Internal Ribosome Entry Site Activity Research Articles

Heterogeneous nuclear ribonucleoprotein A3 binds to the internal ribosomal entry site of enterovirus A71 and affects virus replication in neural cells.

Enterovirus A71 (EV-A71) belongs to the genus Enterovirus of the Picornaviridae family and often causes outbreaks in Asia. EV-A71 infection usually causes hand, foot, and mouth disease and can even affect the central nervous system, causing neurological complications or death. The 5'-untranslated region (5'-UTR) of EV-A71 contains an internal ribosome entry site (IRES) that is responsible for the translation of viral proteins. IRES-transacting factors can interact with the EV-A71 5'-UTR to regulate IRES activity. Heterogeneous nuclear ribonucleoprotein (hnRNP) A3 is a member of the hnRNP A/B protein family of RNA-binding proteins and is involved in RNA transport and modification. We found that hnRNP A3 knockdown promoted the replication of EV-A71 in neural calls. Conversely, increasing the expression of hnRNP A3 within cells inhibits the growth of EV-A71. HnRNP A3 can bind to the EV-A71 5'-UTR, and knockdown of hnRNP A3 enhances the luciferase activity of the EV-A71 5'-UTR IRES. The localization of hnRNP A3 shifts from the nucleus to the cytoplasm of infected cells during viral infection. Additionally, EV-A71 infection can increase the protein expression of hnRNP A3, and the protein level is correlated with efficient viral growth. Based on these findings, we concluded that hnRNP A3 plays a negative regulatory role in EV-A71 replication within neural cells.

Substitutions of stem-loop subdomains in internal ribosome entry site of Senecavirus A: Impacts on rescue of sequence-modifying viruses

Senecavirus A (SVA) has a positive-sense, single-stranded RNA genome. Its 5’ untranslated region harbors an internal ribosome entry site (IRES), comprising ten larger or smaller stem–loop structures (including a pseudoknot) that have been demonstrated to be well conserved. However, it is still unclear whether each stem–loop subdomain, such as a single stem or loop, is also highly conserved. To clarify this issue in the present study, a set of 29 SVA cDNA clones were constructed by site-directed mutagenesis (SDM) on the IRES. The SDM-modified scenarios included (1) stem–formed complementary sequences exchanging with each other, (2) loop transversion, (3) loop transition, and (4) point mutations. All cDNA clones were separately transfected into cells for rescuing viable viruses, whereas only four SVAs of interest could be recovered, and were genetically stable during twenty passages. One progeny grew significantly slower than the other three did. The dual-luciferase reporter assay showed that none of the SDM-modified IRESes significantly inhibited the IRES activity. Our previous study indicated that a single motif from any of the ten stem structures, if completely mutated, would cause the failure of virus recovery. Interestingly, our present study revealed three stem structures, whose individual complementary sequences could exchange with each other to rescue sequence-modifying SVAs. Moreover, one apical loop was demonstrated to have the ability to tolerate its own full-length transition, also having no impact on the recovery of sequence-modifying SVA. The present study suggested that not every stem–loop structure was strictly conserved in its conformation, while the full-length IRES itself was well conserved. This provides a new research direction on interaction between the IRES and many factors.

The Mytilus chilensis Steamer-like Element-1 Retrotransposon Antisense mRNA Harbors an Internal Ribosome Entry Site That Is Modulated by hnRNPK.

LTR-retrotransposons are transposable elements characterized by the presence of long terminal repeats (LTRs) directly flanking an internal coding region. They share genome organization and replication strategies with retroviruses. Steamer-like Element-1 (MchSLE-1) is an LTR-retrotransposon identified in the genome of the Chilean blue mussel Mytilus chilensis. MchSLE-1 is transcribed; however, whether its RNA is also translated and the mechanism underlying such translation remain to be elucidated. Here, we characterize the MchSLE-1 translation mechanism. We found that the MchSLE-1 5' and 3'LTRs command transcription of sense and antisense RNAs, respectively. Using luciferase reporters commanded by the untranslated regions (UTRs) of MchSLE-1, we found that in vitro 5'UTR sense is unable to initiate translation, whereas the antisense 5'UTR initiates translation even when the eIF4E-eIF4G interaction was disrupted, suggesting the presence of an internal ribosomal entry site (IRES). The antisense 5'UTR IRES activity was tested using bicistronic reporters. The antisense 5'UTR has IRES activity only when the mRNA is transcribed in the nucleus, suggesting that nuclear RNA-binding proteins are required to modulate its activity. Indeed, heterogeneous nuclear ribonucleoprotein K (hnRNPK) was identified as an IRES trans-acting factor (ITAF) of the MchSLE-1 IRES. To our knowledge, this is the first report describing an IRES in an antisense mRNA derived from a mussel LTR-retrotransposon.

