3'UTR Diversity: Expanding Repertoire of RNA Alterations in Human mRNAs.

Genomic information stored in the DNA is transcribed to the mRNA and translated to proteins. The 3' untranslated regions (3'UTRs) of the mRNA serve pivotal roles in posttranscriptional gene expression, regulating mRNA stability, translation, and localization. Similar to DNA mutations producing aberrant proteins, RNA alterations expand the transcriptome landscape and change the cellular proteome. Recent global analyses reveal that many genes express various forms of altered RNAs, including 3'UTR length variants. Alternative polyadenylation and alternative splicing are involved in diversifying 3'UTRs, which could act as a hidden layer of eukaryotic gene expression control. In this review, we summarize the functions and regulations of 3'UTRs and elaborate on the generation and functional consequences of 3'UTR diversity. Given that dynamic 3'UTR length control contributes to phenotypic complexity, dysregulated 3'UTR diversity might be relevant to disease development, including cancers. Thus, 3'UTR diversity in cancer could open exciting new research areas and provide avenues for novel cancer theragnostics.

Splicing factor SRSF3 represses translation of p21cip1/waf1 mRNA.

Serine/arginine-rich splicing factor 3 (SRSF3) is an RNA binding protein that most often regulates gene expression at the splicing level. Although the role of SRSF3 in mRNA splicing in the nucleus is well known, its splicing-independent role outside of the nucleus is poorly understood. Here, we found that SRSF3 exerts a translational control of p21 mRNA. Depletion of SRSF3 induces cellular senescence and increases the expression of p21 independent of p53. Consistent with the expression patterns of SRSF3 and p21 mRNA in the TCGA database, SRSF3 knockdown increases the p21 mRNA level and its translation efficiency as well. SRSF3 physically associates with the 3'UTR region of p21 mRNA and the translational initiation factor, eIF4A1. Our study proposes a model in which SRSF3 regulates translation by interacting with eIF4A1 at the 3'UTR region of p21 mRNA. We also found that SRSF3 localizes to the cytoplasmic RNA granule along with eIF4A1, which may assist in translational repression therein. Thus, our results provide a new mode of regulation for p21 expression, a crucial regulator of the cell cycle and senescence, which occurs at the translational level and involves SRSF3.

Nuclear UPF1 Is Associated with Chromatin for Transcription-Coupled RNA Surveillance.

mRNA quality is controlled by multiple RNA surveillance machineries to reduce errors during gene expression processes in eukaryotic cells. Nonsense-mediated mRNA decay (NMD) is a well-characterized mechanism that degrades error-containing transcripts during translation. The ATP-dependent RNA helicase up-frameshift 1 (UPF1) is a key player in NMD that is mostly prevalent in the cytoplasm. However, recent studies on UPF1-RNA interaction suggest more comprehensive roles of UPF1 on diverse forms of target transcripts. Here we used subcellular fractionation and immunofluorescence to understand such complex functions of UPF1. We demonstrated that UPF1 can be localized to the nucleus and predominantly associated with the chromatin. Moreover, we showed that UPF1 associates more strongly with the chromatin when the transcription elongation and translation inhibitors were used. These findings suggest a novel role of UPF1 in transcription elongation-coupled RNA machinery in the chromatin, as well as in translation-coupled NMD in the cytoplasm. Thus, we propose that cytoplasmic UPF1-centric RNA surveillance mechanism could be extended further up to the chromatin-associated UPF1 and cotranscriptional RNA surveillance. Our findings could provide the mechanistic insights on extensive regulatory roles of UPF1 for many cellular RNAs.

Mutation Hotspots in the ß-Catenin Gene: Lessons from the Human Cancer Genome Databases.

