Disruption of PABPN1 phase separation by SNRPD2 drives colorectal cancer cell proliferation and migration through promoting alternative polyadenylation of CTNNBIP1.

Generally shortened 3' UTR due to alternative polyadenylation (APA) is widely observed in cancer, but its regulation mechanisms for cancer are not well characterized. Here, with profiling of APA in colorectal cancer tissues and poly(A) signal editing, we firstly identified that the shortened 3' UTR of CTNNIBP1 in colorectal cancer promotes cell proliferation and migration. We found that liquid-liquid phase separation (LLPS) of PABPN1 is reduced albeit with higher expression in cancer, and the reduction of LLPS leads to the shortened 3' UTR of CTNNBIP1 and promotes cell proliferation and migration. Notably, the splicing factor SNRPD2 upregulated in colorectal cancer, can interact with glutamic-proline (EP) domain of PABPN1, and then disrupt LLPS of PABPN1, which attenuates the repression effect of PABPN1 on the proximal poly(A) sites. Our results firstly reveal a new regulation mechanism of APA by disruption of LLPS of PABPN1, suggesting that regulation of APA by interfering LLPS of 3' end processing factor may have the potential as a new way for the treatment of cancer.

Influenza A virus NS1 effector domain is required for PA-X-mediated host shutoff in infected cells.

Many viruses inhibit general host gene expression to limit innate immune responses and gain preferential access to the cellular translational apparatus for their protein synthesis. This process is known as host shutoff. Influenza A viruses (IAVs) encode two host shutoff proteins: nonstructural protein 1 (NS1) and polymerase acidic X (PA-X). NS1 inhibits host nuclear pre-messenger RNA maturation and export, and PA-X is an endoribonuclease that preferentially cleaves host spliced nuclear and cytoplasmic messenger RNAs. Emerging evidence suggests that in circulating human IAVs NS1 and PA-X co-evolve to ensure optimal magnitude of general host shutoff without compromising viral replication that relies on host cell metabolism. However, the functional interplay between PA-X and NS1 remains unexplored. In this study, we sought to determine whether NS1 function has a direct effect on PA-X activity by analyzing host shutoff in A549 cells infected with wild-type or mutant IAVs with NS1 effector domain deletion. This was done using conventional quantitative reverse transcription polymerase chain reaction techniques and direct RNA sequencing using nanopore technology. Our previous research on the molecular mechanisms of PA-X function identified two prominent features of IAV-infected cells: nuclear accumulation of cytoplasmic poly(A) binding protein (PABPC1) and increase in nuclear poly(A) RNA abundance relative to the cytoplasm. Here we demonstrate that NS1 effector domain function augments PA-X host shutoff and is necessary for nuclear PABPC1 accumulation. By contrast, nuclear poly(A) RNA accumulation is not dependent on either NS1 or PA-X-mediated host shutoff and is accompanied by nuclear retention of viral transcripts. Our study demonstrates for the first time that NS1 and PA-X may functionally interact in mediating host shutoff.IMPORTANCERespiratory viruses including the influenza A virus continue to cause annual epidemics with high morbidity and mortality due to the limited effectiveness of vaccines and antiviral drugs. Among the strategies evolved by viruses to evade immune responses is host shutoff-a general blockade of host messenger RNA and protein synthesis. Disabling influenza A virus host shutoff is being explored in live attenuated vaccine development as an attractive strategy for increasing their effectiveness by boosting antiviral responses. Influenza A virus encodes two proteins that function in host shutoff: the nonstructural protein 1 (NS1) and the polymerase acidic X (PA-X). We and others have characterized some of the NS1 and PA-X mechanisms of action and the additive effects that these viral proteins may have in ensuring the blockade of host gene expression. In this work, we examined whether NS1 and PA-X functionally interact and discovered that NS1 is required for PA-X to function effectively. This work significantly advances our understanding of influenza A virus host shutoff and identifies new potential targets for therapeutic interventions against influenza and further informs the development of improved live attenuated vaccines.

PABPN1 loss-of-function causes APA-shift in oculopharyngeal muscular dystrophy.

