Terminal regions of UAP56 and URH49 are required for their distinct complex formation functioning to an essential role in mRNA processing and export.

UAP56 and URH49 are closely related RNA helicases that function in selective mRNA processing and export pathways to fine-tune gene expression through distinct complex formations. The complex formation of UAP56 and URH49 is believed to play a crucial role in regulating target mRNAs. However, the mechanisms underlying this complex formation have not been fully elucidated. Here we identified the regions essential for the complex formation of both helicases. The terminal regions of UAP56 and the C-terminal region of URH49 were indispensable for their respective complex formation. Further analysis revealed that a specific amino acid at the C-terminus of UAP56 is critical for its complex formation. Alanine substitution of this amino acid impairs its complex formation and subsequently affected its mRNA processing and export activity. Our study provides a deeper understanding of the basis for the complex formation between UAP56 and URH49.

Component of splicing factor SF3b plays a key role in translational control of polyribosomes on the endoplasmic reticulum.

One of the morphological hallmarks of terminally differentiated secretory cells is highly proliferated membrane of the rough endoplasmic reticulum (ER), but the molecular basis for the high rate of protein biosynthesis in these cells remains poorly documented. An important aspect of ER translational control is the molecular mechanism that supports efficient use of targeted mRNAs in polyribosomes. Here, we identify an enhancement system for ER translation promoted by p180, an integral ER membrane protein we previously reported as an essential factor for the assembly of ER polyribosomes. We provide evidence that association of target mRNAs with p180 is critical for efficient translation, and that SF3b4, an RNA-binding protein in the splicing factor SF3b, functions as a cofactor for p180 at the ER and plays a key role in enhanced translation of secretory proteins. A cis-element in the 5' untranslated region of collagen and fibronectin genes is important to increase translational efficiency in the presence of p180 and SF3b4. These data demonstrate that a unique system comprising a p180-SF3b4-mRNA complex facilitates the selective assembly of polyribosomes on the ER.

Epstein-Barr Virus Enhances Cancer-Specific Aberrant Splicing of TSG101 Pre-mRNA.

Tumor viruses gain control of cellular functions when they infect and transform host cells. Alternative splicing is one of the cellular processes exploited by tumor viruses to benefit viral replication and support oncogenesis. Epstein-Barr virus (EBV) participates in a number of cancers, as reported mostly in nasopharyngeal carcinoma (NPC) and Burkitt lymphoma (BL). Using RT-nested-PCR and Northern blot analysis in NPC and BL cells, here we demonstrate that EBV promotes specific alternative splicing of TSG101 pre-mRNA, which generates the TSG101∆154-1054 variant though the agency of its viral proteins, such as EBNA-1, Zta and Rta. The level of TSG101∆154-1054 is particularly enhanced upon EBV entry into the lytic cycle, increasing protein stability of TSG101 and causing the cumulative synthesis of EBV late lytic proteins, such as VCA and gp350/220. TSG101∆154-1054-mediated production of VCA and gp350/220 is blocked by the overexpression of a translational mutant of TSG101∆154-1054 or by the depletion of full-length TSG101, which is consistent with the known role of the TSG101∆154-1054 protein in stabilizing the TSG101 protein. NPC patients whose tumor tissues express TSG101∆154-1054 have high serum levels of anti-VCA antibodies and high levels of viral DNA in their tumors. Our findings highlight the functional importance of TSG101∆154-1054 in allowing full completion of the EBV lytic cycle to produce viral particles. We propose that targeting EBV-induced TSG101 alternative splicing has broad potential as a therapeutic to treat EBV-associated malignancies.

Novel LAMC2 fusion protein has tumor-promoting properties in ovarian carcinoma.