Mutations in the ß-catenin gene (CTNNB1) have been implicated in the pathogenesis of some cancers. The recent development of cancer genome databases has facilitated comprehensive and focused analyses on the mutation status of cancer-related genes. We have used these databases to analyze the CTNNB1 mutations assembled from different tumor types. High incidences of CTNNB1 mutations were detected in endometrial, liver, and colorectal cancers. This finding agrees with the oncogenic role of aberrantly activated ß-catenin in epithelial cells. Elevated frequencies of missense mutations were found in the exon 3 of CTNNB1, which is responsible for encoding the regulatory amino acids at the N-terminal region of the protein. In the case of metastatic colorectal cancers, inframe deletions were revealed in the region spanning exon 3. Thus, exon 3 of CTNNB1 can be considered to be a mutation hotspot in these cancers. Since the N-terminal region of the ß-catenin protein forms a flexible structure, many questions arise regarding the structural and functional impacts of hotspot mutations. Clinical identification of hotspot mutations could provide the mechanistic basis for an oncogenic role of mutant ß-catenin proteins in cancer cells. Furthermore, a systematic understanding of tumor-driving hotspot mutations could open new avenues for precision oncology.

WIG1 is crucial for AGO2-mediated ACOT7 mRNA silencing via miRNA-dependent and -independent mechanisms.

RNA-binding proteins (RBPs) are involved in mRNA splicing, maturation, transport, translation, storage and turnover. Here, we identified ACOT7 mRNA as a novel target of human WIG1. ACOT7 mRNA decay was triggered by the microRNA miR-9 in a WIG1-dependent manner via classic recruitment of Argonaute 2 (AGO2). Interestingly, AGO2 was also recruited to ACOT7 mRNA in a WIG1-dependent manner in the absence of miR-9, which indicates an alternative model whereby WIG1 controls AGO2-mediated gene silencing. The WIG1-AGO2 complex attenuated translation initiation via an interaction with translation initiation factor 5B (eIF5B). These results were confirmed using a WIG1 tethering system based on the MS2 bacteriophage coat protein and a reporter construct containing an MS2-binding site, and by immunoprecipitation of WIG1 and detection of WIG1-associated proteins using liquid chromatography-tandem mass spectrometry. We also identified WIG1-binding motifs using photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation analyses. Altogether, our data indicate that WIG1 governs the miRNA-dependent and the miRNA-independent recruitment of AGO2 to lower the stability of and suppress the translation of ACOT7 mRNA.

SR Proteins: Binders, Regulators, and Connectors of RNA.

Serine and arginine-rich (SR) proteins are RNA-binding proteins (RBPs) known as constitutive and alternative splicing regulators. As splicing is linked to transcriptional and post-transcriptional steps, SR proteins are implicated in the regulation of multiple aspects of the gene expression program. Recent global analyses of SR-RNA interaction maps have advanced our understanding of SR-regulated gene expression. Diverse SR proteins play partially overlapping but distinct roles in transcription-coupled splicing and mRNA processing in the nucleus. In addition, shuttling SR proteins act as adaptors for mRNA export and as regulators for translation in the cytoplasm. This mini-review will summarize the roles of SR proteins as RNA binders, regulators, and connectors from transcription in the nucleus to translation in the cytoplasm.

SRSF3 represses the expression of PDCD4 protein by coordinated regulation of alternative splicing, export and translation.

Gene expression is regulated at multiple steps, such as transcription, splicing, export, degradation and translation. Considering diverse roles of SR proteins, we determined whether the tumor-related splicing factor SRSF3 regulates the expression of the tumor-suppressor protein, PDCD4, at multiple steps. As we have reported previously, knockdown of SRSF3 increased the PDCD4 protein level in SW480 colon cancer cells. More interestingly, here we showed that the alternative splicing and the nuclear export of minor isoforms of pdcd4 mRNA were repressed by SRSF3, but the translation step was unaffected. In contrast, only the translation step of the major isoform of pdcd4 mRNA was repressed by SRSF3. Therefore, overexpression of SRSF3 might be relevant to the repression of all isoforms of PDCD4 protein levels in most types of cancer cell. We propose that SRSF3 could act as a coordinator of the expression of PDCD4 protein via two mechanisms on two alternatively spliced mRNA isoforms.

Wnt activated ß-catenin and YAP proteins enhance the expression of non-coding RNA component of RNase MRP in colon cancer cells.

