer is the most frequently diagnosed cancer and second leading cause of cancer deaths among American men. Current therapies show early antitumor responses, but ultimately lead to treatment resistance, relapse and poorer survival in patients. Alternative RNA splicing, a cell mechanism increasing the proteome diversity by producing multiple transcripts from a single gene, has been associated with prostate cancer development/progression. Reports showed that many aberrant mRNA splice variants are upregulated in prostate cancer, promoting malignancy through enhanced proliferation, metastasis, tumor growth, anti-apoptosis, and/or treatment resistance. Here, we discuss the oncogenic properties of aberrant splicing mechanisms underlying prostate cancer pathogenesis, as well as the uses of the splicing variants as potential diagnostics and treatment targets. Finally, we discuss the pharmacologic and molecular approaches for targeting aberrant splicing mechanisms as effective therapies to correct the splicing errors and overcome the drug resistance, ultimately improving the clinical outcome of prostate cancer patients.

Circular RNAs Repertoire and Expression Profile during Brassica rapa Pollen Development.

Circular RNAs (circRNAs) are covalently closed RNA molecules generated by the back-splicing of exons from linear precursor mRNAs. Though various linear RNAs have been shown to play important regulatory roles in many biological and developmental processes, little is known about the role of their circular counterparts. In this study, we performed high-throughput RNA sequencing to delineate the expression profile and potential function of circRNAs during the five stages of pollen development in Brassica rapa. A total of 1180 circRNAs were detected in pollen development, of which 367 showed stage-specific expression patterns. Functional enrichment and metabolic pathway analysis showed that the parent genes of circRNAs were mainly involved in pollen-related molecular and biological processes such as mitotic and meiotic cell division, DNA processes, protein synthesis, protein modification, and polysaccharide biosynthesis. Moreover, by predicting the circRNA-miRNA network from our differentially expressed circRNAs, we found 88 circRNAs with potential miRNA binding sites, suggesting their role in post-transcriptional regulation of the genes. Finally, we confirmed the back-splicing sites of nine selected circRNAs using divergent primers and Sanger sequencing. Our study presents the systematic analysis of circular RNAs during pollen development and forms the basis of future studies for unlocking complex gene regulatory networks underpinning reproduction in flowering plants.

YY1 and RTCB in mouse uterine decidualization and embryo implantation.

As a multifunctional transcription factor, YY1 regulates the expression of many genes essential for early embryonic development. RTCB is an RNA ligase that plays a role in tRNA maturation and Xbp1 mRNA splicing. YY1 can bind in vitro to the response element in the proximal promoter of Rtcb and regulate Rtcb promoter activity. However, the in vivo regulation and whether these two genes are involved in the mother-fetal dialogue during early pregnancy remain unclear. In this study, we validated that YY1 bound in vivo to the proximal promoter of Rtcb in mouse uterus of early pregnancy. Moreover, via building a variety of animal models, our study suggested that both YY1 and RTCB might play a role in mouse uterus decidualization and embryo implantation during early pregnancy.

Orally administered branaplam does not impact neurogenesis in juvenile mice, rats, and dogs.

Branaplam is a therapeutic agent currently in clinical development for the treatment of infants with type 1 spinal muscular atrophy (SMA). Since preclinical studies showed that branaplam had cell-cycle arrest effects, we sought to determine whether branaplam may affect postnatal cerebellar development and brain neurogenesis. Here, we describe a novel approach for developmental neurotoxicity testing (DNT) of a central nervous system (CNS) active drug. The effects of orally administered branaplam were evaluated in the SMA neonatal mouse model (SMNΔ7), and in juvenile Wistar Hannover rats and Beagle dogs. Histopathological examination and complementary immunohistochemical studies focused on areas of neurogenesis in the cerebellum (mice, rats, and dogs), and the subventricular zone of the striatum and dentate gyrus (rats and dogs) using antibodies directed against Ki67, phosphorylated histone H3, cleaved caspase-3, and glial fibrillary acidic protein. Additionally, image-analysis based quantification of calbindin-D28k and Ki67 was performed in rats and dogs. The patterns of cell proliferation and apoptosis, and neural migration and innervation in the cerebellum and other brain regions of active adult neurogenesis did not differ between branaplam- and control-treated animals. Quantitative image analysis did not reveal any changes in calbindin-D28k and Ki67 expression in rats and dogs. The data show that orally administered branaplam has no impact on neurogenesis in juvenile animals. Application of selected immunohistochemical stainings in combination with quantitative image analysis on a few critical areas of postnatal CNS development offer a reliable approach to assess DNT of CNS-active drug candidates in juvenile animal toxicity studies.