Alternative polyadenylation (APA) at the 3' UTR of transcripts contributes to the cell transcriptome. APA is suppressed by the nuclear RNA-binding protein PABPN1. Aging-associated reduced PABPN1 levels in skeletal muscles lead to muscle wasting. Muscle weakness in oculopharyngeal muscular dystrophy (OPMD) is caused by short alanine expansion in PABPN1 exon1. The expanded PABPN1 forms nuclear aggregates, an OPMD hallmark. Whether the expanded PABPN1 affects APA and how it contributes to muscle pathology is unresolved. To investigate these questions, we developed a procedure including RNA library preparation and a simple pipeline calculating the APA-shift ratio as a readout for PABPN1 activity. Comparing APA-shift results to previously published PAS utilization and APA-shift results, we validated this procedure. The procedure was then applied on the OPMD cell model and on RNA from OPMD muscles. APA-shift was genome-wide in the mouse OPMD model, primarily affecting muscle transcripts. In OPMD individuals, APA-shift was enriched with muscle transcripts. In an OPMD cell model APA-shift was not significant. APA-shift correlated with reduced expression levels of a subset of PABPN1 isoforms, whereas the expression of the expanded PABPN1 did not correlate with APA-shift. PABPN1 activity is not affected by the expression of expanded PABPN1, but rather by reduced PABPN1 expression levels. In muscles, PABPN1 activity initially affects muscle transcripts. We suggest that muscle weakness in OPMD is caused by PABPN1 loss-of-function leading to APA-shift that primarily affects in muscle transcripts.

Dysregulation of RNA-Exosome machinery is directly linked to major cancer hallmarks in prostate cancer: Oncogenic role of PABPN1.

Novel biomarkers and therapeutic strategies for prostate-cancer (PCa) are required to overcome its lethal progression. The dysregulation/implication of the RNA-Exosome-complex (REC; cellular machinery controlling the 3'-5'processing/degradation of most RNAs) in different cancer-types, including PCa, is poorly known. Herein, different cellular/molecular/preclinical approaches with human PCa-samples (tissues and/or plasma of 7 independent cohorts), and in-vitro/in-vivo PCa-models were used to comprehensively characterize the REC-profile and explore its role in PCa. Moreover, isoginkgetin (REC-inhibitor) effects were evaluated on PCa-cells. We demonstrated a specific dysregulation of the REC-components in PCa-tissues, identifying the Poly(A)-Binding-Protein-Nuclear 1 (PABPN1) factor as a critical regulator of major cancer hallmarks. PABPN1 is consistently overexpressed in different human PCa-cohorts and associated with poor-progression, invasion and metastasis. PABPN1 silencing decreased relevant cancer hallmarks in multiple PCa-models (proliferation/migration/tumourspheres/colonies, etc.) through the modulation of key cancer-related lncRNAs (PCA3/FALEC/DLEU2) and mRNAs (CDK2/CDK6/CDKN1A). Plasma PABPN1 levels were altered in patients with metastatic and tumour-relapse. Finally, pharmacological inhibition of REC-activity drastically inhibited PCa-cell aggressiveness. Altogether, the REC is drastically dysregulated in PCa, wherein this novel molecular event/mechanism, especially PABPN1 alteration, may be potentially exploited as a novel prognostic and therapeutic tool for PCa.

PABPN1L is required for maternal mRNA degradation after meiosis resumption.

Poly(A)-binding proteins (PABPs) play roles in mRNA maturation, translational activity, and decay. The functions of PABPs, especially PABPN1 and PABPC1, in somatic cells have been well-studied. However, little is known about the roles of PABPs in oocytes because of the unique mechanisms of mRNA metabolism in oocytes. This study focused on PABPN1L and generated Pabpn1l knockout (KO) mice using the CRISPR/Cas9 system. After mating tests, we found that Pabpn1l KO females were infertile due to the failure of the embryos to develop to the 4-cell stage. RNA-seq analysis revealed aberrant mRNA persistence in Pabpn1l KO-MII oocytes, which indicates impaired mRNA degradation during the germinal vesicle (GV) to MII transition. We also revealed that the exogenous expression of Pabpn1l mRNA in KO-GV oocytes recovered defects of embryonic development. PABPN1L is partly indispensable for female fertility in mice, owing to its necessity for embryonic development, which is supported by mRNA degradation during GV to MII maturation.