RMRP, the RNA component of mitochondrial RNA processing endoribonuclease, is a non-coding RNA (ncRNA) part of the RNase MRP complex functioning in mitochondrial and ribosomal RNA processing. Even though various mutations in the RMRP gene are linked to developmental defects and pathogenesis, its relevance to cancer etiology has not been well established. Here we examined the expression of RMRP and found a significant increase in colorectal and breast cancer patient tissues. So we tested whether the oncogenic signaling pathways, Wnt/ß-catenin and Hippo/YAP pathways, are relevant to the enhanced expression of RMRP in cancer cells because of the predicted ß-catenin/TCF and YAP/TBX5 elements in the upstream regions of the RMRP gene. As expected, Wnt signal activation significantly induced the RMRP transcription thru ß-catenin and YAP transcription factors. More importantly, YAP protein was critical for RMRP transcription by association to the proximal site near the transcription start site of the RMRP gene, a Pol III promoter, along with ß-catenin and TBX5 proteins. We propose that the interplay of Wnt and Hippo signaling pathways could regulate target genes, coding or non-coding, by the ß-catenin/YAP/TBX5 transcription complex in cancer cells.

SERBP1 affects homologous recombination-mediated DNA repair by regulation of CtIP translation during S phase.

DNA double-strand breaks (DSBs) are the most severe type of DNA damage and are primarily repaired by non-homologous end joining (NHEJ) and homologous recombination (HR) in the G1 and S/G2 phase, respectively. Although CtBP-interacting protein (CtIP) is crucial in DNA end resection during HR following DSBs, little is known about how CtIP levels increase in an S phase-specific manner. Here, we show that Serpine mRNA binding protein 1 (SERBP1) regulates CtIP expression at the translational level in S phase. In response to camptothecin-mediated DNA DSBs, CHK1 and RPA2 phosphorylation, which are hallmarks of HR activation, was abrogated in SERBP1-depleted cells. We identified CtIP mRNA as a binding target of SERBP1 using RNA immunoprecipitation-coupled RNA sequencing, and confirmed SERBP1 binding to CtIP mRNA in S phase. SERBP1 depletion resulted in reduction of polysome-associated CtIP mRNA and concomitant loss of CtIP expression in S phase. These effects were reversed by reconstituting cells with wild-type SERBP1, but not by SERBP1 ΔRGG, an RNA binding defective mutant, suggesting regulation of CtIP translation by SERBP1 association with CtIP mRNA. These results indicate that SERBP1 affects HR-mediated DNA repair in response to DNA DSBs by regulation of CtIP translation in S phase.

HuR represses Wnt/ß-catenin-mediated transcriptional activity by promoting cytoplasmic localization of ß-catenin.

ß-Catenin is the key transcriptional activator of canonical Wnt signaling in the nucleus; thus, nuclear accumulation of ß-catenin is a critical step for expressing target genes. ß-Catenin accumulates in the nucleus of cancer cells where it activates oncogenic target genes. Hu antigen R (HuR) is a RNA binding protein that regulates multiple post-transcriptional processes including RNA stability. Thus, cytoplasmic HuR protein may be involved in tumorigenesis by stabilizing oncogenic transcripts, but the molecular mechanism remains unclear. Here, we observed that Wnt/ß-catenin signaling induced export of the HuR protein, whereas HuR overexpression promoted accumulation of the ß-catenin protein in the cytoplasm. Thus, Wnt/ß-catenin-mediated transcriptional activity in the nucleus was reduced by overexpressing HuR. These results suggest novel and uncharacterized cytoplasmic ß-catenin functions related to HuR-mediated RNA metabolism in cancer cells.

Correlation of breast cancer subtypes, based on estrogen receptor, progesterone receptor, and HER2, with functional imaging parameters from 68Ga-RGD PET/CT and ¹8F-FDG PET/CT.