Heterogeneous nuclear ribonucleoprotein K promotes cap-independent translation initiation of retroviral mRNAs.

Translation initiation of the human immunodeficiency virus-type 1 (HIV-1) genomic mRNA (vRNA) is cap-dependent or mediated by an internal ribosome entry site (IRES). The HIV-1 IRES requires IRES-transacting factors (ITAFs) for function. In this study, we evaluated the role of the heterogeneous nuclear ribonucleoprotein K (hnRNPK) as a potential ITAF for the HIV-1 IRES. In HIV-1-expressing cells, the depletion of hnRNPK reduced HIV-1 vRNA translation. Furthermore, both the depletion and overexpression of hnRNPK modulated HIV-1 IRES activity. Phosphorylations and protein arginine methyltransferase 1 (PRMT1)-induced asymmetrical dimethylation (aDMA) of hnRNPK strongly impacted the protein's ability to promote the activity of the HIV-1 IRES. We also show that hnRNPK acts as an ITAF for the human T cell lymphotropic virus-type 1 (HTLV-1) IRES, present in the 5'UTR of the viral sense mRNA, but not for the IRES present in the antisense spliced transcript encoding the HTLV-1 basic leucine zipper protein (sHBZ). This study provides evidence for a novel role of the host hnRNPK as an ITAF that stimulates IRES-mediated translation initiation for the retroviruses HIV-1 and HTLV-1.

Internal ribosomal entry site-mediated translational activity of nitric oxide synthase 2

ABSTRACT The internal ribosome entry site (IRES) is a unique structure found in the 5’ untranslated region (5'-UTR) of specific messenger RNAs (mRNAs) that allows ribosomes to bind and initiate translation without the need for a cap structure. In this study, we investigated the presence and functional properties of the IRES activity of nitric oxide synthase 2 (NOS2) mRNA, which encodes an enzyme that produces nitric oxide in response to various stimuli such as inflammation. Nitric oxide is a signaling molecule that plays a crucial role in various physiological processes, including immune responses and neuronal signaling. Our results showed the existence of IRES activity in the 5'-UTR of Nos2 mRNA in various cell types. IRES-mediated translation of NOS2 mRNA was higher in neuronal cells and its activity increased in response to lipopolysaccharide (LPS). Despite inhibition of cap-dependent translation, nitrite production was partially maintained. These results demonstrate the presence of IRES activity in the 5'-UTR of NOS2 mRNA and suggest that IRES-mediated translation plays a key role in controlling nitric oxide production in response to LPS, an inflammatory stimulus.

Variable eIF4E-binding sites and their synergistic effect on cap-independent translation in a novel IRES of wheat yellow mosaic virus RNA2 isolates

Some viral proteins are translated cap-independently via the internal ribosome entry site (IRES), which maintains conservative characteristic among different isolates of the same virus species. However, IRES activity showed a 7-fold variance in RNA2 of wheat yellow mosaic virus (WYMV) HC and LYJN isolates in this study. Based on RNA structure probing and mutagenesis assay, the loosened middle stem of H1 and the hepta-nucleotide top loop of H2 in the LYJN isolate synergistically ensured higher IRES activity than that in the HC isolate. In addition, the conserved top loop of H1 ensured basic IRES activity in HC and LYJN isolates. WYMV RNA2 5′-UTR specifically interacted with the wheat eIF4E, accomplished by the top loop of H1 in the HC isolate or the top loop of H1 and H2 in the LYJN isolate. The high IRES activity of the WYMV RNA2 LYJN isolate was regulated by two eIF4E-binding sites, which showed a synergistic effect mediated by the proximity of the H1 and H2 top loops owing to the flexibility of the middle stem in H1. This report presents a novel evolution pattern of IRES, which altered the number of eIF4E-binding sites to regulate IRES activity.