PABPN1 functions as a predictive biomarker in colorectal carcinoma.

PURPOSE: PABPN1 acts as a modulator of poly(A) tail length and alternative polyadenylation. This research was aimed to explore the role of PABPN1 in colorectal cancer (CRC). METHODS: Public databases were performed to analyze expression, location, roles of prognosis and tumor immunity and interaction with RNAs and proteins of PABPN1. To investigate PABPN1 expression in tissues, 78 CRC specimens were collected to conduct IHC, and 30 pairs of frozen CRC and corresponding adjacent normal tissues were used to conduct qRT-PCR and WB. In addition, in vitro experiments were then carried out to identify the role of PABPN1 in CRC. RESULTS: Compared with normal tissues, PABPN1 expression was significant higher in CRC. Its high level predicted poor outcome of CRC. Th1 and Treg had significant negative relationships not only with PABPN1 expression, but also with six molecules interacting with PABPN1, including IFT172, KIAA0895L, RECQL4, WDR6, PABPC1 and NCBP1. In addition, PABPN1 had negative relationships with quite a few immune markers, such as CSF1R, IL-10, CCL2 and so on. In cellular experiments, silencing PABPN1 inhibited proliferation and promoted apoptosis in HCT-116 CRC cells. CONCLUSION: In summary, PABPN1 might become a novel biomarker and correlate with tumor immunity in CRC.

The polyA tail facilitates splicing of last introns with weak 3' splice sites via PABPN1.

The polyA tail of mRNAs is important for many aspects of RNA metabolism. However, whether and how it regulates pre-mRNA splicing is still unknown. Here, we report that the polyA tail acts as a splicing enhancer for the last intron via the nuclear polyA binding protein PABPN1 in HeLa cells. PABPN1-depletion induces the retention of a group of introns with a weaker 3' splice site, and they show a strong 3'-end bias and mainly locate in nuclear speckles. The polyA tail is essential for PABPN1-enhanced last intron splicing and functions in a length-dependent manner. Tethering PABPN1 to nonpolyadenylated transcripts also promotes splicing, suggesting a direct role for PABPN1 in splicing regulation. Using TurboID-MS, we construct the PABPN1 interactome, including many spliceosomal and RNA-binding proteins. Specifically, PABPN1 can recruit RBM26&27 to promote splicing by interacting with the coiled-coil and RRM domain of RBM27. PABPN1-regulated terminal intron splicing is conserved in mice. Together, our study establishes a novel mode of post-transcriptional splicing regulation via the polyA tail and PABPN1.

Alternative Splicing of lncRNAs From SNHG Family Alters snoRNA Expression and Induces Chemoresistance in Hepatoblastoma.

BACKGROUND & AIMS: Hepatoblastoma (HB) is a common pediatric malignant liver tumor that is characterized by a low level of genetic mutations. Alternative splicing (AS) has been shown to be closely associated with cancer progression, especially in tumors with a low mutational burden. However, the role of AS in HB remains unknown. METHODS: Transcriptome sequencing was performed on 5 pairs of HB tissues and matched non-tumor tissues to delineate the AS landscape in HB. AS events were validated in 92 samples from 46 patients. RNA pull-down and RNA immunoprecipitation assays were carried out to identify splicing factors that regulate the AS of small nucleolar RNA host genes (SNHG). Patient-derived organoids (PDOs) were established to investigate the role of the splicing factor polyadenylate-binding nuclear protein 1 (PABPN1). RESULTS: This study uncovered aberrant alternative splicing in HB, including lncRNAs from SNHG family that undergo intron retention in HB. Further investigations revealed that PABPN1, a significantly upregulated RNA binding protein, interacts with splicing machinery in HB, inducing the intron retention of these SNHG RNAs and the downregulation of intronic small nucleolar RNAs (snoRNAs). Functionally, PABPN1 acts as an oncofetal splicing regulator in HB by promoting cell proliferation and DNA damage repair via inducing the intron retention of SNHG19. Knock-down of PABPN1 increases the cisplatin sensitivity of HB PDOs. CONCLUSIONS: Our findings revealed the role of intron retention in regulating snoRNA expression in hepatoblastoma, explained detailed regulatory mechanism between PABPN1 and the intron retention of SNHG RNAs, and provided insight into the development of new HB treatment options.

