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.

Common Host Factors Involved in the Function of the Foot-and-mouth Disease Virus and Classical Swine Fever Virus Internal Ribosomal Entry Sites

Foot-and-mouth disease virus (FMDV) and classical swine fever virus (CSFV) both possess positive strand RNA genomes and an internal ribosomal entry site (IRES) element within their 5¢-untranslated regions. To identify common host factors involved with the activity of these IRESes, we utilized cell lines expressing a bicistronic luciferase reporter plasmid, which contained an FMDV-IRES or CSFV-IRES element between the Renilla and firefly luciferase genes. First, we treated FMDV-IRES cells with French maritime pine extract, Pycnogenol® (PYC), and evaluated its suppressive effect on FMDV-IRES activity, as anti-viral effect of PYC was reported so far. We next performed microarray analysis to identify host factors affected by PYC, and confirmed host-factor-IRES interaction by applying host factor-specific siRNAs. We found that polycystic kidney disease 1-like 3 (PKD1L3) and ubiquitin specific peptidase 31 (USP31) are involved in FMDV-IRES activity. Moreover, silencing of these factors also significantly suppressed CSFV-IRES activity. Accordingly, we suggest that PKD1L3 and USP31 are host factors that are involved in the function of the FMDV and CSFV-IRES elements.

N6-methyladenosine modification of HCV RNA genome regulates cap-independent IRES-mediated translation via YTHDC2 recognition

Hepatitis C virus (HCV) infections are associated with the risk of progression to fibrosis, cirrhosis, and hepatocellular carcinoma. The HCV RNA genome is translated by an internal ribosome entry site (IRES)-dependent mechanism. The structure and function of the HCV IRES have been investigated by both biological and biophysical criteria. Recently, the role of N6-methyladenosine (m6A) in cellular RNA and viral transcripts has been intensely investigated. The HCV RNA genome is m6A-methylated, and this modification regulates the viral life cycle. In this study, we investigated the role of m6A modification of the HCV genome in the IRES-dependent translation function by mutating m6A consensus motifs (DRACH) within the IRES element in stem–loop III and IV regions and studied their effect on translation initiation. There are several DRACH motifs within the IRES element. Of these, the DRACH motif at nucleotide (nt) 329-333, located about 7 nt upstream of initiator AUG (iAUG) codon, regulates IRES-mediated translation initiation. Mutational analysis showed that m6A methylation of the adenosine at nt 331 is essential for the IRES-dependent translation. m6A reader protein YTHDC2, containing the RNA helicase domain, recognizes m6A-methylated adenosine at nt 331 and, in concert with the cellular La antigen, supports HCV IRES-dependent translation. The RNA helicase dead YTHDC2 (E332Q) mutant failed to stimulate HCV translation initiation. This report highlights the functional roles of m6A modification and YTHDC2 in the HCV IRES-dependent translation initiation, thus offering alternative therapeutic avenues to interfere with the infectious process.