Laminins are heterotrimeric ECM proteins composed of α, ß, and γ chains. The γ2 chain (Lm-γ2) is a frequently expressed monomer and its expression is closely associated with cancer progression. Laminin-γ2 contains an epidermal growth factor (EGF)-like domain in its domain III (DIII or LEb). Matrix metalloproteinases can cleave off the DIII region of Lm-γ2 that retains the ligand activity for EGF receptor (EGFR). Herein, we show that a novel short form of Lm-γ2 (Lm-γ2F) containing DIII is generated without requiring MMPs and chromosomal translocation between LAMC2 on chromosome 1 and NR6A1 gene locus on chromosome 9 in human ovarian cancer SKOV3 cells. Laminin-γ2F is expressed as a truncated form lacking domains I and II, which are essential for its association with Lm-α3 and -ß3 chains of Lm-332. Secreted Lm-γ2F can act as an EGFR ligand activating the EGFR/AKT pathways more effectively than does the Lm-γ2 chain, which in turn promotes proliferation, survival, and motility of ovarian cancer cells. LAMC2-NR6A1 translocation was detected using in situ hybridization, and fusion transcripts were expressed in ovarian cancer cell tissues. Overexpression and suppression of fusion transcripts significantly increased and decreased the tumorigenic growth of cells in mouse models, respectively. To the best of our knowledge, this is the first report regarding a fusion gene of ECM showing that translocation of LAMC2 plays a crucial role in the malignant growth and progression of ovarian cancer cells and that the consequent product is a promising therapeutic target against ovarian cancers.

The Exon Junction Complex Core Represses Cancer-Specific Mature mRNA Re-splicing: A Potential Key Role in Terminating Splicing.

Using TSG101 pre-mRNA, we previously discovered cancer-specific re-splicing of mature mRNA that generates aberrant transcripts/proteins. The fact that mRNA is aberrantly re-spliced in various cancer cells implies there must be an important mechanism to prevent deleterious re-splicing on the spliced mRNA in normal cells. We thus postulated that mRNA re-splicing is controlled by specific repressors, and we searched for repressor candidates by siRNA-based screening for mRNA re-splicing activity. We found that knock-down of EIF4A3, which is a core component of the exon junction complex (EJC), significantly promoted mRNA re-splicing. Remarkably, we could recapitulate cancer-specific mRNA re-splicing in normal cells by knock-down of any of the core EJC proteins, EIF4A3, MAGOH, or RBM8A (Y14), implicating the EJC core as the repressor of mRNA re-splicing often observed in cancer cells. We propose that the EJC core is a critical mRNA quality control factor to prevent over-splicing of mature mRNA.

Rbm38 Reduces the Transcription Elongation Defect of the SMEK2 Gene Caused by Splicing Deficiency.

Pre-mRNA splicing is an essential mechanism for ensuring integrity of the transcriptome in eukaryotes. Therefore, splicing deficiency might cause a decrease in functional proteins and the production of nonfunctional, aberrant proteins. To prevent the production of such aberrant proteins, eukaryotic cells have several mRNA quality control mechanisms. In addition to the known mechanisms, we previously found that transcription elongation is attenuated to prevent the accumulation of pre-mRNA under splicing-deficient conditions. However, the detailed molecular mechanism behind the defect in transcription elongation remains unknown. Here, we showed that the RNA binding protein Rbm38 reduced the transcription elongation defect of the SMEK2 gene caused by splicing deficiency. This reduction was shown to require the N- and C-terminal regions of Rbm38, along with an important role being played by the RNA-recognition motif of Rbm38. These findings advance our understanding of the molecular mechanism of the transcription elongation defect caused by splicing deficiency.

Global analysis of pre-mRNA subcellular localization following splicing inhibition by spliceostatin A.