PURPOSE: Imaging biomarkers from functional imaging modalities were assessed as potential surrogate markers of disease status. Specifically, in this prospective study, we investigated the relationships between functional imaging parameters and histological prognostic factors and breast cancer subtypes. METHODS: In total, 43 patients with large or locally advanced invasive ductal carcinoma (IDC) were analyzed (47.6 ± 7.5 years old). (68)Ga-Labeled arginine-glycine-aspartic acid (RGD) and (18)F-fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) were performed. The maximum and average standardized uptake values (SUVmax and SUVavg) from RGD PET/CT and SUVmax and SUVavg from FDG PET/CT were the imaging parameters used. For histological prognostic factors, estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression was identified using immunohistochemistry (IHC) or fluorescent in situ hybridization (FISH). Four breast cancer subtypes, based on ER/PR and HER2 expression (ER/PR+,Her2-, ER/PR+,Her2+, ER/PR-,Her2+, and ER/PR-,Her2-), were considered. RESULTS: Quantitative FDG PET parameters were significantly higher in the ER-negative group (15.88 ± 8.73 vs 10.48 ± 6.01, p = 0.02 for SUVmax; 9.40 ± 5.19 vs 5.92 ± 4.09, p = 0.02 for SUVavg) and the PR-negative group (8.37 ± 4.94 vs 4.79 ± 3.93, p = 0.03 for SUVavg). Quantitative RGD PET parameters were significantly higher in the HER2-positive group (2.42 ± 0.59 vs 2.90 ± 0.75, p = 0.04 for SUVmax; 1.60 ± 0.38 vs 1.95 ± 0.53, p = 0.04 for SUVavg) and showed a significant positive correlation with the HER2/CEP17 ratio (r = 0.38, p = 0.03 for SUVmax and r = 0.46, p < 0.01 for SUVavg). FDG PET parameters showed significantly higher values in the ER/PR-,Her2- subgroup versus the ER/PR+,Her2- or ER/PR+,Her2+ subgroups, while RGD PET parameters showed significantly lower values in the ER/PR-,Her2- subgroup versus the other subgroups. There was no correlation between FDG and RGD PET parameters in the overall group. Only the ER/PR-,Her2- subgroup showed a significant positive correlation between FDG and RGD PET parameters (r = 0.59, p = 0.03 for SUVmax). CONCLUSION: (68)Ga-RGD and (18)F-FDG PET/CT are promising functional imaging modalities for predicting biomarkers and molecular phenotypes in breast cancer patients.

ß-Catenin and peroxisome proliferator-activated receptor-δ coordinate dynamic chromatin loops for the transcription of vascular endothelial growth factor A gene in colon cancer cells.

Vascular endothelial growth factor A (VEGFA) mRNA is regulated by ß-catenin and peroxisome proliferator activated receptor δ (PPAR-δ) activation in colon cancer cells, but the detailed mechanism remains to be elucidated. As chromatin loops are generally hubs for transcription factors, we tested here whether ß-catenin could modulate chromatin looping near the VEGFA gene and play any important role for PPAR-δ activated VEGFA transcription. First, we identified the far upstream site as an important site for VEGFA transcription by luciferase assay and chromatin immunoprecipitation in colorectal carcinoma HCT116 cells. Chromatin conformation capture analysis also revealed the chromatin loops formed by the ß-catenin bindings on these sites near the VEGFA gene. Dynamic association and dissociation of ß-catenin/TCF-4/PPAR-δ on the far upstream site and ß-catenin/NF-κB p65 on the downstream site were also detected depending on PPAR-δ activation. Interestingly, ß-catenin-mediated chromatin loops were relieved by PPAR-δ activation, suggesting a regulatory role of ß-catenin for VEGFA transcription. Based on these data, we propose a model for PPAR-δ-activated VEGFA transcription that relies on ß-catenin-mediated chromatin looping as a prerequisite for the activation. Our findings could extend to other ß-catenin regulated target genes and could provide a general mechanism and novel paradigm for ß-catenin-mediated oncogenesis.

ß-Catenin recognizes a specific RNA motif in the cyclooxygenase-2 mRNA 3'-UTR and interacts with HuR in colon cancer cells.

