Minimum internal ribosome entry site required for poliovirus infectivity.

Translation initiation by internal ribosome binding is a recently discovered mechanism of eukaryotic viral and cellular protein synthesis in which ribosome subunits interact with the mRNAs at internal sites in the 5' untranslated RNA sequences and not with the 5' methylguanosine cap structure present at the extreme 5' ends of mRNA molecules. Uncapped poliovirus mRNAs harbor internal ribosome entry sites (IRES) in their long and highly structured 5' noncoding regions. Such IRES sequences are required for viral protein synthesis. In this study, a novel poliovirus was isolated whose genomic RNA contains two gross deletions removing approximately 100 nucleotides from the predicted IRES sequences within the 5' noncoding region. The deletions originated from previously in vivo-selected viral revertants displaying non-temperature-sensitive phenotypes. Each revertant had a different predicted stem-loop structure within the 5' noncoding region of their genomic RNAs deleted. The mutant poliovirus (Se1-5NC-delta DG) described in this study contains both stem-loop deletions in a single RNA genome, thereby creating a minimum IRES. Se1-5NC-delta DG exhibited slow growth and a pinpoint plaque phenotype following infection of HeLa cells, delayed onset of protein synthesis in vivo, and defective initiation during in vitro translation of the mutated poliovirus mRNAs. Interestingly, the peak levels of viral RNA synthesis in cells infected with Se1-5NC-delta DG occurred at slightly later times in infection than those achieved by wild-type poliovirus, but these mutant virus RNAs accumulated in the host cells during the late phases of virus infection. UV cross-linking assays with the 5' noncoding regions of wild-type and mutated RNAs were carried out in cytoplasmic extracts from HeLa cells and neuronal cells and in reticulocyte lysates to identify the cellular factors that interact with the putative IRES elements. The cellular proteins that were cross-linked to the minimum IRES may represent factors playing an essential role in internal translation initiation of poliovirus mRNAs.     

Comparison of computational methods for identifying translation initiation sites in EST data

Background

Expressed Sequence Tag (EST) sequences are generally single-strand, single-pass sequences, only 200–600 nucleotides long, contain errors resulting in frame shifts, and represent different parts of their parent cDNA. If the cDNAs contain translation initiation sites, they may be suitable for functional genomics studies. We have compared five methods to predict translation initiation sites in EST data: first-ATG, ESTScan, Diogenes, Netstart, and ATGpr.

Results

A dataset of 100 EST sequences, 50 with and 50 without, translation initiation sites, was created. Based on analysis of this dataset, ATGpr is found to be the most accurate for predicting the presence versus absence of translation initiation sites. With a maximum accuracy of 76%, ATGpr more accurately predicts the position or absence of translation initiation sites than NetStart (57%) or Diogenes (50%). ATGpr similarly excels when start sites are known to be present (90%), whereas NetStart achieves only 60% overall accuracy. As a baseline for comparison, choosing the first ATG correctly identifies the translation initiation site in 74% of the sequences. ESTScan and Diogenes, consistent with their intended use, are able to identify open reading frames, but are unable to determine the precise position of translation initiation sites.

Conclusions

ATGpr demonstrates high sensitivity, specificity, and overall accuracy in identifying start sites while also rejecting incomplete sequences. A database of EST sequences suitable for validating programs for translation initiation site prediction is now available. These tools and materials may open an avenue for future improvements in start site prediction and EST analysis.

     

Genomic RNA of mengovirus. VI. Translation of its two cistrons in lysates of interferon-treated cells.

The addition of low levels (40 ng/ml) of the synthetic double-stranded polyribonucleotide poly I:C to lysates of interferon-treated L-cells resulted in a strong inhibition (70 to 75%) of the in vitro translation of mengovirus RNA. Under these conditions, the rates of incorporation of [35S]methionine or formyl-[35S]methionine were depressed to a comparable extent. The sequences of mengovirus RNA recognized by ribosomes of interferon-treated cells at initiation of translation were compared with those present in initiation complexes formed by ribosomes of untreated controls. Fingerprint analysis revealed that the same sequences of mengovirus RNA were protected against nuclease attack by the 80S and the 40S initiation complexes formed in vitro in lysates of control or interferon-treated L-cells. Mengovirus RNA-coded proteins were labeled at their N-terminal end with formyl-[35S]methionine and digested to completion with trypsin. The resulting fragments were separated by high-voltage paper electrophoresis. Two different formyl-[35S]methionine-labeled N termini were resolved. Further analyses supported the notion that the two radioactive peaks originated in the initiation of translation at two different sites. This pattern did not change when mengovirus RNA was translated in lysates of interferon-treated cells.     