PABPN1 aggregation is driven by Ala expansion and poly(A)-RNA binding, leading to CFIm25 sequestration that impairs alternative polyadenylation.

Poly(A)-binding protein nuclear 1 (PABPN1) is an RNA-binding protein localized in nuclear speckles, while its alanine (Ala)-expanded variants accumulate as intranuclear aggregates in oculopharyngeal muscular dystrophy. The factors that drive PABPN1 aggregation and its cellular consequences remain largely unknown. Here, we investigated the roles of Ala stretch and poly(A) RNA in the phase transition of PABPN1 using biochemical and molecular cell biology methods. We have revealed that the Ala stretch controls its mobility in nuclear speckles, and Ala expansion leads to aggregation from the dynamic speckles. Poly(A) nucleotide is essential to the early-stage condensation that thereby facilitates speckle formation and transition to solid-like aggregates. Moreover, the PABPN1 aggregates can sequester CFIm25, a component of the pre-mRNA 3'-UTR processing complex, in an mRNA-dependent manner and consequently impair the function of CFIm25 in alternative polyadenylation. In conclusion, our study elucidates a molecular mechanism underlying PABPN1 aggregation and sequestration, which will be beneficial for understanding PABPN1 proteinopathy.

The RNA-binding protein ZC3H11A interacts with the nuclear poly(A)-binding protein PABPN1 and alters polyadenylation of viral transcripts.

Nuclear mRNA metabolism is regulated by multiple proteins, which either directly bind to RNA or form multiprotein complexes. The RNA-binding protein ZC3H11A is involved in nuclear mRNA export, NF-κB signaling, and is essential during mouse embryo development. Furthermore, previous studies have shown that ZC3H11A is important for nuclear-replicating viruses. However, detailed biochemical characterization of the ZC3H11A protein has been lacking. In this study, we established the ZC3H11A protein interactome in human and mouse cells. We demonstrate that the nuclear poly(A)-binding protein PABPN1 interacts specifically with the ZC3H11A protein and controls ZC3H11A localization into nuclear speckles. We report that ZC3H11A specifically interacts with the human adenovirus type 5 (HAdV-5) capsid mRNA in a PABPN1-dependent manner. Notably, ZC3H11A uses the same zinc finger motifs to interact with PABPN1 and viral mRNA. Further, we demonstrate that the lack of ZC3H11A alters the polyadenylation of HAdV-5 capsid mRNA. Taken together, our results suggest that the ZC3H11A protein may act as a novel regulator of polyadenylation of nuclear mRNA.

TERRA stability is regulated by RALY and polyadenylation in a telomere-specific manner.

Telomeric repeat-containing RNA (TERRA) is a long non-coding RNA transcribed from telomeres that plays key roles in telomere maintenance. A fraction of TERRA is polyadenylated, and the presence of the poly(A) tail influences TERRA localization and stability. However, the mechanisms of TERRA biogenesis remain mostly elusive. Here, we show that the stability of TERRA transcripts is regulated by the RNA-binding protein associated with lethal yellow mutation (RALY). RALY depletion results in lower TERRA levels, impaired localization of TERRA at telomeres, and ultimately telomere damage. Importantly, we show that TERRA polyadenylation is telomere specific and that RALY preferentially stabilizes non-polyadenylated TERRA transcripts. Finally, we report that TERRA interacts with the poly(A)-binding protein nuclear 1 (PABPN1). Altogether, our results indicate that TERRA stability is regulated by the interplay between RALY and PABPN1, defined by the TERRA polyadenylation state. Our findings also suggest that different telomeres may trigger distinct TERRA-mediated responses.

PABPN1 regulates mRNA alternative polyadenylation to inhibit bladder cancer progression.

BACKGROUND: About 10-20% of patients with bladder cancer (BC) progress to muscle-invasive diseases, of which the underlying key molecular events have yet to be addressed. RESULTS: Here, we identified poly(A) binding protein nuclear 1 (PABPN1), a general factor of alternative polyadenylation (APA), was downregulated in BC. Overexpression and knockdown of PABPN1 significantly decreased and increased BC aggressiveness, respectively. Mechanistically, we provide evidence that the preference of PABPN1-bound polyadenylation signals (PASs) depends on the relative location between canonical and non-canonical PASs. PABPN1 shapes inputs converging on Wnt signaling, cell cycle, and lipid biosynthesis. CONCLUSIONS: Together, these findings provide insights into how PABPN1-mediated APA regulation contributes to BC progression, and suggest that pharmacological targeting PABPN1 might have therapeutic potential in patients with BC.