Unconventional viral gene expression mechanisms as therapeutic targets.

Unlike the human genome that comprises mostly noncoding and regulatory sequences, viruses have evolved under the constraints of maintaining a small genome size while expanding the efficiency of their coding and regulatory sequences. As a result, viruses use strategies of transcription and translation in which one or more of the steps in the conventional gene-protein production line are altered. These alternative strategies of viral gene expression (also known as gene recoding) can be uniquely brought about by dedicated viral enzymes or by co-opting host factors (known as host dependencies). Targeting these unique enzymatic activities and host factors exposes vulnerabilities of a virus and provides a paradigm for the design of novel antiviral therapies. In this Review, we describe the types and mechanisms of unconventional gene and protein expression in viruses, and provide a perspective on how future basic mechanistic work could inform translational efforts that are aimed at viral eradication.

Periplocin and cardiac glycosides suppress the unfolded protein response.

The unfolded protein response (UPR) controls protein homeostasis through transcriptional and translational regulation. However, dysregulated UPR signaling has been associated with the pathogenesis of many human diseases. Therefore, the compounds modulating UPR may provide molecular insights for these pathologies in the context of UPR. Here, we screened small-molecule compounds that suppress UPR, using a library of Myanmar wild plant extracts. The screening system to track X-box binding protein 1 (XBP1) splicing activity revealed that the ethanol extract of the Periploca calophylla stem inhibited the inositol-requiring enzyme 1 (IRE1)-XBP1 pathway. We isolated and identified periplocin as a potent inhibitor of the IRE1-XBP1 axis. Periplocin also suppressed other UPR axes, protein kinase R-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6). Examining the structure-activity relationship of periplocin revealed that cardiac glycosides also inhibited UPR. Moreover, periplocin suppressed the constitutive activation of XBP1 and exerted cytotoxic effects in the human multiple myeloma cell lines, AMO1 and RPMI8226. These results reveal a novel suppressive effect of periplocin or the other cardiac glycosides on UPR regulation, suggesting that these compounds will contribute to our understanding of the pathological or physiological importance of UPR.

RNA-Targeting Splicing Modifiers: Drug Development and Screening Assays.

RNA splicing is an essential step in producing mature messenger RNA (mRNA) and other RNA species. Harnessing RNA splicing modifiers as a new pharmacological modality is promising for the treatment of diseases caused by aberrant splicing. This drug modality can be used for infectious diseases by disrupting the splicing of essential pathogenic genes. Several antisense oligonucleotide splicing modifiers were approved by the U.S. Food and Drug Administration (FDA) for the treatment of spinal muscular atrophy (SMA) and Duchenne muscular dystrophy (DMD). Recently, a small-molecule splicing modifier, risdiplam, was also approved for the treatment of SMA, highlighting small molecules as important warheads in the arsenal for regulating RNA splicing. The cellular targets of these approved drugs are all mRNA precursors (pre-mRNAs) in human cells. The development of novel RNA-targeting splicing modifiers can not only expand the scope of drug targets to include many previously considered "undruggable" genes but also enrich the chemical-genetic toolbox for basic biomedical research. In this review, we summarized known splicing modifiers, screening methods for novel splicing modifiers, and the chemical space occupied by the small-molecule splicing modifiers.

OsPPR939, a nad5 splicing factor, is essential for plant growth and pollen development in rice.

