Translation regulation

Surfactant protein A (SP-A), a molecule with roles in lung innate immunity and surfactant-related functions, is encoded by two genes in humans: SFTPA1 (SP-A1) and SFTPA2 (SP-A2). The mRNAs from these genes differ in their 5′-untranslated regions (5′-UTR) due to differential splicing. The 5′-UTR variant ACD′ is exclusively found in transcripts of SP-A1, but not in those of SP-A2. Its unique exon C contains two upstream AUG codons (uAUGs) that may affect SP-A1 translation efficiency. The first uAUG (u1) is in frame with the primary start codon (p), but the second one (u2) is not. The purpose of this study was to assess the impact of uAUGs on SP-A1 expression. We employed RT-qPCR to determine the presence of exon C-containing SP-A1 transcripts in human RNA samples. We also used in vitro techniques including mutagenesis, reporter assays, and toeprinting analysis, as well as in silico analyses to determine the role of uAUGs. Exon C-containing mRNA is present in most human lung tissue sam...

A characteristic mechanism of gene expression regulation during seed germination is the selective translation of mRNAs. Previous findings indicate that the two cap-binding complexes eIF4F (with eIF4E and eIF4G subunits) and eIF(iso)4F [with eIF(iso)4E and eIF(iso)4G subunits] are differentially expressed during maize seed germination. In addition, several studies in vitro have suggested that these factors may participate in selective mRNA translation. The translational activities of eIF4E and eIF(iso)4E were tested in vitro using transcripts from two different sets: dry (0 h) and 24-h-imbibed maize embryonic axes. In vitro translation of these mRNA pools in the presence of the recombinant eIF4E or eIF(iso)4E, and the native cap-binding complexes from dry- or 24-h-imbibed axes, produced different profiles of proteins which were visualized by two-dimensional protein gels and autoradiography. The data indicated that eIF(iso)4E was particularly required for translation of the stored mRN...

OLA1, an Obg-family GTPase, has been implicated in eIF2-mediated translational control, but its physiological functions remain obscure. Here, we report that mouse embryos lacking OLA1 have stunted growth, delayed development leading to immature organs—especially lungs—at birth, and frequent perinatal lethality. Proliferation of primary Ola1 -/- mouse embryonic fibroblasts (MEFs) is impaired due to defective cell cycle progression, associated with reduced cyclins D1 and E1, attenuated Rb phosphorylation, and increased p21 Cip1/Waf1 . Accumulation of p21 in Ola1 -/- MEFs is due to enhanced mRNA translation and can be prevented by either reconstitution of OLA1 expression or treatment with an eIF2α-dephosphorylation inhibitor, suggesting that OLA1 regulates p21 through a translational mechanism involving eIF2. With immunohistochemistry, overexpression of p21 protein was detected in Ola1-null embryos with reduced cell proliferation. Moreover, we have generated p21 -/- Ola1 -/- mice and found that knockout of p21 can partially rescue the growth retardation defect of Ola1 -/- embryos but fails to rescue them from developmental delay and the lethality. These data demonstrate, for the first time, that OLA1 is required for normal progression of mammalian development. OLA1 plays an important role in promoting cell proliferation at least in part through suppression of p21 and organogenesis via factors yet to be discovered.

Surfactant protein A (SP-A), a molecule with roles in lung innate immunity and surfactant-related functions, is encoded by two genes in humans: SFTPA1 (SP-A1) and SFTPA2 (SP-A2). The mRNAs from these genes differ in their 5'-untranslated regions (5'-UTR) due to differential splicing. The 5'-UTR variant ACD' is exclusively found in transcripts of SP-A1, but not in those of SP-A2. Its unique exon C contains two upstream AUG codons (uAUGs) that may affect SP-A1 translation efficiency. The first uAUG (u1) is in frame with the primary start codon (p), but the second one (u2) is not. The purpose of this study was to assess the impact of uAUGs on SP-A1 expression. We employed RT-qPCR to determine the presence of exon C-containing SP-A1 transcripts in human RNA samples. We also used in vitro techniques including mutagenesis, reporter assays, and toeprinting analysis, as well as in silico analyses to determine the role of uAUGs. Exon C-containing mRNA is present in most human lung tissue samples and its expression can, under certain conditions, be regulated by factors such as dexamethasone or endotoxin. Mutating uAUGs resulted in increased luciferase activity. The mature protein size was not affected by the uAUGs, as shown by a combination of toeprint and in silico analysis for Kozak sequence, secondary structure, and signal peptide and in vitro translation in the presence of microsomes. In conclusion, alternative splicing may introduce uAUGs in SP-A1 transcripts, which in turn negatively affect SP-A1 translation, possibly affecting SP-A1/SP-A2 ratio, with potential for clinical implication.

The eukaryotic initiation factor 3 (eIF3) plays an important role in translation initiation, acting as a docking site for several eIFs that assemble on the 40S ribosomal subunit. Here, we use mass spectrometry to probe the subunit interactions within the human eIF3 complex. Our results show that the 13-subunit complex can be maintained intact in the gas phase, enabling us