PABPN1 prevents the nuclear export of an unspliced RNA with a constitutive transport element and controls human gene expression via intron retention.

Intron retention is a type of alternative splicing where one or more introns remain unspliced in a polyadenylated transcript. Although many viral systems are known to translate proteins from mRNAs with retained introns, restriction mechanisms generally prevent export and translation of incompletely spliced mRNAs. Here, we provide evidence that the human nuclear poly(A)-binding protein, PABPN1, functions in such restrictions. Using a reporter construct in which nuclear export of an incompletely spliced mRNA is enhanced by a viral constitutive transport element (CTE), we show that PABPN1 depletion results in a significant increase in export and translation from the unspliced CTE-containing transcript. Unexpectedly, we find that inactivation of poly(A)-tail exosome targeting by depletion of PAXT components had no effect on export and translation of the unspliced reporter mRNA, suggesting a mechanism largely independent of nuclear RNA decay. Interestingly, a PABPN1 mutant selectively defective in stimulating poly(A) polymerase elongation strongly enhanced the expression of the unspliced, but not of intronless, reporter transcripts. Analysis of RNA-seq data also revealed that PABPN1 controls the expression of many human genes via intron retention. Notably, PABPN1-dependent intron retention events mostly affected 3'-terminal introns and were insensitive to PAXT and NEXT deficiencies. Our findings thus disclose a role for PABPN1 in restricting nuclear export of intron-retained transcripts and reinforce the interdependence between terminal intron splicing, 3' end processing, and polyadenylation.

A Case of Oculopharyngeal Muscular Dystrophy Caused by a Novel PABPN1 c.34G > T (p.Gly12Trp) Point Mutation without Polyalanine Expansion.

Immediately after the initial methionine codon, the PABPN1 gene encodes a stretch of 10 alanines, 1 glycine, and 2 alanines. Oculopharyngeal muscular dystrophy (OPMD) is caused by the expansion of the first 10 alanine stretches. The only exception is the missense mutation of glycine at the 12th residue into alanine, which elongates the stretch to 13 alanines by connecting the first and second stretch with the addition of one alanine in between, indicating that the expansion or elongation of the alanine stretch results in OPMD. We report a 77-year-old man with the novel missense mutation c.34G > T (p.Gly12Trp) in PABPN1 gene whose clinicopathological findings were compatible with OPMD. He presented with slowly progressive bilateral ptosis, dysphagia, and symmetrical proximal dominant muscle weakness. Magnetic resonance imaging revealed selective fat replacement of the tongue, bilateral adductor magnus, and soleus muscles. Immunohistochemistry studies of the muscle biopsy sample revealed PABPN1-posibive aggregates in the myonuclei which have been reported to be specific to OPMD. This is the first OPMD case caused by neither the expansion nor the elongation of alanine stretch. The present case suggests that OPMD may be caused not only by triplet repeats but also by point mutations.

Pharyngeal pathology in a mouse model of oculopharyngeal muscular dystrophy is associated with impaired basal autophagy in myoblasts.

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset dominant disease that primarily affects craniofacial muscles. Despite the fact that the genetic cause of OPMD is known to be expansion mutations in the gene encoding the nuclear polyadenosine RNA binding protein PABPN1, the molecular mechanisms of pathology are unknown and no pharmacologic treatments are available. Due to the limited availability of patient tissues, several animal models have been employed to study the pathology of OPMD. However, none of these models have demonstrated functional deficits in the muscles of the pharynx, which are predominantly affected by OPMD. Here, we used a knock-in mouse model of OPMD, Pabpn1 +/A17 , that closely genocopies patients. In Pabpn1 +/A17 mice, we detected impaired pharyngeal muscle function, and impaired pharyngeal satellite cell proliferation and fusion. Molecular studies revealed that basal autophagy, which is required for normal satellite cell function, is higher in pharynx-derived myoblasts than in myoblasts derived from limb muscles. Interestingly, basal autophagy is impaired in cells derived from Pabpn1 +/A17 mice. Pabpn1 knockdown in pharyngeal myoblasts failed to recapitulate the autophagy defect detected in Pabpn1 +/A17 myoblasts suggesting that loss of PABPN1 function does not contribute to the basal autophagy defect. Taken together, these studies provide the first evidence for pharyngeal muscle and satellite cell pathology in a mouse model of OPMD and suggest that aberrant gain of PABPN1 function contributes to the craniofacial pathology in OPMD.

