Cytoplasmic relocalization of heterogeneous nuclear ribonucleoprotein A1 controls translation initiation of specific mRNAs.

Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is a nucleocytoplasmic shuttling protein that regulates gene expression through its action on mRNA metabolism and translation. The cytoplasmic redistribution of hnRNP A1 is a regulated process during viral infection and cellular stress. Here, we show that hnRNP A1 is an internal ribosome entry site (IRES) trans-acting factor that binds specifically to the 5' untranslated region of both the human rhinovirus-2 and the human apoptotic peptidase activating factor 1 (apaf-1) mRNAs, thereby regulating their translation. Furthermore, the cytoplasmic redistribution of hnRNP A1 after rhinovirus infection leads to enhanced rhinovirus IRES-mediated translation, whereas the cytoplasmic relocalization of hnRNP A1 after UVC irradiation limits the UVC-triggered translational activation of the apaf-1 IRES. Therefore, this study provides a direct demonstration that IRESs behave as translational enhancer elements regulated by specific trans-acting mRNA binding proteins in given physiological conditions. Our data highlight a new way to regulate protein synthesis in eukaryotes through the subcellular relocalization of a nuclear mRNA-binding protein.

Testosterone regulates FGF-2 expression during testis maturation by an IRES-dependent translational mechanism.

Spermatogenesis is a complex process involving cell proliferation, differentiation, and apoptosis. Fibroblast growth factor 2 (FGF-2) is involved in testicular function, but its role in spermatogenesis has not been fully documented. The control of FGF-2 expression particularly occurs at the translational level, by an internal ribosome entry site (IRES)-dependent mechanism driving the use of alternative initiation codons. To study IRES activity regulation in vivo, we have developed transgenic mice expressing a bicistronic construct coding for two luciferase genes. Here, we show that the FGF-2 IRES is age-dependently activated in mouse testis, whereas EMCV and c-myc IRESs are not. Real-time PCR confirms that this regulation is translational. By using immunohistological techniques, we demonstrate that FGF-2 IRES stimulation occurs in adult, but not in immature, type-A spermatogonias. This is correlated with activation of endogenous FGF-2 expression in spermatogonia; whereas FGF-2 mRNA transcription is known to decrease in adult testis. Interestingly, the FGF-2 IRES activation is triggered by testosterone and is partially inhibited by siRNA directed against the androgen receptor. Two-dimensional analysis of proteins bound to the FGF-2 mRNA 5'UTR after UV cross-linking reveals that testosterone treatment correlates with the binding of several proteins. These data suggest a paracrine loop where IRES-dependent FGF-2 expression, stimulated by Sertoli cells in response to testosterone produced by Leydig cells, would in turn activate Leydig function and testosterone production. In addition, nuclear FGF-2 isoforms could be involved in an intracrine function of FGF-2 in the start of spermatogenesis, mitosis, or meiosis initiation. This report demonstrates that mRNA translation regulation by an IRES-dependent mechanism participates in a physiological process.

Heterogeneous nuclear ribonucleoprotein A1 is a novel internal ribosome entry site trans-acting factor that modulates alternative initiation of translation of the fibroblast growth factor 2 mRNA.

Alternative initiation of translation of the human fibroblast growth factor 2 (FGF-2) mRNA at five in-frame CUG or AUG translation initiation codons requires various RNA cis-acting elements, including an internal ribosome entry site (IRES). Here we describe the purification of a trans-acting factor controlling FGF-2 mRNA translation achieved by several biochemical purification approaches. We have identified the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) as a factor that binds to the FGF-2 5'-leader RNA and that also complements defective FGF-2 translation in vitro in rabbit reticulocyte lysate. Recombinant hnRNP A1 stimulates in vitro translation at the four IRES-dependent initiation codons but has no effect on the cap-dependent initiation codon. Consistent with a role of hnRNP A1 in the control of alternative initiation of translation, short interfering RNA-mediated knock down of hnRNP A1 specifically inhibits translation at the four IRES-dependent initiation codons. Furthermore, hnRNP A1 binds to the FGF-2 IRES, implicating this interaction in the control of alternative initiation of translation.

Selective inhibitory DNA aptamers of the human RNase H1.

Human RNase H1 binds double-stranded RNA via its N-terminal domain and RNA-DNA hybrid via its C-terminal RNase H domain, the latter being closely related to Escherichia coli RNase HI. Using SELEX, we have generated a set of DNA sequences that can bind efficiently (K(d) values ranging from 10 to 80 nM) to the human RNase H1. None of them could fold into a simple perfect double-stranded DNA hairpin confirming that double-stranded DNA does not constitute a trivial ligand for the enzyme. Only two of the 37 DNA aptamers selected were inhibitors of human RNase H1 activity. The two inhibitory oligomers, V-2 and VI-2, were quite different in structure with V-2 folding into a large, imperfect but stable hairpin loop. The VI-2 structure consists of a central region unimolecular quadruplex formed by stacking of two guanine quartets flanked by the 5' and 3' tails that form a stem of six base pairs. Base pairing between the 5' and 3' tails appears crucial for conferring the inhibitory properties to the aptamer. Finally, the inhibitory aptamers were capable of completely abolishing the action of an antisense oligonucleotide in a rabbit reticulocyte lysate supplemented with human RNase H1, with IC50 ranging from 50 to 100 nM.