KEY MESSAGE: P-subfamily PPR protein OsPPR939, which can be phosphorylated by OsS6K1, regulates plant growth and pollen development by involving in the splicing of mitochondrial nad5 introns 1, 2, and 3. In land plants, pentatricopeptide repeat (PPR) proteins play key roles in mitochondrial group II intron splicing, but how these nucleus-encoded proteins are imported into mitochondria is unknown. To date, a few PPR proteins have been characterized in rice (Oryza sativa). Here, we demonstrate that the mitochondrion-localized P-subfamily PPR protein OsPPR939 is required for the splicing of nad5 introns 1, 2, and 3 in rice. Complete knockout or partial disruption of OsPPR939 function resulted in different degrees of growth retardation and pollen sterility. The dramatically reduced splicing efficiency of these introns in osppr939-4 and osppr939-5 led to reduced mitochondrial complex I abundance and activity and enhanced expression of alternative respiratory pathway genes. Complementation with OsPPR939 rescued the defective plant morphology of osppr939-4 and restored its decreased splicing efficiency of nad5 introns 1, 2, and 3. Therefore, OsPPR939 plays crucial roles in plant growth and pollen development by splicing mitochondrial nad5 introns 1, 2, and 3. More importantly, the 12th amino acid Ser in the N-terminal targeting sequence of OsPPR939 is phosphorylated by OsS6K1, and truncated OsPPR939 with a non-phosphorylatable S12A mutation in its presequence could not be imported into mitochondria, suggesting that phosphorylation of this amino acid plays an important role in the mitochondrial import of OsPPR939. To our knowledge, the 12th residue Ser on OsPPR939 is the first experimentally proven phosphorylation site in PPR proteins. Our results provide a basis for investigating the regulatory mechanism of PPR proteins at the post-translational level.

The protein kinase Ire1 impacts pathogenicity of Candida albicans by regulating homeostatic adaptation to endoplasmic reticulum stress.

The unfolded protein response (UPR), crucial for the maintenance of endoplasmic reticulum (ER) homeostasis, is tied to the regulation of multiple cellular processes in pathogenic fungi. Here, we show that Candida albicans relies on an ER-resident protein, inositol-requiring enzyme 1 (Ire1) for sensing ER stress and activating the UPR. Compromised Ire1 function impacts cellular processes that are dependent on functional secretory homeostasis, as inferred from transcriptional profiling. Concordantly, an Ire1-mutant strain exhibits pleiotropic roles in ER stress response, antifungal tolerance, cell wall regulation and virulence-related traits. Hac1 is the downstream target of C. albicans Ire1 as it initiates the unconventional splicing of the 19 bp intron from HAC1 mRNA during tunicamycin-induced ER stress. Ire1 also activates the UPR in response to perturbations in cell wall integrity and cell membrane homeostasis in a manner that does not necessitate the splicing of HAC1 mRNA. Furthermore, the Ire1-mutant strain is severely defective in hyphal morphogenesis and biofilm formation as well as in establishing a successful infection in vivo. Together, these findings demonstrate that C. albicans Ire1 functions to regulate traits that are essential for virulence and suggest its importance in responding to multiple stresses, thus integrating various stress signals to maintain ER homeostasis.

The integral spliceosomal component CWC15 is required for development in Arabidopsis.

Efficient mRNA splicing is a prerequisite for protein biosynthesis and the eukaryotic splicing machinery is evolutionarily conserved among species of various phyla. At its catalytic core resides the activated splicing complex Bact consisting of the three small nuclear ribonucleoprotein complexes (snRNPs) U2, U5 and U6 and the so-called NineTeen complex (NTC) which is important for spliceosomal activation. CWC15 is an integral part of the NTC in humans and it is associated with the NTC in other species. Here we show the ubiquitous expression and developmental importance of the Arabidopsis ortholog of yeast CWC15. CWC15 associates with core components of the Arabidopsis NTC and its loss leads to inefficient splicing. Consistent with the central role of CWC15 in RNA splicing, cwc15 mutants are embryo lethal and additionally display strong defects in the female haploid phase. Interestingly, the haploid male gametophyte or pollen in Arabidopsis, on the other hand, can cope without functional CWC15, suggesting that developing pollen might be more tolerant to CWC15-mediated defects in splicing than either embryo or female gametophyte.

Proteomics of SARS-CoV-2-infected host cells reveals therapy targets.

A new coronavirus was recently discovered and named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Infection with SARS-CoV-2 in humans causes coronavirus disease 2019 (COVID-19) and has been rapidly spreading around the globe1,2. SARS-CoV-2 shows some similarities to other coronaviruses; however, treatment options and an understanding of how SARS-CoV-2 infects cells are lacking. Here we identify the host cell pathways that are modulated by SARS-CoV-2 and show that inhibition of these pathways prevents viral replication in human cells. We established a human cell-culture model for infection with a clinical isolate of SARS-CoV-2. Using this cell-culture system, we determined the infection profile of SARS-CoV-2 by translatome3 and proteome proteomics at different times after infection. These analyses revealed that SARS-CoV-2 reshapes central cellular pathways such as translation, splicing, carbon metabolism, protein homeostasis (proteostasis) and nucleic acid metabolism. Small-molecule inhibitors that target these pathways prevented viral replication in cells. Our results reveal the cellular infection profile of SARS-CoV-2 and have enabled the identification of drugs that inhibit viral replication. We anticipate that our results will guide efforts to understand the molecular mechanisms that underlie the modulation of host cells after infection with SARS-CoV-2. Furthermore, our findings provide insights for the development of therapies for the treatment of COVID-19.