AGEs induce high expression of Dll4 via endoplasmic reticulum stress PERK signaling-mediated internal ribosomal entry site mechanism in macrophages

Background and aimAdvanced glycation end products (AGEs)- exposed macrophages was characterized by Delta-like ligand 4 (Dll4) high expressed and has been shown to participate in diabetes-related atherosclerosis. This study was aimed to investigate the translational regulatory mechanism of Dll4 high expression in macrophages exposed to AGEs. MethodsHuman Dll4 5′ untranslated region (5′UTR) sequence was cloned and inserted into a bicistronic reporter plasmid. Human THP-1 macrophages transfected with the bicistronic reporter plasmids were exposed to AGEs. Dual-luciferase assay was used to detect internal ribosome entry site (IRES) activity contained in Dll4 5′UTR. Small interference RNA transfection was used to knock-down specific gene expression. Localization of protein was analyzed. ResultsAGEs exposure significantly induced IRES activity in Dll4 5′ UTR in human macrophages. Internal potential promoter and ribosome read-through mechanisms were excluded. Inhibition of endoplasmic reticulum stress and specific silencing of protein kinase R-like endoplasmic reticulum kinase (PERK)/eukaryotic initiation factor 2α (eIF2α) signaling pathway activation reduced IRES activity in Dll4 5′ UTR in human macrophages. Dll4 5′ UTR IRES activity was also inhibited by targeted silencing of heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1). Moreover, specific inhibition of PERK/eIF2α signaling pathway led to deactivation of hnRNPA1, resulting to reduction of AGEs- induced Dll4 5’ UTR IRES activity in human macrophages. ConclusionsAGEs induced Dll4 5′ UTR IRES activity in human macrophages which was dependent on endoplasmic reticulum stress PERK/eIF2α signaling pathway. hnRNPA1 acted the role as an ITAF was also indispensable for AGEs-induced Dll4 5′UTR IRES activity in human macrophages.

The intrinsically disordered region of eIF5B stimulates IRES usage and nucleates biological granule formation

Cells activate stress response pathways to survive adverse conditions. Such responses involve the inhibition of global cap-dependent translation. This inhibition is a block that essential transcripts must escape via alternative methods of translation initiation, e.g., an internal ribosome entry site (IRES). IRESs have distinct structures and generally require a limited repertoire of translation factors. Cellular IRESs have been identified in many critical cellular stress response transcripts. We previously identified cellular IRESs in the murine insulin receptor (Insr) and insulin-like growth factor 1 receptor (Igf1r) transcripts and demonstrated their resistance to eukaryotic initiation factor 4F (eIF4F) inhibition. Here, we find that eIF5B preferentially promotes Insr, Igf1r, and hepatitis C virus IRES activity through a non-canonical mechanism that requires its highly charged and disordered N terminus. We find that the N-terminal region of eIF5B can drive cytoplasmic granule formation. This eIF5B granule is triggered by cellular stress and is sufficient to specifically promote IRES activity.

RB1 5́UTR contains an IRES related to cell cycle control and cancer progression

Retinoblastoma gene1 (RB1) is the first tumor suppressor gene that stands as the guardian of the gate of the G1 period and plays a central role in proliferation and differentiation. However, no reports focused on the possible internal ribosome entry site (IRES) function of the RB1 gene flanking sequence. In this study, we constructed a bicistronic reporter with the RB1 5′untranslated region (5́UTR) inserted between two reporter coding regions. We found RB1 5′UTR harbors an IRES and has higher activity in cancer cell lines than normal cells. Besides, RB1 IRES acquired the highest activity in the G0/G1 phase of the cell cycle, and the RB1 5′UTR mutation collected from retinoblastoma decreased IRES activity compared with RB1 5′UTR wild-type. These data indicated that RB1 IRES is a mechanism of stress regulation and is related to cell cycle control and cancer progression.

Translation of paired box 6 (PAX6) mRNA is IRES-mediated and inhibited by cymarin in breast cancer cells.