Spliceostatin A (SSA) is a methyl ketal derivative of FR901464, a potent antitumor compound isolated from a culture broth of Pseudomonas sp no. 2663. These compounds selectively bind to the essential spliceosome component SF3b, a subcomplex of the U2 snRNP, to inhibit pre-mRNA splicing. However, the mechanism of SSA's antitumor activity is unknown. It is noteworthy that SSA causes accumulation of a truncated form of the CDK inhibitor protein p27 translated from CDKN1B pre-mRNA, which is involved in SSA-induced cell-cycle arrest. However, it is still unclear whether pre-mRNAs are uniformly exported from the nucleus following SSA treatment. We performed RNA-seq analysis on nuclear and cytoplasmic fractions of SSA-treated cells. Our statistical analyses showed that intron retention is the major consequence of SSA treatment, and a small number of intron-containing pre-mRNAs leak into the cytoplasm. Using a series of reporter plasmids to investigate the roles of intronic sequences in the pre-mRNA leakage, we showed that the strength of the 5' splice site affects pre-mRNA leakage. Additionally, we found that the level of pre-mRNA leakage is related to transcript length. These results suggest that the strength of the 5' splice site and the length of the transcripts are determinants of the pre-mRNA leakage induced by SF3b inhibitors.

Cancer-Specifically Re-Spliced TSG101 mRNA Promotes Invasion and Metastasis of Nasopharyngeal Carcinoma.

TSG101 (Tumor susceptibility 101) gene and its aberrantly spliced isoform, termed TSG101∆154-1054, are tightly linked to tumorigenesis in various cancers. The aberrant TSG101∆154-1054 mRNA is generated from cancer-specific re-splicing of mature TSG101 mRNA. The TSG101∆154-1054 protein protects the full-length TSG101 protein from ubiquitin-mediated degradation, implicating TSG101∆154-1054 protein in the progression of cancer. Here, we confirmed that the presence of TSG101∆154-1054 mRNA indeed caused an accumulation of the TSG101 protein in biopsies of human nasopharyngeal carcinoma (NPC), which was recapitulated by the overexpression of TSG101∆154-1054 in the NPC cell line TW01. We demonstrate the potential function of the TSG101∆154-1054 protein in the malignancy of human NPC with scratch-wound healing and transwell invasion assays. By increasing the stability of the TSG101 protein, TSG101∆154-1054 specifically enhanced TSG101-mediated TW01 cell migration and invasion, suggesting the involvement in NPC metastasis in vivo. This finding sheds light on the functional significance of TSG101∆154-1054 generation via re-splicing of TSG101 mRNA in NPC metastasis and hints at its potential importance as a therapeutic target.

Splicing activator RNPS1 suppresses errors in pre-mRNA splicing: A key factor for mRNA quality control.

Human RNPS1 protein was first identified as a pre-mRNA splicing activator in vitro and RNPS1 regulates alternative splicing in cellulo. RNPS1 was also known as a peripheral factor of the exon junction complex (EJC). Here we show that cellular knockdown of RNPS1 induced a reduction of the wild-type aurora kinase B (AURKB) protein due to the induced aberrant pre-mRNA splicing events, indicating that the fidelity of AURKB pre-mRNA splicing was reduced. The major aberrant AURKB mRNA was derived from the upstream pseudo 5' and 3' splice sites in intron 5, which resulted in the production of the non-functional truncated AURKB protein. AURKB, is an essential mitotic factor, whose absence is known to cause multiple nuclei, and this multinucleation phenotype was recapitulated in RNPS1-knockdown cells. Importantly this RNPS1-knockdown phenotype was rescued by ectopic expression of AURKB, implying it is a major functional target of RNPS1. We found RNPS1 protein, not as a component of the EJC, binds directly to a specific element in the AURKB exon upstream of the authentic 5' splice site, and this binding is required for normal splicing. RNPS1-knockdown induces a parallel aberrant splicing pattern in a fully distinct pre-mRNA, MDM2, suggesting that RNPS1 is a global guardian of splicing fidelity. We conclude that RNPS1 is a key factor for the quality control of mRNAs that is essential for the phenotypes including cell division.

MALAT1 long non-coding RNA in cancer.

A recent massive parallel sequencing analysis has shown the fact that more than 80% of the human genome is transcribed into RNA. Among many kinds of the non-protein coding RNAs, we focus on the metastasis associated lung adenocarcinoma transcript 1 (MALAT1) that is a long non-coding RNA upregulated in metastatic carcinoma cells. Two molecular functions of MALAT1 have been proposed, one is the control of alternative splicing and the other is the transcriptional regulation. In this review, we document the molecular characteristics and functions of MALAT1 and shed light on the implication in the molecular pathology of various cancers. This article is part of a Special Issue entitled: Clues to long noncoding RNA taxonomy1, edited by Dr. Tetsuro Hirose and Dr. Shinichi Nakagawa.