Cap-independent polysomal association of natural mRNAs encoding c-myc, BiP, and eIF4G conferred by internal ribosome entry sites.

Sequence elements that can function as internal ribosome entry sites (IRES) have been identified in 5' noncoding regions of certain uncapped viral and capped cellular mRNA molecules. However, it has remained largely unknown whether IRES elements are functional when located in their natural capped mRNAs. Therefore, the polysomal association and translation of several IRES-containing cellular mRNAs was tested under conditions that severely inhibited cap-dependent translation, that is, after infection with poliovirus. It was found that several known IRES-containing mRNAs, such as BiP and c-myc, were both associated with the translation apparatus and translated in infected cells when cap-dependent translation of most host-cell mRNAs was blocked, indicating that the IRES elements were functional in their natural mRNAs. Curiously, the mRNAs that encode eukaryotic initiation factor 4GI (eIF4GI) and 4GII (eIF4GII), two proteins with high identity and similar functions in the initiation of cap-dependent translation, were both associated with polysomes in infected cells. The 5'-end sequences of eIF4GI mRNA were isolated from a cDNA expression library and shown to function as an internal ribosome entry site when placed into a dicistronic mRNA. These findings suggest that eIF4G proteins can be synthesized at times when 5' cap-dependent mRNA translation is blocked, supporting the notion that eIF4G proteins are needed in both 5' cap-independent and 5' cap-dependent translational initiation mechanisms.     

Internal ribosomal entry site-mediated translation initiation in equine rhinitis A virus: similarities to and differences from that of foot-and-mouth disease virus

Equine rhinitis A virus (ERAV) has recently been classified as an aphthovirus, a genus otherwise comprised of the different serotypes of Foot-and-mouth disease virus (FMDV). FMDV initiates translation via a type II internal ribosomal entry site (IRES) and utilizes two in-frame AUG codons to produce the leader proteinases Lab and Lb. Here we show that the ERAV 5' nontranslated region also possesses the core structures of a type II IRES. The functional activity of this region was characterized by transfection of bicistronic plasmids into BHK-21 cells. In this system the core type II structures, stem-loops D to L, in addition to a stem-loop (termed M) downstream of the first putative initiation codon, are required for translation of the second reporter gene. In FMDV, translation of Lb is more efficient than that of Lab despite the downstream location of the Lb AUG codon. The ERAV genome also has putative initiation sites in positions similar to those utilized in FMDV, except that in ERAV these are present as two AUG pairs (AUGAUG). Using the bicistronic expression system, we detected initiation from both AUG pairs, although in contrast to FMDV, the first site is strongly favored over the second. Mutational analysis of the AUG codons indicated that AUG2 is the major initiation site, although AUG1 can be accessed, albeit inefficiently, in the absence of AUG2. Further mutational analysis indicated that codons downstream of AUG2 appear to be accessed by a mechanism other than leaky scanning. Furthermore, we present preliminary evidence that it is possible for ribosomes to access downstream of the two AUG pairs. This study reveals important differences in IRES function between aphthoviruses.     

In vitro translation of poliovirus RNA: utilization of internal initiation sites in reticulocyte lysate.

The translation of poliovirus RNA in rabbit reticulocyte lysate was examined. Translation of poliovirus RNA in this cell-free system resulted in an electrophoretic profile of poliovirus-specific proteins distinct from that observed in vivo or after translation in poliovirus-infected HeLa cell extract. A group of proteins derived from the P3 region of the polyprotein was identified by immunoprecipitation, time course, and N-formyl-[35S]methionine labeling studies to be the product of the initiation of protein synthesis at an internal site(s) located within the 3'-proximal RNA sequences. Utilization of this internal initiation site(s) on poliovirus RNA was abolished when reticulocyte lysate was supplemented with poliovirus-infected HeLa cell extract. Authentic P1-1a was also synthesized in reticulocyte lysate, indicating that correct 5'-proximal initiation of translation occurs in that system. We conclude that the deficiency of a component(s) of the reticulocyte lysate necessary for 5'-proximal initiation of poliovirus protein synthesis resulted in the ability of ribosomes to initiate translation on internal sequences. This aberrant initiation could be corrected by factors present in the HeLa cell extract. Apparently, under certain conditions, ribosomes are capable of recognizing internal sequences as authentic initiation sites.     

