Terminal sequences do not contain the rate-limiting decay determinants of E. coli cat mRNA.

The mechanism of E. coli chloramphenicol acetyltransferase (cat) mRNA decay was investigated. Alteration of the 5' untranslated terminus does not appear to have an effect on the turnover rate of the mRNA. Similarly, changes at the 3' terminus of the message, including the addition of a stable stem and loop structure, do not affect the half-life of the message. The data suggest that 5' and 3' terminal untranslated sequences do not contain the rate-limiting determinants for cat message decay. Decay rates for various segments of the cat mRNA were measured and indicate that all regions of the message have similar stabilities. The current model of cat mRNA degradation involves a rate-limiting endonucleolytic decay event that occurs internal to the message followed by degradation of the cleavage products.     

Ribosome stacking defines CGS1 mRNA degradation sites during nascent peptide-mediated translation arrest

Expression of the Arabidopsis CGS1 gene that codes for cystathionine gamma-synthase is feedback-regulated at the step of mRNA degradation in response to S-adenosyl-L-methionine (AdoMet). This regulation occurs during translation and involves AdoMet-induced temporal translation arrest prior to the mRNA degradation. Here, we have identified multiple intermediates of CGS1 mRNA degradation with different 5' ends that are separated by approximately 30 nucleotides. Longer intermediates were found to be produced as the number of ribosomes loaded on mRNA was increased. Sucrose density gradient centrifugation experiments showed that the shortest mRNA degradation intermediate was associated with monosomes, whereas longer degradation intermediates were associated with multiple ribosomes. Immunoblot analyses revealed a ladder of premature polypeptides whose molecular weights corresponded to products of ribosomes in a stalled stack. An increase in smaller premature polypeptides was observed as the number of ribosomes loaded on mRNA increased. These results show that AdoMet induces the stacking of ribosomes on CGS1 mRNA and that multiple mRNA degradation sites probably correspond to each stacked ribosome.     

The 5'' untranslated region of mRNA for ribosomal protein S19 is involved in its translational regulation during Xenopus development.

During Xenopus development, the synthesis of ribosomal proteins is regulated at the translational level. To identify the region of the ribosomal protein mRNAs responsible for their typical translational behavior, we constructed a fused gene in which the upstream sequences (promoter) and the 5' untranslated sequence (first exon) of the gene coding for Xenopus ribosomal protein S19 were joined to the coding portion of the procaryotic chloramphenicol acetyltransferase (CAT) gene deleted of its own 5' untranslated region. This fused gene was introduced in vivo by microinjection into Xenopus fertilized eggs, and its activity was monitored during embryogenesis. By analyzing the pattern of appearance of CAT activity and the distribution of the S19-CAT mRNA between polysomes and messenger ribonucleoproteins, it was concluded that the 35-nucleotide-long 5' untranslated region of the S19 mRNA is able to confer to the fused S19-CAT mRNA the translational behavior typical of ribosomal proteins during Xenopus embryo development.     

Poly(rC) binding proteins and the 5' cloverleaf of uncapped poliovirus mRNA function during de novo assembly of polysomes

Poliovirus (PV) mRNA is unusual because it possesses a 5'-terminal monophosphate rather than a 5'-terminal cap. Uncapped mRNAs are typically degraded by the 5' exonuclease XRN1. A 5'-terminal cloverleaf RNA structure interacts with poly(rC) binding proteins (PCBPs) to protect uncapped PV mRNA from 5' exonuclease (K. E. Murray, A. W. Roberts, and D. J. Barton, RNA 7:1126-1141, 2001). In this study, we examined de novo polysome formation using HeLa cell-free translation-replication reactions. PV mRNA formed polysomes coordinate with the time needed for ribosomes to traverse the viral open reading frame (ORF). Nascent PV polypeptides cofractionated with viral polysomes, while mature PV proteins were released from the polysomes. Alterations in the size of the PV ORF correlated with alterations in the size of polysomes with ribosomes present every 250 to 500 nucleotides of the ORF. Eukaryotic initiation factor 4GI (eIF4GI) was cleaved rapidly as viral polysomes assembled and the COOH-terminal portion of eIF4GI cofractionated with viral polysomes. Poly(A) binding protein, along with PCBP 1 and 2, also cofractionated with viral polysomes. A C24A mutation that inhibits PCBP-5'-terminal cloverleaf RNA interactions inhibited the formation and stability of nascent PV polysomes. Kinetic analyses indicated that the PCBP-5' cloverleaf RNA interaction was necessary to protect PV mRNA from 5' exonuclease immediately as ribosomes initially traversed the viral ORF, before viral proteins could alter translation factors within nascent polysomes or contribute to ribonucleoprotein complexes at the termini of the viral mRNA.     