ETV6-LPXN fusion transcript generated by t(11;12)(q12.1;p13) in a patient with relapsing acute myeloid leukemia with NUP98-HOXA9.

ETV6, which encodes an ETS family transcription factor, is frequently rearranged in human leukemias. We show here that a patient with acute myeloid leukemia with t(7;11)(p15;p15) gained, at the time of relapse, t(11;12)(q12.1;p13) with a split ETV6 FISH signal. Using 3'-RACE PCR analysis, we found that ETV6 was fused to LPXN at 11q12.1, which encodes leupaxin. ETV6-LPXN, an in-frame fusion between exon 4 of ETV6 and exon 2 of LPXN, did not transform the interleukin-3-dependent 32D myeloid cell line to cytokine independence; however, an enhanced proliferative response was observed when these cells were treated with G-CSF without inhibition of granulocytic differentiation. The 32D and human leukemia cell lines each transduced with ETV6-LPXN showed enhanced migration towards the chemokine CXCL12. We show here for the first time that LPXN is a fusion partner of ETV6 and present evidence indicating that ETV6-LPXN plays a crucial role in leukemia progression through enhancing the response to G-CSF and CXCL12.

The Exon Junction Complex Controls the Efficient and Faithful Splicing of a Subset of Transcripts Involved in Mitotic Cell-Cycle Progression.

The exon junction complex (EJC) that is deposited onto spliced mRNAs upstream of exon-exon junctions plays important roles in multiple post-splicing gene expression events, such as mRNA export, surveillance, localization, and translation. However, a direct role for the human EJC in pre-mRNA splicing has not been fully understood. Using HeLa cells, we depleted one of the EJC core components, Y14, and the resulting transcriptome was analyzed by deep sequencing (RNA-Seq) and confirmed by RT-PCR. We found that Y14 is required for efficient and faithful splicing of a group of transcripts that is enriched in short intron-containing genes involved in mitotic cell-cycle progression. Tethering of EJC core components (Y14, eIF4AIII or MAGOH) to a model reporter pre-mRNA harboring a short intron showed that these core components are prerequisites for the splicing activation. Taken together, we conclude that the EJC core assembled on pre-mRNA is critical for efficient and faithful splicing of a specific subset of short introns in mitotic cell cycle-related genes.

Endogenous Multiple Exon Skipping and Back-Splicing at the DMD Mutation Hotspot.

Duchenne muscular dystrophy (DMD) is a severe muscular disorder. It was reported that multiple exon skipping (MES), targeting exon 45-55 of the DMD gene, might improve patients' symptoms because patients who have a genomic deletion of all these exons showed very mild symptoms. Thus, exon 45-55 skipping treatments for DMD have been proposed as a potential clinical cure. Herein, we detected the expression of endogenous exons 44-56 connected mRNA transcript of the DMD using total RNAs derived from human normal skeletal muscle by reverse transcription polymerase chain reaction (RT-PCR), and identified a total of eight types of MES products around the hotspot. Surprisingly, the 5' splice sites of recently reported post-transcriptional introns (remaining introns after co-transcriptional splicing) act as splicing donor sites for MESs. We also tested exon combinations to generate DMD circular RNAs (circRNAs) and determined the preferential splice sites of back-splicing, which are involved not only in circRNA generation, but also in MESs. Our results fit the current circRNA-generation model, suggesting that upstream post-transcriptional introns trigger MES and generate circRNA because its existence is critical for the intra-intronic interaction or for extremely distal splicing.

NUP214-RAC1 and RAC1-COL12A1 Fusion in Complex Variant Translocations Involving Chromosomes 6, 7 and 9 in an Acute Myeloid Leukemia Case with DEK-NUP214.