Proteomic analysis of the cyanobacterium Anabaena sp. strain PCC7120 with two-dimensional gel electrophoresis and amino-terminal sequencing

A protein-gene linkage map of the cyanobacterium Anabaena sp. strain PCC7120 was successfully constructed for 123 relatively abundant proteins. The total proteins extracted from the cell were resolved by two-dimensional electrophoresis, and the amino-terminal sequences of the protein spots were determined. By comparing the determined amino-terminal sequences with the entire genome sequence, the putative translation initiation sites of 87 genes were successfully assigned on the genome. The elucidated sequence features surrounding the translation initiation sites were as follows: (1) GTG and TTG in addition to the ATG were used as rare initiation codons; (2) the core sequences (GAGG, GGAG and AGGA) of the Shine-Dalgarno sequence were identified in the appropriate position preceding the 51 initiation sites (58.6%); (3) the nucleotides at the two regions, from -35 to -33, and from -19 to -17 (relative to the first nucleotide in the initiation codon) were preferentially adenines or thymines; (4) the nucleotides at the region from -14 to -8 were preferentially purines; (5) the nucleotide at position -1 was biased towards non-guanine (96.6%); (6) the nucleotide at the position +5 was preferentially cytosine (63.2%). It was evident that removal of the translation initiator methionine was dependent on the side-chain bulkiness of the penultimate amino acid residue. The predicted putative signal peptide sequences were also indicated. Besides confirming the existence of many predicted proteins, the data will serve as a starting point for the study of signals important in post-translational processing and nucleotide sequences important in the initiation of translation.     

Inefficient translation of T7 late mRNA by Bacillus subtilis ribosomes. Implications for species-specific translation

Bacillus subtilis 30 S subunits inefficiently recognize initiation sites in mRNAs from Gram-negative bacteria, but they are able to efficiently recognize initiation sites in mRNA derived from Gram-positive bacteria. McLaughlin et al. (McLaughlin, J. R., Murray, C. L., and Rabinowitz, J. C. (1981) J. Biol. Chem. 256, 11283-11291) have suggested that B. subtilis ribosomes require a strong Shine-Dalgarno sequence for translation initiation. To test whether this criterion is sufficient to explain the translational specificity of B. subtilis ribosomes, T7 late mRNA, which contains strong Shine-Dalgarno sequences before many of the late genes (Dunn, J. J., and Studier, F. W. (1983) J. Mol. Biol. 166, 477-535), was translated in vitro with both Escherichia coli and B. subtilis ribosomes. The identification of several of the in vitro products upon gel electrophoresis indicated that B. subtilis ribosomes recognize correct translation initiation sites in late T7 mRNA, but they do not translate these products efficiently. Competition experiments demonstrated that late T7 mRNA does not inhibit B. subtilis ribosomal translation of B. subtilis derived mRNA (from the bacteriophage phi 29). It is concluded that strong Shine-Dalgarno sequences may be necessary in B. subtilis translation initiation sites; however, additional determinants of initiation which differ from those found in the translation initiation sites of E. coli mRNAs must exist.     

The positive regulatory function of the 5''-proximal open reading frames in GCN4 mRNA can be mimicked by heterologous, short coding sequences.

Translational control of GCN4 expression in the yeast Saccharomyces cerevisiae is mediated by multiple AUG codons present in the leader of GCN4 mRNA, each of which initiates a short open reading frame of only two or three codons. Upstream AUG codons 3 and 4 are required to repress GCN4 expression in normal growth conditions; AUG codons 1 and 2 are needed to overcome this repression in amino acid starvation conditions. We show that the regulatory function of AUG codons 1 and 2 can be qualitatively mimicked by the AUG codons of two heterologous upstream open reading frames (URFs) containing the initiation regions of the yeast genes PGK and TRP1. These AUG codons inhibit GCN4 expression when present singly in the mRNA leader; however, they stimulate GCN4 expression in derepressing conditions when inserted upstream from AUG codons 3 and 4. This finding supports the idea that AUG codons 1 and 2 function in the control mechanism as translation initiation sites and further suggests that suppression of the inhibitory effects of AUG codons 3 and 4 is a general consequence of the translation of URF 1 and 2 sequences upstream. Several observations suggest that AUG codons 3 and 4 are efficient initiation sites; however, these sequences do not act as positive regulatory elements when placed upstream from URF 1. This result suggests that efficient translation is only one of the important properties of the 5' proximal URFs in GCN4 mRNA. We propose that a second property is the ability to permit reinitiation following termination of translation and that URF 1 is optimized for this regulatory function.     

Atypical nucleosome spacing of rat neuronal identifier elements in non-neuronal chromatin.