Paired box 6 (PAX6) is a member of the PAX family and plays an essential role in cancer cell cycle progression, colony formation, proliferation and invasion. Its expression is upregulated in many cancers including breast cancer, but the process of PAX6 mRNA translation has rarely been studied. We found that PAX6 translation level increased in MCF-7 breast cancer cells treated with the chemotherapeutic drug adriamycin (ADM), which might be attributable to internal ribosome entry site (IRES)-mediated translation. By modifying a bicistronic luciferase plasmid that is widely used to examine IRES activity, we found that the 469-base 5'-UTR of PAX6 mRNA contains an IRES element and that core IRES activity is located between nucleotides 159 and 333. Moreover, PAX6 IRES activity was induced during ADM treatment, which may be the main reason for the elevated level of PAX6 protein. We also found that cymarin, a cardiac glycoside, acts as an inhibitor of PAX6 protein expression by impairing its IRES-mediated translation. Furthermore, MCF-7 cell proliferation was suppressed during treatment with cymarin. These results provide novel insights into the translation mechanism of PAX6 in breast cancer cells and suggest that cymarin is a promising candidate for the treatment of breast cancer via targeting the expression of PAX6.

Enhanced bypass of PD-L1 translation reduces the therapeutic response to mTOR kinase inhibitors

Increased PD-L1 expression in cancer cells is known to enhance immunosuppression, but the mechanism underlying PD-L1 upregulation is incompletely characterized. We show that PD-L1 expression is upregulated through internal ribosomal entry site (IRES)-mediated translation upon mTORC1 inhibition. We identify an IRES element in the PD-L1 5'-UTR that permits cap-independent translation and promotes continuous production of PD-L1 protein despite effective inhibition of mTORC1. eIF4A is found to be a key PD-L1 IRES-binding protein that enhances PD-L1 IRES activity and protein production in tumor cells treated with mTOR kinase inhibitors (mTORkis). Notably, treatment with mTORkis invivo elevates PD-L1 levels and reduces the number of tumor-infiltrating lymphocytes in immunogenic tumors, but anti-PD-L1 immunotherapy restores antitumor immunity and enhances the therapeutic efficacy of mTORkis. These findings report a molecular mechanism for regulating PD-L1 expression through bypassing mTORC1-mediated cap-dependent translation and provide a rationale for targeting PD-L1 immune checkpoint to improve mTOR-targeted therapy.

M6A-modification of cyclin D1 and c-myc IRESs in glioblastoma controls ITAF activity and resistance to mTOR inhibition

A major mechanism conferring resistance to mTOR inhibitors is activation of a salvage pathway stimulating internal ribosome entry site (IRES)-mediated mRNA translation, driving the synthesis of proteins promoting resistance of glioblastoma (GBM). Previously, we found this pathway is stimulated by the requisite IRES-trans-acting factor (ITAF) hnRNP A1, which itself is subject to phosphorylation and methylation events regulating cyclin D1 and c-myc IRES activity. Here we describe the requirement for m6A-modification of IRES RNAs for efficient translation and resistance to mTOR inhibition. DRACH-motifs within these IRES RNAs upon m6A modification resulted in enhanced IRES activity via increased hnRNP A1-binding following mTOR inhibitor exposure. Inhibitor exposure stimulated the expression of m6A-methylosome components resulting in increased activity in GBM. Silencing of METTL3-14 complexes reduced IRES activity upon inhibitor exposure and sensitized resistant GBM lines. YTHDF3 associates with m6A-modified cyclin D1 or c-myc IRESs, regulating IRES activity, and mTOR inhibitor sensitivity in vitro and in xenograft experiments. YTHDF3 interacted directly with hnRNP A1 and together stimulated hnRNP A1-dependent nucleic acid strand annealing activity. These data demonstrate that m6A-methylation of IRES RNAs regulate GBM responses to this class of inhibitors.

Advances and Breakthroughs in IRES-Directed Translation and Replication of Picornaviruses.