DEK-NUP214 gene fusion in acute myeloid leukemia (AML) is associated with poor prognosis. It is most often a sole translocation and more rarely observed as complex chromosomal forms. We describe an AML case with complex karyotype abnormalities involving chromosome bands 6p23, 6q13, 7p22, and 9q34. RNA sequencing analysis revealed that exon 17 of NUP214 (9q34) was fused to exon 2 of RAC1 (7p22). We also detected that the 5'-end of intron 1 of RAC1 was fused with the antisense strand of intron 5 of COL12A1 (6q13). RT-PCR analysis confirmed the expression of DEK-NUP214, NUP214-RAC1, RAC1-COL12A1, NUP214, and RAC1. These results suggest that the 5'- and 3'-ends of NUP214 from the breakpoint in the same locus were fused to RAC1 and DEK, respectively, and the 5'-end of RAC1 was fused to COL12A1. The reading frame of NUP214 was not matched with RAC1; however, high expression of the RAC1 protein was detected by Western blotting. This study identifies the variant complex fusion genesNUP214-RAC1 and RAC1- COL12A1 in a case of AML.

Identification and Validation of Evolutionarily Conserved Unusually Short Pre-mRNA Introns in the Human Genome.

According to the length distribution of human introns, there is a large population of short introns with a threshold of 65 nucleotides (nt) and a peak at 85 nt. Using human genome and transcriptome databases, we investigated the introns shorter than 66 nt, termed ultra-short introns, the identities of which are scarcely known. Here, we provide for the first time a list of bona fide human ultra-short introns, which have never been characterized elsewhere. By conducting BLAST searches of the databases, we screened 22 introns (37-65 nt) with conserved lengths and sequences among closely related species. We then provide experimental and bioinformatic evidence for the splicing of 15 introns, of which 12 introns were remarkably G-rich and 9 introns contained completely inefficient splice sites and/or branch sites. These unorthodox characteristics of ultra-short introns suggest that there are unknown splicing mechanisms that differ from the well-established mechanism.

Identification of cis- and trans-acting factors involved in the localization of MALAT-1 noncoding RNA to nuclear speckles.

MALAT-1 noncoding RNA is localized to nuclear speckles despite its mRNA-like characteristics. Here, we report the identification of several key factors that promote the localization of MALAT-1 to nuclear speckles and also provide evidence that MALAT-1 is involved in the regulation of gene expression. Heterokaryon assays revealed that MALAT-1 does not shuttle between the nucleus and cytoplasm. RNAi-mediated repression of the nuclear speckle proteins, RNPS1, SRm160, or IBP160, which are well-known mRNA processing factors, resulted in the diffusion of MALAT-1 to the nucleoplasm. We demonstrated that MALAT-1 contains two distinct elements directing transcripts to nuclear speckles, which were also capable of binding to RNPS1 in vitro. Depletion of MALAT-1 represses the expression of several genes. Taken together, our results suggest that RNPS1, SRm160, and IBP160 contribute to the localization of MALAT-1 to nuclear speckles, where MALAT-1 could be involved in regulating gene expression.

Re-splicing of mature mRNA in cancer cells promotes activation of distant weak alternative splice sites.

Transcripts of the human tumor susceptibility gene 101 (TSG101) are aberrantly spliced in many cancers. A major aberrant splicing event on the TSG101 pre-mRNA involves joining of distant alternative 5' and 3' splice sites within exon 2 and exon 9, respectively, resulting in the extensive elimination of the mRNA. The estimated strengths of the alternative splice sites are much lower than those of authentic splice sites. We observed that the equivalent aberrant mRNA could be generated from an intron-less TSG101 gene expressed ectopically in breast cancer cells. Remarkably, we identified a pathway-specific endogenous lariat RNA consisting solely of exonic sequences, predicted to be generated by a re-splicing between exon 2 and exon 9 on the spliced mRNA. Our results provide evidence for a two-step splicing pathway in which the initial constitutive splicing removes all 14 authentic splice sites, thereby bringing the weak alternative splice sites into close proximity. We also demonstrate that aberrant multiple-exon skipping of the fragile histidine triad (FHIT) pre-mRNA in cancer cells occurs via re-splicing of spliced FHIT mRNA. The re-splicing of mature mRNA can potentially generate mutation-independent diversity in cancer transcriptomes. Conversely, a mechanism may exist in normal cells to prevent potentially deleterious mRNA re-splicing events.