RNA-binding proteins regulate multiple steps of RNA metabolism through both dynamic and combined binding. In addition to its crucial roles in cell adhesion and Wnt-activated transcription in cancer cells, ß-catenin regulates RNA alternative splicing and stability possibly by binding to target RNA in cells. An RNA aptamer was selected for specific binding to ß-catenin to address RNA recognition by ß-catenin more specifically. Here, we characterized the structural properties of the RNA aptamer as a model and identified a ß-catenin RNA motif. Similar RNA motif was found in cellular RNA, Cyclooxygenase-2 (COX-2) mRNA 3'-untranslated region (3'-UTR). More significantly, the C-terminal domain of ß-catenin interacted with HuR and the Armadillo repeat domain associated with RNA to form the RNA-ß-catenin-HuR complex in vitro and in cells. Furthermore, the tertiary RNA-protein complex was predominantly found in the cytoplasm of colon cancer cells; thus, it might be related to COX-2 protein level and cancer progression. Taken together, the ß-catenin RNA aptamer was valuable for deducing the cellular RNA aptamer and identifying novel and oncogenic RNA-protein networks in colon cancer cells.

Selection and characterization of tenascin C targeting peptide.

Since tenascin C is a factor expressed highly in the tumor-associated matrix, it would be a desirable first step for targeting the tumor-specific microenvironment. In fact, a high level of tenascin C expression has been reported in most solid tumors, including lung cancer, colon cancer and glioblastoma. Therefore, the targeted binding of tenascin C in tumor stroma would inhibit tumor metastasis by modulating cancer cell growth and migration. We isolated a peptide that bound to tenascin C by phage display peptide library selection, and the selected peptide specifically recognized tenascin C protein in xenograft mouse tissue. We also observed exclusive staining of tenascin C by the selected peptide in tumor patient tissues. Moreover, the peptide reduced tenascin C-induced cell rounding and migration. We propose that the tenascin C targeting peptide may be useful as a specific anti-cancer diagnostic and therapeutic tool for most human solid tumors.

Annexin A2 binds RNA and reduces the frameshifting efficiency of infectious bronchitis virus.

Annexin A2 (ANXA2) is a protein implicated in diverse cellular functions, including exocytosis, DNA synthesis and cell proliferation. It was recently proposed to be involved in RNA metabolism because it was shown to associate with some cellular mRNA. Here, we identified ANXA2 as a RNA binding protein (RBP) that binds IBV (Infectious Bronchitis Virus) pseudoknot RNA. We first confirmed the binding of ANXA2 to IBV pseudoknot RNA by ultraviolet crosslinking and showed its binding to RNA pseudoknot with ANXA2 protein in vitro and in the cells. Since the RNA pseudoknot located in the frameshifting region of IBV was used as bait for cellular RBPs, we tested whether ANXA2 could regulate the frameshfting of IBV pseudoknot RNA by dual luciferase assay. Overexpression of ANXA2 significantly reduced the frameshifting efficiency from IBV pseudoknot RNA and knockdown of the protein strikingly increased the frameshifting efficiency. The results suggest that ANXA2 is a cellular RBP that can modulate the frameshifting efficiency of viral RNA, enabling it to act as an anti-viral cellular protein, and hinting at roles in RNA metabolism for other cellular mRNAs.

In vitro selection of RNA aptamer and specific targeting of ErbB2 in breast cancer cells.

The ErbB family (also referred to as HER/neu or HER) of receptor tyrosine kinases plays major roles in the formation and progression of human tumors. Amplification and/or overexpression of ErbB2 have been reported in numerous cancers, including breast, ovarian, stomach, bladder, salivary, and lung cancers. As ErbB2 has been used as a target for the treatment of advanced cancer, RNA aptamers for the extracellular domain of the ErbB2 were selected from a RNA library consisting of 2'-fluorine-modified RNA transcripts. After 15 cycles of selection, high-affinity RNA aptamer was isolated. Binding patterns of the selected RNA aptamer clones were evaluated to choose RNA aptamers that were specific to the extracellular domain of ErbB2 protein. RNA aptamer 15-8 was the best candidate and its minimal version (mini-aptamer) was chemically synthesized. Surface plasmon resonance measurement showed that the mini-aptamer specifically bound to the ErbB2 protein with high affinity and specificity. To evaluate its potential as an ErbB2-targeting molecule in breast cancer cells, specific recognition of the mini-aptamer was confirmed with various breast cancer cell lines. We propose that the selected RNA aptamer is a potential cancer imaging agent by targeting malignant cells overexpressing the ErbB2 receptor.