Viruses lack the properties to replicate independently due to the limited resources encoded in their genome; therefore, they hijack the host cell machinery to replicate and survive. Picornaviruses get the prerequisite for effective protein synthesis through specific sequences known as internal ribosome entry sites (IRESs). In the past 2 decades, significant progress has been made in identifying different types of IRESs in picornaviruses. This review will discuss the past and current findings related to the five different types of IRESs and various internal ribosome entry site trans-acting factors (ITAFs) that either promote or suppress picornavirus translation and replication. Some IRESs are inefficient and thus require ITAFs. To achieve their full efficiency, they recruit various ITAFs, which enable them to translate more effectively and efficiently, except type IV IRES, which does not require any ITAFs. Although there are two kinds of ITAFs, one promotes viral IRES-dependent translation, and the second type restricts. Picornaviruses IRESs are classified into five types based on their use of sequence, ITAFs, and initiation factors. Some ITAFs regulate IRES activity by localizing to the viral replication factories in the cytoplasm. Also, some drugs, chemicals, and herbal extracts also regulate viral IRES-dependent translation and replication. Altogether, this review will elaborate on our understanding of the past and recent advancements in the IRES-dependent translation and replication of picornaviruses. IMPORTANCE The family Picornaviridae is divided into 68 genera and 158 species. The viruses belonging to this family range from public health importance, such as poliovirus, enterovirus A71, and hepatitis A virus, to animal viruses of great economic importance, such as foot-and-mouth disease virus. The genomes of picornaviruses contain 5' untranslated regions (5' UTRs), which possess crucial and highly structured stem-loops known as IRESs. IRES assemble the ribosomes and facilitate the cap-independent translation. Virus-host interaction is a hot spot for researchers, which warrants deep insight into understanding viral pathogenesis better and discovering new tools and ways for viral restriction to improve human and animal health. The cap-independent translation in the majority of picornaviruses is modulated by ITAFs, which bind to various IRES regions to initiate the translation. The discoveries of ITAFs substantially contributed to understanding viral replication behavior and enhanced our knowledge about virus-host interaction more effectively than ever before. This review discussed the various types of IRESs found in Picornaviridae, past and present discoveries regarding ITAFs, and their mechanism of action. The herbal extracts, drugs, and chemicals, which indicated their importance in controlling viruses, were also summarized. In addition, we discussed the movement of ITAFs from the nucleus to viral replication factories. We believe this review will stimulate researchers to search for more novel ITAFs, drugs, herbal extracts, and chemicals, enhancing the understanding of virus-host interaction.

Polypyrimidine-Tract-Binding Protein Isoforms Differentially Regulate the Hepatitis C Virus Internal Ribosome Entry Site.

Translation initiation of the hepatitis C virus (HCV) mRNA depends on an internal ribosome entry site (IRES) that encompasses most of the 5'UTR and includes nucleotides of the core coding region. This study shows that the polypyrimidine-tract-binding protein (PTB), an RNA-binding protein with four RNA recognition motifs (RRMs), binds to the HCV 5'UTR, stimulating its IRES activity. There are three isoforms of PTB: PTB1, PTB2, and PTB4. Our results show that PTB1 and PTB4, but not PTB2, stimulate HCV IRES activity in HuH-7 and HEK293T cells. In HuH-7 cells, PTB1 promotes HCV IRES-mediated initiation more strongly than PTB4. Mutations in PTB1, PTB4, RRM1/RRM2, or RRM3/RRM4, which disrupt the RRM's ability to bind RNA, abrogated the protein's capacity to stimulate HCV IRES activity in HuH-7 cells. In HEK293T cells, PTB1 and PTB4 stimulate HCV IRES activity to similar levels. In HEK293T cells, mutations in RRM1/RRM2 did not impact PTB1's ability to promote HCV IRES activity; and mutations in PTB1 RRM3/RRM4 domains reduced, but did not abolish, the protein's capacity to stimulate HCV IRES activity. In HEK293T cells, mutations in PTB4 RRM1/RRM2 abrogated the protein's ability to promote HCV IRES activity, and mutations in RRM3/RRM4 have no impact on PTB4 ability to enhance HCV IRES activity. Therefore, PTB1 and PTB4 differentially stimulate the IRES activity in a cell type-specific manner. We conclude that PTB1 and PTB4, but not PTB2, act as IRES transacting factors of the HCV IRES.

Redefining GBA gene structure unveils the ability of Cap-independent, IRES-dependent gene regulation

Glucosylceramide is the primary molecule of glycosphingolipids, and its metabolic regulation is crucial for life. Defects in the catabolizing enzyme, glucocerebrosidase (GCase), cause a lysosomal storage disorder known as Gaucher disease. However, the genetic regulation of GCase has not been fully understood. Here we show the redefined structure of the GCase coding gene (GBA), and clarify the regulatory mechanisms of its transcription and translation. First, alternative uses of the two GBA gene promoters were identified in fibroblasts and HL60-derived macrophages. Intriguingly, both GBA transcripts and GCase activities were induced in macrophages but not in neutrophils. Second, we observed cap-independent translation occurs via unique internal ribosome entry site activities in first promoter-driven GBA transcripts. Third, the reciprocal expression was observed in GBA and miR22-3p versus GBAP1 transcripts before and after HL60-induced macrophage differentiation. Nevertheless, these findings clearly demonstrate novel cell-type-specific GBA gene expression regulatory mechanisms, providing new insights into GCase biology.

