The polyamines, putrescine, spermidine, and spermine, are essential polycations, intimately involved in the regulation of cellular proliferation. Although polyamines exert dynamic effects on the conformation of nucleic acids and macromolecular synthesis in vitro, their specific functions in vivo are poorly understood. We investigated the cellular function of polyamines by overexpression of a key catabolic enzyme, spermidine/spermine N ¹ -acetyltransferase 1 (SAT1) in mammalian cells. Transient cotransfection of HeLa cells with GFP and SAT1 vectors suppressed GFP protein expression without lowering its mRNA level, an indication that the block in GFP expression was not at transcription, but at translation. Fluorescence single-cell imaging also revealed specific inhibition of endogenous protein synthesis in the SAT1 overexpressing cells, without any inhibition of synthesis of DNA or RNA. Overexpression of SAT1 using a SAT1 adenovirus led to rapid depletion of cellular spermidine and spermine, total inhibition of protein synthesis, and growth arrest within 24 h. The SAT1 effect is most likely due to depletion of spermidine and spermine, because stable polyamine analogs that are not substrates for SAT1 restored GFP and endogenous protein synthesis. Loss of polysomes with increased 80S monosomes in the polyamine-depleted cells suggests a direct role for polyamines in translation initiation. Our data provide strong evidence for a primary function of polyamines, spermidine and spermine, in translation in mammalian cells.

Iron‐responsive elements (IREs) are regulatory RNA elements which are characterized by a phylogenetically defined sequence‐structure motif. Their biological function is to provide a specific binding site for the IRE‐binding protein (IRE‐BP). Iron starvation of cells induces high affinity binding of the cytoplasmic IRE‐BP to an IRE which has at least two different known biological consequences, repression of ferritin mRNA translation and stabilization of the transferrin receptor transcript. We report the identification of a novel, evolutionarily conserved IRE motif in the 5′ UTR of murine and human erythroid‐specific delta‐aminolevulinic acid synthase (eALAS) mRNA which encodes the first, and possibly rate limiting, enzyme of the heme biosynthetic pathway. We demonstrate the function of the eALAS IRE as a specific binding site for the IRE‐BP by gel retardation analyses and by in vitro translation experiments. In addition, we show that the 5′ UTR of eALAS mRNA is sufficient to mediate iron‐dependent translational regulation in vivo. These findings strongly suggest involvement of the IRE‐IRE‐BP system in the control of heme biosynthesis during erythroid differentiation.

The multifaceted control of gene expression requires tight coordination of regulatory mechanisms at transcriptional and post-transcriptional level. Here, we studied the interdependence of transcription initiation, splicing and polyadenylation events on single mRNA molecules by full-length mRNA sequencing. In MCF-7 breast cancer cells, we find 2700 genes with interdependent alternative transcription initiation, splicing and polyadenylation events, both in proximal and distant parts of mRNA molecules, including examples of coupling between transcription start sites and polyadenylation sites. The analysis of three human primary tissues (brain, heart and liver) reveals similar patterns of interdependency between transcription initiation and mRNA processing events. We predict thousands of novel open reading frames from full-length mRNA sequences and obtained evidence for their translation by shotgun proteomics. The mapping database rescues 358 previously unassigned peptides and improves the assignment of others. By recognizing sample-specific amino-acid changes and novel splicing patterns, full-length mRNA sequencing improves proteogenomics analysis of MCF-7 cells. Our findings demonstrate that our understanding of transcriptome complexity is far from complete and provides a basis to reveal largely unresolved mechanisms that coordinate transcription initiation and mRNA processing.

The phylogenetically conserved eukaryotic translation initiation factor 5A (eIF5A) is the only known cellular protein to contain the post‐translationally derived amino acid hypusine [N^ε‐(4‐amino‐2‐hydroxybutyl)lysine]. Both eIF5A and its hypusine modification are essential for sustained cell proliferation. Normally only one eIF5A protein is expressed in human cells. Recently, we identified a second human EIF5A gene that would encode an isoform (eIF5A‐2) of 84% sequence identity. Overexpression of eIF5A‐2 mRNA in certain human cancer cells, in contrast to weak normal expression limited to human testis and brain, suggests EIF5A2 as a potential oncogene. However, eIF5A‐2 protein has not been described in human or mammalian cells heretofore. Here, we describe the identification of eIF5A‐2 protein in human colorectal and ovarian cancer lines, SW‐480 and UACC‐1598, that overexpress eIF5A‐2 mRNAs. Functional characterization of the human isoforms revealed that either human EIF5A gene can complement growth of a yeast strain in which the yeast EIF5A genes were disrupted. This indicates functional similarity of the human isoforms in yeast and suggests that eIF5A‐2 has an important role in eukaryotic cell survival similar to that of the ubiquitous eIF5A‐1. Detectable structural differences were also noted, including lack of immunological cross‐reactivity, formation of different complexes with deoxyhypusine synthase, and K_m values (1.5 ± 0.2 vs. 8.3 ± 1.4 µm for eIF5A‐1 and ‐2, respectively) as substrates for deoxyhypusine synthase in vitro. These physical characteristics and distinct amino acid sequences in the C‐terminal domain together with differences in gene expression patterns imply differentiated, tissue‐specific functions of the eIF5A‐2 isoform in the mammalian organism and in cancer.

Most mutations in the sequence of the RNA hairpin that specifically binds MS2 coat protein either reduce the binding affinity or have no effect. However, one RNA mutation, a uracil to cytosine change in the loop, has the unusual property of increasing the binding affinity to the protein by nearly 100-fold. Guided by the structure of the protein–RNA complex, we used a series of protein mutations and RNA modifications to evaluate the thermodynamic basis for the improved affinity: The tight binding of the cytosine mutation is due to ( i ) the amino group of the cytosine residue making an intra-RNA hydrogen bond that increases the propensity of the free RNA to adopt the structure seen in the complex and ( ii ) the increased affinity of hydrogen bonds between the protein and a phosphate two bases away from the cytosine residue. The data are in good agreement with a recent comparison of the cocrystal structures of the two complexes, where small differences in the two structures are seen at the thermodynamically important sites.