Messenger RNA competition in living Xenopus oocytes.

When calf lens crystallin mRNA and rabbit globin mRNA are competing for factors limiting protein synthesis in living Xenopus oocytes, no mRNA species is preferentially selected for translation. Differences in the intrinsic translational efficiency of the mRNA species exist, but the relative efficiencies are the same at high and low mRNA concentrations. mRNAs already being translated, in particular endogenous oocyte mRNAs, are less sensitive to competitive inhibition by injected mRNAs. As injected mRNAs gradually become incorporated into the protein-synthesizing machinery of the oocyte, they acquire the same status as the oocyte's own active mRNAs. Exogenous mRNAs this become endogenous mRNAs. These results, together with previous estmates of the translational efficiency of injected heterologous mRNA species, are compatible with the assumption that a large proportion of the endogenous mRNAs is not competing for the translational apparatus of the oocyte and, therefore, probably is present in the temporarily inactivated form.     

Translation of aberrant mRNAs lacking a termination codon or with a shortened 3'-UTR is repressed after initiation in yeast

A novel mRNA surveillance for mRNA lacking a termination codon (nonstop mRNA) has been proposed in which Ski7p is thought to recognize stalled ribosomes at the 3' end of mRNA. Here we report our analysis of translation and decay of nonstop mRNAs in Saccharomyces cerevisiae. Although the reduction of nonstop mRNAs was only 4.5-fold, a level that is sufficient for residual protein synthesis, translation products of nonstop mRNAs were hardly detectable. We show that nonstop mRNAs were associated with polysomes, but not with Pab1p. We also show that ribosomes translating nonstop mRNA formed stable and heavy polysome complexes with mRNA. These data suggest that ribosome stalling at the 3' end of nonstop mRNA may block further rounds of translation, hence repressing protein synthesis. Furthermore, it was found that the 5' --> 3' decay pathway was accelerated for nonstop mRNA decay in the absence of Ski7p. We also found that translation of aberrant mRNAs with a shortened 3'-UTR was repressed, suggesting that an improper spatial distance between the termination codon and the 3' end of mRNA results in translation repression.     

Synthesis and turnover of polysomal mRNAs in sea urchin embryos

The synthesis and turnover kinetics of polysomal mRNA have been measured in sea urchin embryos. Polysomes were isolated from stages ranging between mesenchyme blastula and late gastrula Strongylocentrotus purpuratus embryos which had been exposed to exogenous ³H-guanosine. The amount of radioactivity incorporated into messenger and ribosomal RNAs was determined separately as a function of time, and the precursor pool specific activity was measured in the same embryos. Synthesis and decay rate constants were extracted from the data by a leastsquares procedure. Per embryo, the rate of mRNA synthesis was calculated to be about 0.13 pg min^?1, while the rate of rRNA synthesis is about 0.022 pg min^?1. The newly synthesized mRNA turns over with a half-time of 5.7 hr. The data support only a single decay rate for the mRNA, but small fractions of mRNA decaying at different rates cannot be excluded. Previous studies have shown that a minor fraction of the mRNA includes the least abundant, most highly diverse set of messages (“complex class” mRNAs). To determine whether mRNAs of the complex class are synthesized and degraded at similar rates, labeled mRNA was measured in hybrids formed in mRNA excess reactions with single copy DNA. These experiments showed that complex class mRNAs represent an approximately proportional amount of the new mRNA synthesis, and turn over at the same average rate as does the bulk of the mRNA. Most of the mRNAs in the embryo polysomes are newly synthesized, rather than maternal. This statement refers both to complex class mRNAs and to prevalent mRNAs. Considering the sequence homology between embryo and oocyte mRNAs shown earlier, these results indicate that many of the same structural genes active during oogenesis are being transcribed in embryos at these stages.     

Expression of the thyroid hormone receptor gene, erbAalpha, in B lymphocytes: alternative mRNA processing is independent of differentiation but correlates with antisense RNA levels.

The erbAalpha gene encodes two alpha-thyroid hormone receptor isoforms, TRalpha1 and TRalpha2, which arise from alternatively processed mRNAs, erbAalpha1 (alpha1) and erb alpha2 (alpha2). The splicing and alternative polyadenylation patterns of these mRNAs resemble that of mRNAs encoding different forms of immunoglobulin heavy chains, which are regulated at the level of alternative processing during B cell differentiation. This study examines the levels of erbAalpha mRNA in eight B cell lines representing four stages of differentiation in order to determine whether regulation of the alternatively processed alpha1 and alpha2 mRNAs parallels the processing of immunoglobulin heavy chain mRNAs. Results show that the pattern of alpha1 and alpha2 mRNA expression is clearly different from that observed for immunoglobulin heavy chain mRNAs. B cell lines display characteristic ratios of alpha1/alpha2 mRNA at distinct stages of differentiation. Furthermore, expression of an overlapping gene, Rev-ErbAalpha (RevErb), was found to correlate strongly with an increase in the ratio of alpha1/alpha2 mRNA. These results suggest that alternative processing of erbAalpha mRNAs is regulated by a mechanism which is distinct from that regulating immunoglobulin mRNA. The correlation between RevErb and erbAalpha mRNA is consistent with negative regulation of alpha2 via antisense interactions with the complementary RevErb mRNA.     