Persistent anti-tumor effects via recombinant adeno-associated virus encoding herpes thymidine kinase gene monitored by PET-imaging.

Despite the well-documented advantages of the recombinant adeno-associated virus (rAAV) as a gene delivery vehicle, including its non-pathogenic and long-term therapeutic gene expression, there have been very limited studies on its potential for producing persistent anti-tumor effects, particularly in vivo. To address this issue, we constructed rAAV vectors encoding herpes simplex virus 1-thymidine kinase (HSV-TK) or its mutant form (sc39TK) as therapeutic genes, and GFP as a control gene. Effective rAAV-mediated gene delivery was readily observed in human cancer cells using immunocytochemistry and Western blotting. Cell survival analysis following prodrug ganciclovir treatment implied that both preferential and superior cytotoxicity was achieved by rAAV-sc39TK introduction. Persistent anti-tumor effects in vivo were investigated in Balb/c nude mice bearing human cancer cells treated with either rAAV-sc39TK or -GFP. Severe tumor growth inhibition was clearly observed only in the case of sc39TK with ganciclovir treatment. Non-invasive micro-PET imaging using 18F-FHBG directly correlated with persistent anti-tumor effects by sc39TK. Therefore, the present study provides evidence that rAAV-mediated persistent therapeutic gene expression can occur, resulting in long-term anti-tumor activities and that these events can be readily monitored using micro-PET imaging.

SR proteins induce alternative exon skipping through their activities on the flanking constitutive exons.

SR proteins are well known to promote exon inclusion in regulated splicing through exonic splicing enhancers. SR proteins have also been reported to cause exon skipping, but little is known about the mechanism. We previously characterized SRSF1 (SF2/ASF)-dependent exon skipping of the CaMKIIδ gene during heart remodeling. By using mouse embryo fibroblasts derived from conditional SR protein knockout mice, we now show that SR protein-induced exon skipping depends on their prevalent actions on a flanking constitutive exon and requires collaboration of more than one SR protein. These findings, coupled with other established rules for SR proteins, provide a theoretical framework to understand the complex effect of SR protein-regulated splicing in mammalian cells. We further demonstrate that heart-specific CaMKIIδ splicing can be reconstituted in fibroblasts by downregulating SR proteins and upregulating a RBFOX protein and that SR protein overexpression impairs regulated CaMKIIδ splicing and neuronal differentiation in P19 cells, illustrating that SR protein-dependent exon skipping may constitute a key strategy for synergism with other splicing regulators in establishing tissue-specific alternative splicing critical for cell differentiation programs.

Acquisition of selective antitumoral effects of recombinant adeno-associated virus by genetically inserting tumor-targeting peptides into capsid proteins.

Recombinant adeno-associated virus serotype 5 (rAAV5) is considered to be a promising gene transfer vehicle. However, preferential gene delivery to the tumor remains a requirement for cancer treatment. We generated rAAV5 mutants bearing tumor marker-binding peptides and analyzed their properties as viral vectors, as well as their transduction efficiencies and preferential antitumoral potencies. All of the mutants were successfully produced. Transduction analyses showed that rAAV5 mutants harboring tumor-homing peptides, including RGD and TnC, transduced human cancer cells expressing corresponding receptors on their surfaces. RGDS peptides and TnC antibodies significantly suppressed transduction by rAAV5-RGD and rAAV5-TnC. Cytotoxicity was evident upon transfer of HSV-TK to cells by re-targeted rAAV5. These results provide evidence that rAAV5 vectors, genetically armed with tumor-targeting ligands, preferentially infect human cancer cells harboring the corresponding receptors, thereby inducing antitumoral effects. Further optimization of rAAV5 mutant viruses should thus facilitate practical exploitation of these vectors for gene-based cancer treatment.