Structural differences between the closely related RNA helicases, UAP56 and URH49, fashion distinct functional apo-complexes.

mRNA export is an essential pathway for the regulation of gene expression. In humans, closely related RNA helicases, UAP56 and URH49, shape selective mRNA export pathways through the formation of distinct complexes, known as apo-TREX and apo-AREX complexes, and their subsequent remodeling into similar ATP-bound complexes. Therefore, defining the unidentified components of the apo-AREX complex and elucidating the molecular mechanisms underlying the formation of distinct apo-complexes is key to understanding their functional divergence. In this study, we identify additional apo-AREX components physically and functionally associated with URH49. Furthermore, by comparing the structures of UAP56 and URH49 and performing an integrated analysis of their chimeric mutants, we exhibit unique structural features that would contribute to the formation of their respective complexes. This study provides insights into the specific structural and functional diversification of these two helicases that diverged from the common ancestral gene Sub2.

SAP30BP interacts with RBM17/SPF45 to promote splicing in a subset of human short introns.

Human pre-mRNA splicing requires the removal of introns with highly variable lengths, from tens to over a million nucleotides. Therefore, mechanisms of intron recognition and splicing are likely not universal. Recently, we reported that splicing in a subset of human short introns with truncated polypyrimidine tracts depends on RBM17 (SPF45), instead of the canonical splicing factor U2 auxiliary factor (U2AF) heterodimer. Here, we demonstrate that SAP30BP, a factor previously implicated in transcriptional control, is an essential splicing cofactor for RBM17. In vitro binding and nuclear magnetic resonance analyses demonstrate that a U2AF-homology motif (UHM) in RBM17 binds directly to a newly identified UHM-ligand motif in SAP30BP. We show that this RBM17-SAP30BP interaction is required to specifically recruit RBM17 to phosphorylated SF3B1 (SF3b155), a U2 small nuclear ribonucleoprotein (U2 snRNP) component in active spliceosomes. We propose a mechanism for splicing in a subset of short introns, in which SAP30BP guides RBM17 in the assembly of active spliceosomes.

RBM17 Expression Is Associated With the Efficacy of ICI Monotherapy in NSCLC With Low PD-L1 Expression.

BACKGROUND/AIM: Immune checkpoint inhibitors (ICIs) are currently a standard treatment tool for non-small cell lung cancer (NSCLC). RNA-binding motif protein 17 (RBM17), a splicing factor, is frequently over-expressed in NSCLC, but little is known about the role of RBM17 in the efficacy of ICIs for NSCLC. Thus, we investigated the correlation between RBM17 expression and ICI efficacy in NSCLC. PATIENTS AND METHODS: Biopsy or surgical specimens were collected from patients with advanced or recurrent NSCLC who received ICI monotherapy or chemo-immunotherapy in a first-line setting. RBM17 expression was examined using immunohistochemistry. The correlation between the efficacy of ICI monotherapy or chemo-immunotherapy and RBM17 expression was evaluated. RESULTS: Among the 218 cases, 115 (52.8%) cases were positive for RBM17 expression. RBM17 expression was not associated with the objective response rate (ORR) or progression-free survival (PFS) in either of the ICI monotherapy or chemo-immunotherapy groups. However, among those with a low PD-L1 expression level (PD-L1 <50%; n=86), RBM17 expression was significantly associated with a better ORR (p=0.045) and a better PFS (p<0.001) in the ICI monotherapy group, and was significantly associated with a poor ORR in the chemo-immunotherapy group (p=0.041). CONCLUSION: RBM17 might be a useful predictive marker for a higher efficacy of ICI monotherapy in NSCLC patients with a low PD-L1 expression level.