A Silent Exonic Mutation in a Rice Integrin-α FG-GAP Repeat-Containing Gene Causes Male-Sterility by Affecting mRNA Splicing.

Pollen development plays crucial roles in the life cycle of higher plants. Here we characterized a rice mutant with complete male-sterile phenotype, pollen-less 1 (pl1). pl1 exhibited smaller anthers with arrested pollen development, absent Ubisch bodies, necrosis-like tapetal hypertrophy, and smooth anther cuticular surface. Molecular mapping revealed a synonymous mutation in the fourth exon of PL1 co-segregated with the mutant phenotype. This mutation disrupts the exon-intron splice junction in PL1, generating aberrant mRNA species and truncated proteins. PL1 is highly expressed in the tapetal cells of developing anther, and its protein is co-localized with plasma membrane (PM) and endoplasmic reticulum (ER) signal. PL1 encodes an integrin-α FG-GAP repeat-containing protein, which has seven ß-sheets and putative Ca2+-binding motifs and is broadly conserved in terrestrial plants. Our findings therefore provide insights into both the role of integrin-α FG-GAP repeat-containing protein in rice male fertility and the influence of exonic mutation on intronic splice donor site selection.

The Yin and Yang of RNA surveillance in B lymphocytes and antibody-secreting plasma cells.

The random V(D)J recombination process ensures the diversity of the primary immunoglobulin (Ig) repertoire. In two thirds of cases, imprecise recombination between variable (V), diversity (D), and joining (J) segments induces a frameshift in the open reading frame that leads to the appearance of premature termination codons (PTCs). Thus, many B lineage cells harbour biallelic V(D)J-rearrangements of Ig heavy or light chain genes, with a productively-recombined allele encoding the functional Ig chain and a nonproductive allele potentially encoding truncated Ig polypeptides. Since the pattern of Ig gene expression is mostly biallelic, transcription initiated from nonproductive Ig alleles generates considerable amounts of primary transcripts with out-of-frame V(D)J junctions. How RNA surveillance pathways cooperate to control the noise from nonproductive Ig genes will be discussed in this review, focusing on the benefits of nonsense- mediated mRNA decay (NMD) activation during B-cell development and detrimental effects of nonsense-associated altered splicing (NAS) in terminally differentiated plasma cells. [BMB Reports 2019; 52(12): 671-678].

Sex-Dimorphic Behavioral Alterations and Altered Neurogenesis in U12 Intron Splicing-Defective Zrsr1 Mutant Mice.

Mutant mice with respect to the splicing factor Zrsr1 present altered spermatogenesis and infertility. To investigate whether Zrsr1 is involved in the homeostatic control that the hypothalamus exerts over reproductive functions, we first analyzed both differential gene and isoform expression and alternative splicing alterations in Zrsr1 mutant (Zrsr1mu) hypothalamus; second, we analyzed the spontaneous and social behavior of Zrsr1mu mice; and third, we analyzed adult cell proliferation and survival in the Zrsr1mu hypothalamus. The Zrsr1mu hypothalamus showed altered expression of genes and isoforms related to the glutathione metabolic process, synaptonemal complex assembly, mRNA transport, and altered splicing events involving the enrichment of U12-type intron retention (IR). Furthermore, increased IR in U12-containing genes related with the prolactin, progesterone, and gonadotropin-releasing hormone (GnRH) reproductive signaling pathway was observed. This was associated with a hyperactive phenotype in both males and females, with an anxious phenotype in females, and with increased social interaction in males, instead of the classical aggressive behavior. In addition, Zrsr1mu females but not males exhibited reduced cell proliferation in both the hypothalamus and the subventricular zone. Overall, these results suggest that Zrsr1 expression and function are relevant to organization of the hypothalamic cell network controlling behavior.