AUUUA is not sufficient to promote poly(A) shortening and degradation of an mRNA: the functional sequence within AU-rich elements may be UUAUUUA(U/A)(U/A).

AU-rich elements (AREs) in the 3' untranslated regions of several cytokine and oncogene mRNAs have been shown to function as signals for rapid mRNA degradation, and it is assumed that the many other cytokine and oncogene mRNAs that contain AU-rich sequences in the 3' untranslated region are similarly targeted for rapid turnover. We have used a chimeric gene composed mostly of growth hormone sequences with expression driven by the c-fos promoter to investigate the minimal sequence required to act as a functional destabilizing element and to monitor the effect of these sequences on early steps in the degradation pathway. We find that neither AUUUA, UAUUUA, nor AUUUAU can function as a destabilizing element. However, the sequence UAUUUAU, when present in three copies, is sufficient to destabilize a chimeric mRNA. We propose that this sequence functions by virtue of being a sufficient portion of the larger sequence, UUAUUUA(U/A)(U/A), that we propose forms the optimal binding site for a destabilizing factor. The destabilizing effect depends on the number of copies of this proposed binding site and their degree of mismatch in the first two and last two positions, with mismatches in the AUUUA sequence not being tolerated. We found a strict correlation between the effect of an ARE on degradation rate and the effect on the rate of poly(A) shortening, consistent with deadenylation being the first and rate-limiting step in degradation, and the step stimulated by destabilizing AREs. Deadenylation was observed to occur in at least two phases, with an oligo(A) intermediate transiently accumulating, consistent with the suggestion that the degradation processes may be similar in yeast and mammalian cells. AREs that are especially U rich and contain no UUAUUUA(U/A)(U/A) motifs failed to influence the degradation rate or the deadenylation rate, either when downstream of suboptimal destabilizing AREs or when alone.     

Dynamics and processivity of 40S ribosome scanning on mRNA in yeast

The eukaryotic 40S ribosomal subunit locates the translation initiation codon on an mRNA via the so-called scanning process that follows 40S binding to the capped 5' end. This key step in translation is required for the expression of almost all eukaryotic genes, yet the mechanism and dynamics of scanning are unknown. We have performed quantitative studies in vivo and in vitro of the movement of yeast 40S ribosomes along 5' untranslated regions (UTRs) of different lengths. 40S subunits perform cap-dependent scanning with high processivity for more than 1700 nucleotides in cells of Saccharomyces cerevisiae. Moreover, the observed rates of expression indicate that scanning is performed by an untethered 40S subunit that has been released from the 5' cap complex. Unexpectedly, the capability to maintain scanning competence on a long 5' UTR is more dependent on the Ded1/Dbp1 type of helicase than on eIF4A or eIF4B. In a yeast cell-free extract, scanning shows reduced processivity, with an estimated net 5'-->3' rate of approximately 10 nucleotides per second at 26 degrees C. We have developed a biased bidirectional walking model of ribosomal scanning that provides a framework for understanding the above observations as well as other known quantitative and qualitative features of this process.     

Initiation of translation can occur only in a restricted region of the CYC1 mRNA of Saccharomyces cerevisiae.