Experimental evidence for occurrence of putative copy-choice recombination between two Senecavirus A genomes

Senecavirus A (SVA), formerly known as Seneca Valley virus, belongs to the genus Senecavirus in the family Picornaviridae. SVA has a single-stranded, positive-sense RNA genome, which is actually an mRNA that initiates translation via its own internal ribosome entry site (IRES). The SVA IRES has been demonstrated to be the hepatitis C virus (HCV)-like IRES, containing eight stem-loop domains: domain (D)II, DIIIa, DIIIb, DIIIc, DIIId1, DIIId2, DIIIe and DIIIf. In this study, stem-forming motifs (SFMs) in the eight domains were independently subjected to site-directed mutagenesis (SDM) to construct eight SVA minigenomes for dual-luciferase reporter assay. The result suggested that except the DII, the other seven domains were closely evolved in the IRES activity. Subsequently, a full-length SVA cDNA clone tagged with a reporter gene was genetically modified to construct eight SFM-mutated ones, separately transfected into BSR-T7/5 cells in an attempt to rescue replication-competent SVAs. Nevertheless, no virus was successfully rescued from its own cDNA clone, implying each of the putative domains necessary in SVA IRES for viral replication. Further, we attempted to rescue replication-competent SVA via pairwise transfection of cDNA clones. Out of 28 combinations of co-transfection, four were demonstrated to be able to rescue replication-competent SVAs. Sanger sequencing showed that all four viruses had the wild-type IRES genotype, suggesting the occurrence of putative copy-choice recombination between two IRES-modifying genomes.

Multiple Cis-acting Polypyrimidine Tract Elements Regulate a Cooperative Mechanism for Triticum Mosaic Virus Internal Ribosomal Entry Site Activity.

Diverse elements within the 5′ untranslated region of an mRNA can influence the translation efficiency at the main AUG codon. We previously identified a core picornaviral like Y16X11-AUG motif with 16-nt polypyrimidine CU tract separated by an 11-nt spacer sequence from the 13th AUG codon, which is recognized as the preferred initiation site within the Triticum mosaic virus (TriMV) internal ribosome entry site (IRES) element. The motif is proposed to function as an internal ribosomal landing site at the designated start codon. Here, we exposed the cooperative role of multiple CU-rich segments flanking the TriMV YX-AUG motif to reach and drive internal initiation of translation at the preferred start site. We propose that these auxiliary domains may enhance the ribosome capacity and their delivery at proximity of the correct initiation site. These polypyrimidine tracts can be modulated with a cryptic AUG in a position-dependent manner to replace the native YX-AUG motif, and thus uncovering a new layer of control of start codon selection. In line with these observations, mass spectrometry analysis of proteins directly interacting with translationally impaired TriMV IRES mutants that bear these motifs indicated an enrichment in 40S and 60S ribosomal related proteins, revealing a new function of polypyrimidine tracts to regulate IRES-driven translation. Accessibility of these RNA regions for in trans interaction was validated by SHAPE analysis of the entire TriMV leader sequence and supported by the ability of anti-sense oligonucleotides designed to block the CU tracts accessibility to impair IRES activity. This is the first evidence that defines the core modular domains required for ribosomal recruitment and start codon selection in a complex, multi-AUG viral 5′ UTR for translation in plants.

Critical Role for Cap-Independent c-MYC Translation in Progression of Multiple Myeloma.