Assessment of PABPN1 nuclear inclusions on a large cohort of patients and in a human xenograft model of oculopharyngeal muscular dystrophy.

Oculopharyngeal muscular dystrophy (OPMD) is a rare muscle disease characterized by an onset of weakness in the pharyngeal and eyelid muscles. The disease is caused by the extension of a polyalanine tract in the Poly(A) Binding Protein Nuclear 1 (PABPN1) protein leading to the formation of intranuclear inclusions or aggregates in the muscle of OPMD patients. Despite numerous studies stressing the deleterious role of nuclear inclusions in cellular and animal OPMD models, their exact contribution to human disease is still unclear. In this study, we used a large and unique collection of human muscle biopsy samples to perform an in-depth analysis of PABPN1 aggregates in relation to age, genotype and muscle status with the final aim to improve our understanding of OPMD physiopathology. Here we demonstrate that age and genotype influence PABPN1 aggregates: the percentage of myonuclei containing PABPN1 aggregates increases with age and the chaperone HSP70 co-localize more frequently with PABPN1 aggregates with a larger polyalanine tract. In addition to the previously described PRMT1 and HSP70 co-factors, we identified new components of PABPN1 aggregates including GRP78/BiP, RPL24 and p62. We also observed that myonuclei containing aggregates are larger than myonuclei without. When comparing two muscles from the same patient, a similar amount of aggregates is observed in different muscles, except for the pharyngeal muscle where fewer aggregates are observed. This could be due to the peculiar nature of this muscle which has a low level of PAPBN1 and contains regenerating fibers. To confirm the fate of PABPN1 aggregates in a regenerating muscle, we generated a xenograft model by transplanting human OPMD muscle biopsy samples into the hindlimb of an immunodeficient mouse. Xenografts from subjects with OPMD displayed regeneration of human myofibers and PABPN1 aggregates were rapidly present-although to a lower extent-after muscle fiber regeneration. Our data obtained on human OPMD samples add support to the dual non-exclusive models in OPMD combining toxic PABPN1 intranuclear inclusions together with PABPN1 loss of function which altogether result in this late-onset and muscle selective disease.

Novel read-through fusion transcript Bcl2l2-Pabpn1 in glioblastoma cells.

Read-through fusion transcripts have recently been identified as chimeric RNAs and have since been linked to tumour growth in some cases. Many fusion genes generated by chromosomal rearrangements have been described in glioblastoma. However, read-through fusion transcripts between neighbouring genes in glioblastoma remain unexplored. We performed paired-end RNA-seq of rat C6 glioma cells and normal cells and discovered a read-through fusion transcript Bcl2l2-Pabpn1 in which exon 3 of Bcl-2-like protein 2 (Bcl2l2) fused to exon 2 of Polyadenylate-binding protein 1 (Pabpn1). This fusion transcript was found in both human glioblastoma and normal cells. Unlike other fusions reported in glioblastoma, Bcl2l2-Pabpn1 appeared to result from RNA processing rather than genomic rearrangement. Bcl2l2-Pabpn1 fusion transcript encoded a fusion protein with BH4, BCL and RRM domains. Functionally, Bcl2l2-Pabpn1 knockdown by targeting its fusion junction decreased its expression, and suppressed cell proliferation, migration and invasion in vitro. Mechanistically, Bcl2l2-Pabpn1 blocked Bax activity and activated PI3K/AKT pathway to promote glioblastoma progression. Together, our work characterized a glioblastoma-associated Bcl2l2-Pabpn1 fusion transcript shared by humans and rats.