The steady-state levels and half-lives of CYC1 mRNAs were estimated in a series of mutant strains of Saccharomyces cerevisiae containing (i) TAA nonsense codons, (ii) ATG initiator codons, or (iii) the sequence ATA ATG ACT TAA (denoted ATG-TAA) at various positions along the CYC1 gene, which encodes iso-1-cytochrome c. These mutational alterations were made in backgrounds lacking all internal in-frame and out-of-frame ATG triplets or containing only one ATG initiator codon at the normal position. The results revealed a "sensitive" region encompassing approximately the first half of the CYC1 mRNA, in which nonsense codons caused Upf1-dependent degradation. This result and the stability of CYC1 mRNAs lacking all ATG triplets, as well as other results, suggested that degradation occurs unless elements associated with this sensitive region are covered with 80S ribosomes, 40S ribosomal subunits, or ribonucleoprotein particle proteins. While elongation by 80S ribosomes could be prematurely terminated by TAA codons, the scanning of 40S ribosomal units could not be terminated solely by TAA codons but could be disrupted by the ATG-TAA sequence, which caused the formation and subsequent prompt release of 80S ribosomes. The ATG-TAA sequence caused degradation of the CYC1 mRNA only when it was in the region spanning nucleotide positions -27 to +37 but not in the remaining 3' distal region, suggesting that translation could initiate only in this restricted initiation region. CYC1 mRNA distribution on polyribosomes confirmed that only ATG codons within the initiation region were translated at high efficiency. This initiation region was not entirely dependent on the distance from the 5' cap site and was not obviously dependent on the short-range secondary structure but may simply reflect an open structural requirement for initiation of translation of the CYC1 mRNA.     

Properties of the exonuclease activity that degrades H4 histone mRNA

We have described a cell-free system for studying mRNA degradation (Ross, J., and Kobs, G. (1986) J. Mol. Biol. 188, 579-593). Using that system we found that human H4 histone mRNA was degraded in a 3' to 5' direction by an exonucleolytic activity. Here we investigate several properties of the crude system and of the exonuclease. A RNase inhibitor, such as that from placenta, was required to block nonspecific ribonucleases and thereby to permit different mRNAs to be degraded at different rates. The histone mRNA exonuclease required divalent cation (magnesium) but not exogenously added ATP or GTP. It functioned efficiently at monovalent cation concentrations ranging from 0.5 to 200 mM. It was bound to ribosomes isolated from cells lysed in low salt buffers. However, it was eluted in active form from the ribosomes by exposing them to 0.3 M KCl. The enzyme rapidly degraded deproteinized, 32P-labeled histone mRNA prepared enzymatically.     

Double-stranded RNA causes synthesis of 2',5'-oligo(A) and degradation of messenger RNA in interferon-treated cells

Interferon-treated HeLa cells were incubated with [3H]uridine to label mRNA and were then exposed to the double-stranded RNA poly(inosinic acid).poly(cytidylic acid) (In.Cn). The incubation with In.Cn greatly enhanced the decay of mRNA. When the cells were incubated in this way in the presence of cycloheximide, which blocks ribosome movement along mRNA, extensive polysome degradation was detected in interferon-treated cells. Products of degradation of mRNA were recovered from monosomes which were presumably formed as a result of endonucleolytic breaks of mRNA. This endonucleolytic activity was correlated with the formation of 2',5'-oligo(A) by an enzyme induced by interferon and activated by double-stranded RNA; the 2',5'-oligo(A) was previously shown to activate an endonuclease in cell extracts. The 2',5'-oligo(A) levels in cells were measured by a competition-binding assay. Details of the procedure used are described, including synthesis of highly radioactive (2'-5')pppA3[32P]cytidine 3',5'-diphosphate, separation of 2',5'-oligo(A) binding from degrading activities, and specificity of the assay.     

Saccharomyces cerevisiae translational activator Cbs1p is associated with translationally active mitochondrial ribosomes

In the yeast Saccharomyces cerevisiae, mitochondrial translation of most, if not all, mitochondrially encoded genes is regulated by an individual set of gene-specific activators. Translation of the COB mRNA encoding cytochrome b requires the function of two nuclearly encoded proteins, Cbs1p and Cbs2p. Genetic data revealed that the 5'-untranslated region of COB mRNA is the target of both proteins. Recently, we provided evidence for an interaction of Cbs2p with mitochondrial ribosomes. We demonstrate here by means of blue native gel electrophoresis, density gradient centrifugation and tandem affinity purification that a portion of Cbs1p is also associated with mitochondrial ribosomes. In addition, we demonstrate that the amount of ribosome-associated Cbs1p is elevated in the presence of chloramphenicol, which is known to stall ribosomes on mRNAs. In the presence of puromycin, which strips off the mRNA and nascent protein chains from ribosomes, Cbs1p is no longer associated with ribosomes. Our data indicate that the observed interaction is mediated by ribosome-bound mRNA, thus restricting the association to ribosomes actively translating cytochrome b.