Dysregulated c-myc is a determinant of multiple myeloma progression. Translation of c-myc can be achieved by an mTOR-mediated, cap-dependent mechanism or a cap-independent mechanism where a sequence in the 5'UTR of mRNA, termed the internal ribosome entry site (IRES), recruits the 40S ribosomal subunit. This mechanism requires the RNA-binding factor hnRNP A1 (A1) and becomes critical when cap-dependent translation is inhibited during endoplasmic reticulum (ER) stress. Thus, we studied the role of A1 and the myc IRES in myeloma biology. A1 expression correlated with enhanced c-myc expression in patient samples. Expression of A1 in multiple myeloma lines was mediated by c-myc itself, suggesting a positive feedback circuit where myc induces A1 and A1 enhances myc translation. We then deleted the A1 gene in a myc-driven murine myeloma model. A1-deleted multiple myeloma cells demonstrated downregulated myc expression and were inhibited in their growth in vivo. Decreased myc expression was due to reduced translational efficiency and depressed IRES activity. We also studied the J007 inhibitor, which prevents A1's interaction with the myc IRES. J007 inhibited myc translation and IRES activity and diminished myc expression in murine and human multiple myeloma lines as well as primary samples. J007 also inhibited tumor outgrowth in mice after subcutaneous or intravenous challenge and prevented osteolytic bone disease. When c-myc was ectopically reexpressed in A1-deleted multiple myeloma cells, tumor growth was reestablished. These results support the critical role of A1-dependent myc IRES translation in myeloma.

G-Quadruplex Regulation of VEGFA mRNA Translation by RBM4.

In eukaryotes, mRNAs translation is mainly mediated in a cap-dependent or cap-independent manner. The latter is primarily initiated at the internal ribosome entry site (IRES) in the 5′-UTR of mRNAs. It has been reported that the G-quadruplex structure (G4) in the IRES elements could regulate the IRES activity. We previously confirmed RBM4 (also known as LARK) as a G4-binding protein in human. In this study, to investigate whether RBM4 is involved in the regulation of the IRES activity by binding with the G4 structure within the IRES element, the IRES-A element in the 5′-UTR of vascular endothelial growth factor A (VEGFA) was constructed into a dicistronic reporter vector, psiCHECK2, and the effect of RBM4 on the IRES activity was tested in 293T cells. The results showed that the IRES insertion significantly increased the FLuc expression activity, indicating that this G4-containing IRES was active in 293T cells. When the G4 structure in the IRES was disrupted by base mutation, the IRES activity was significantly decreased. The IRES activity was notably increased when the cells were treated with G4 stabilizer PDS. EMSA results showed that RBM4 specifically bound the G4 structure in the IRES element. The knockdown of RBM4 substantially reduced the IRES activity, whereas over-expressing RBM4 increased the IRES activity. Taking all results together, we demonstrated that RBM4 promoted the mRNA translation of VEGFA gene by binding to the G4 structure in the IRES.

Phenotypic Spectrum of Dystrophinopathy Due to Duchenne Muscular Dystrophy Exon 2 Duplications.

To describe the phenotypic spectrum of dystrophinopathy in a large cohort of individuals with DMD exon 2 duplications (Dup2), who may be particularly amenable to therapies directed at restoring expression of either full-length dystrophin or nearly full-length dystrophin through utilization of the DMD exon 5 internal ribosome entry site (IRES). In this retrospective observational study, we analyzed data from large genotype-phenotype databases (the United Dystrophinopathy Project [UDP] and the Italian DMD network) and classified participants into Duchenne muscular dystrophy (DMD), intermediate muscular dystrophy (IMD), or Becker muscular dystrophy (BMD) phenotypes. Log-rank tests for time-to-event variables were used to compare age at loss of ambulation (LOA) in participants with Dup2 vs controls without Dup2 in the UDP database and for comparisons between steroid-treated vs steroid-naive participants with Dup2. Among 66 participants with Dup2 (UDP = 40, Italy = 26), 61% were classified as DMD, 9% as IMD, and 30% as BMD. Median age at last observation was 15.4 years (interquartile range 8.79-26.0) and 75% had been on corticosteroids for at least 6 months. Age at LOA differed significantly between participants with Dup2 DMD and historical controls without Dup2 DMD (p < 0.001). Valid spirometry was limited but suggested a delay in the typical age-related decline in forced vital capacity and 24 of 55 participants with adequate cardiac data had cardiomyopathy. Some patients with Dup2 display a milder disease course than controls without Dup2 DMD, and prolonged ambulation with corticosteroids suggests the potential of IRES activation as a molecular mechanism. As Dup2-targeted therapies reach clinical applications, this information is critical to aid in the interpretation of the efficacy of new treatments.