Exosome-Mediated Transfer of miR-1323 from Cancer-Associated Fibroblasts Confers Radioresistance of C33A Cells by Targeting PABPN1 and Activating Wnt/ß-Catenin Signaling Pathway in Cervical Cancer.

Plenty of pieces of evidence suggest that the resistance to radiotherapy greatly influences the therapeutic effect in cervical cancer (CCa). MicroRNAs (miRNAs) have been reported to regulate cellular processes by acting as tumor suppressors or promoters, thereby driving radioresistance or radiosensitivity. Meanwhile, it has been reported that microRNA-1323 (miR-1323) widely participates in cancer progression and radiotherapy effects. However, the role of miR-1323 is still not clear in CCa. Hence, in this study, we are going to investigate the molecular mechanism of miR-1323 in CCa cells. In the beginning, miR-1323 was found aberrantly upregulated in CCa cells via RT-qPCR assay. Functional assays indicated that miR-1323 was transferred by cancer-associated fibroblasts-secreted (CAFs-secreted) exosomes and miR-1323 downregulation suppressed cell proliferation, migration, invasion, and increased cell radiosensitivity in CCa. Mechanism assays demonstrated that miR-1323 targeted poly(A)-binding protein nuclear 1 (PABPN1). Besides, PABPN1 recruited insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) to regulate glycogen synthase kinase 3 beta (GSK-3ß) and influenced Wnt/ß-catenin signaling pathway. Therefore, rescue experiments were implemented to validate that PABPN1 overexpression rescued the inhibited cancer development and radioresistance induced by the miR-1323 inhibitor. In conclusion, miR-1323 was involved in CCa progression and radioresistance which might provide a novel insight for CCa treatment.

Characterization of a Read-through Fusion Transcript, BCL2L2-PABPN1, Involved in Porcine Adipogenesis.

cis-Splicing of adjacent genes (cis-SAGe) has been involved in multiple physiological and pathological processes in humans. However, to the best of our knowledge, there is no report of cis-SAGe in adipogenic regulation. In this study, a cis-SAGe product, BCL2L2-PABPN1 (BP), was characterized in fat tissue of pigs with RT-PCR and RACE method. BP is an in-frame fusion product composed of 333 aa and all the functional domains of both parents. BP is highly conserved among species and rich in splicing variants. BP was found to promote proliferation and inhibit differentiation of primary porcine preadipocytes. A total of 3074/44 differentially expressed mRNAs (DEmRs)/known miRNAs (DEmiRs) were identified in porcine preadipocytes overexpressing BP through RNA-Seq analysis. Both DEmRs and target genes of DEmiRs were involved in various fat-related pathways with MAPK and PI3K-Akt being the top enriched. PPP2CB, EGFR, Wnt5A and EHHADH were hub genes among the fat-related pathways identified. Moreover, ssc-miR-339-3p was found to be critical for BP regulating adipogenesis through integrated analysis of mRNA and miRNA data. The results highlight the role of cis-SAGe in adipogenesis and contribute to further revealing the mechanisms underlying fat deposition, which will be conductive to human obesity control.

Alternative Polyadenylation Utilization Results in Ribosome Assembly and mRNA Translation Deficiencies in a Model for Muscle Aging.

Aging-associated muscle wasting is regulated by multiple molecular processes, whereby aberrant mRNA processing regulation induces muscle wasting. The poly(A)-binding protein nuclear 1 (PABPN1) regulates polyadenylation site (PAS) utilization, in the absence of PABPN1 the alternative polyadenylation (APA) is utilized. Reduced PABPN1 levels induce muscle wasting where the expression of cellular processes regulating protein homeostasis, the ubiquitin-proteasome system, and translation, are robustly dysregulated. Translation is affected by mRNA levels, but PABPN1 impact on translation is not fully understood. Here we show that a persistent reduction in PABPN1 levels led to a significant loss of translation efficiency. RNA-sequencing of rRNA-depleted libraries from polysome traces revealed reduced mRNA abundance across ribosomal fractions, as well as reduced levels of small RNAs. We show that the abundance of translated mRNAs in the polysomes correlated with PAS switches at the 3'-UTR. Those mRNAs are enriched in cellular processes that are essential for proper muscle function. This study suggests that the effect of PABPN1 on translation efficiency impacts protein homeostasis in aging-associated muscle atrophy.