Rat neuronal identifier (ID) elements are located in chromatin regions that are organized in nucleosomal structures in both neuronal and non-neuronal cells. A subpopulation of ID sequences in chromatin of liver and kidney cells are relatively resistant to micrococcal nuclease digestion and are organized in nucleosomes exhibiting an atypically short repeat length. Other repetitive elements do not show this organization.     

Alternative splicing within the elk-1 5' untranslated region serves to modulate initiation events downstream of the highly conserved upstream open reading frame 2

The 5' untranslated region (UTR) plays a central role in the regulation of mammalian translation initiation. Key components include RNA structure, upstream AUGs (uAUGs), upstream open reading frames (uORFs), and internal ribosome entry site elements that can interact to modulate the readout. We previously reported the characterization of two alternatively spliced 5' UTR isoforms of the human elk-1 gene. Both contain two uAUGs and a stable RNA stem-loop, but the long form (5' UTR(L)) was more repressive than the short form (5' UTR(S)) for initiation at the ELK-1 AUG. We now demonstrate that ELK-1 expression arises by a combination of leaky scanning and reinitiation, with the latter mediated by the small uORF2 conserved in both spliced isoforms. In HEK293T cells, a considerable fraction of ribosomes scans beyond the ELK-1 AUG in a reinitiation mode. These are sequestered by a series of out-of-frame AUG codons that serve to prevent access to a second in-frame AUG start site used to express short ELK-1 (sELK-1), an N-terminally truncated form of ELK-1 that has been observed only in neuronal cells. We present evidence that all these events are fine-tuned by the nature of the 5' UTR and the activity of the α subunit of eukaryotic initiation factor 2 and provide insights into the neuronal specificity of sELK-1 expression.     

Phosphorylation of type-1 inositol 1,4,5-trisphosphate receptors by cyclic nucleotide-dependent protein kinases: a mutational analysis of the functionally important sites in the S2+ and S2- splice variants

Inositol 1,4,5-trisphosphate receptors (InsP3R) are the major route of intracellular calcium release in eukaryotic cells and as such are pivotal for stimulation of Ca2+-dependent effectors important for numerous physiological processes. Modulation of this release has important consequences for defining the particular spatio-temporal characteristics of Ca2+ signals. In this study, regulation of Ca2+ release by phosphorylation of type-1 InsP3R (InsP3R-1) by cAMP (PKA)- and cGMP (PKG)-dependent protein kinases was investigated in the two major splice variants of InsP3R-1. InsP3R-1 was expressed in DT-40 cells devoid of endogenous InsP3R. In cells expressing the neuronal, S2+ splice variant of the InsP3R-1, Ca2+ release was markedly enhanced when either PKA or PKG was activated. The sites of phosphorylation were investigated by mutation of serine residues present in two canonical phosphorylation sites present in the protein. Potentiated Ca2+ release was abolished when serine 1755 was mutated to alanine (S1755A) but was unaffected by a similar mutation of serine 1589 (S1589A). These data demonstrate that Ser-1755 is the functionally important residue for phosphoregulation by PKA and PKG in the neuronal variant of the InsP3R-1. Activation of PKA also resulted in potentiated Ca2+ release in cells expressing the non-neuronal, S2- splice variant of the InsP3R-1. However, the PKA-induced potentiation was still evident in S1589A or S1755A InsP3R-1 mutants. The effect was abolished in the double (S1589A/S1755A) mutant, indicating both sites are phosphorylated and contribute to the functional effect. Activation of PKG had no effect on Ca2+ release in cells expressing the S2- variant of InsP3R-1. Collectively, these data indicate that phosphoregulation of InsP3R-1 has dramatic effects on Ca2+ release and defines the molecular sites phosphorylated in the major variants expressed in neuronal and peripheral tissues.     

Ultrastructure of neuropeptide-Y immunoreactive elements in the superior colliculus of cat

Whereas the presence of neuropeptide-Y (NPY) in the superior colliculus (SC) has been established, its participation in the ultrastructural organisation of the neuronal networks in the SC has not been studied. Accordingly, in the present paper light and electron microscopic NPY immunohistochemical studies were performed on the SC of cat. NPY fibres were found to be present predominantly in the superficial grey layer (SGL) of the SC, though a few small NPY cells were found in both the deeper and the upper layers. Ultrastructural observations revealed that the NPY nerve endings establish almost exclusively axo-dendritic synaptic contacts in the SGL of the SC. Thus, the presumably inhibitory impact of the NPY terminals is exerted through the dendrites of the SGL neurons, and not directly to the retinal axons, as thought previously.