Translation of nonSTOP mRNA is repressed post-initiation in mammalian cells

We investigated the fate of aberrant mRNAs lacking in-frame termination codons (called nonSTOP mRNA) in mammalian cells. We found that translation of nonSTOP mRNA was considerably repressed although a corresponding reduction of mRNA was not observed. The repression appears to be post-initiation since (i) repressed nonSTOP mRNAs were associated with polysomes, (ii) translation of IRES-initiated and uncapped nonSTOP mRNA were repressed, and (iii) protein production from nonSTOP mRNA associating with polysomes was significantly reduced when used to program an in vitro run-off translation assay. NonSTOP mRNAs distributed into lighter polysome fractions compared to control mRNAs encoding a stop codon, and a significant amount of heterogeneous polypeptides were produced during in vitro translation of nonSTOP RNAs, suggesting premature termination of ribosomes translating nonSTOP mRNA. Moreover, a run-off translation assay using hippuristanol and RNAse protection assays suggested the presence of a ribosome stalled at the 3' end of nonSTOP mRNAs. Taken together, these data indicate that ribosome stalling at the 3' end of nonSTOP mRNAs can block translation by preventing upstream translation events.     

Spatial organization of the synthesis of cytoskeletal proteins

The cytoskeleton of most cells is complex and spatially diverse. The mRNAs for some cytoskeletal proteins are localized, suggesting that synthesis of these proteins may occur at sites appropriate for function or assembly. mRNA concentrations were first observed for several oocyte and embryonic mRNAs. Some insight has been gained into the mechanisms that help to position these mRNAs. More surprising to some, many cytoskeletal mRNAs are also localized. Among them are mRNAs for actin, tubulin, intermediate filaments, and a variety of associated proteins. Different mRNAs in the same cell can be located in different places; the same mRNA can be located in different places; the same mRNA can be located differently at different times of development. For example, we observed vimentin mRNA in developing chicken muscle cultures by fluorescent in situ hybridization. We found that vimentin mRNA takes on a variety of positions during myogenesis, ending up located with its cognate protein at costameres. This last pattern is significant because it is too finely structured to have a function in the soluble phase and probably reflects contranslational assembly of this particular protein. Analogies can be made between oocyte or embryonic positions (animal/vegetal poles, oocyte cortex, and interior) and somatic cell positions (anterior/posterior and cell cortex/cell center). These analogies may point to conserved mechanisms for moving and retaining mRNA. Localization of cytoskeletal synthesis, through the mRNA or by other means, may prove as important for assembling and maintaining differentiated cytoskeletal structures and somatic cells as mRNA location is for organizing the embryo. Mechanisms that permit mRNA localization are likely to be conserved.     

Identification of cis elements which direct the localization of maternal mRNAs to the posterior pole of ascidian embryos

During ascidian embryogenesis, some mRNAs show clear localization at the posterior-most region. These postplasmic mRNAs are divided into two groups (type I and type II) according to their pattern of localization. To elucidate how these localization patterns are achieved, we attempted to identify the localization elements of these mRNAs. When in vitro synthesized postplasmic mRNAs were introduced into eggs, these mRNAs showed posterior localization similar to the endogenous mRNAs. The posterior localization of these mRNAs was mediated by their 3' untranslated regions (3' UTRs), as is the case for several localized Drosophila and Xenopus mRNAs. We identified smaller fragments of the 3' UTRs of HrWnt-5 and HrPOPK-1 mRNAs (type I) and HrPet-3 mRNA (type II) that were sufficient to direct green fluorescent protein mRNA to the posterior pole. For the localization of HrWnt-5 mRNA, two UG dinucleotide repetitive elements were essential. Motifs similar to these small elements also exist within the HrPOPK-1 mRNA localization element and 3' UTR of HrZF-1 mRNA, suggesting the conservation of localization elements among type I mRNAs. In contrast, the smallest sequence that suffices for the posterior localization of HrPet-3 (a type II mRNA) has different features from those of type I mRNAs; indeed, it does not have an identifiable critical element. This difference may distinguish type II mRNAs from type I mRNAs. These findings, especially the identification of the small localization element of HrWnt-5 mRNA, provide new insights into the localization of mRNAs during ascidian embryogenesis.     

Temperature-dependent translation of leaderless and canonical mRNAs in Escherichia coli

Leaderless mRNAs beginning with a 5'-terminal start codon occur in all biological systems. In this work, we have studied the comparative translational efficiency of leaderless and leadered mRNAs as a function of temperature by in vitro translation competition assays with Escherichia coli extracts. At low temperature (25 degrees C) leaderless mRNAs were found to be translated comparatively better than mRNAs containing an internal canonical ribosome binding site, whereas at high temperature (42 degrees C) the translational efficiency of canonical mRNAs is by far superior to that of leaderless mRNA. The inverse correlation between temperature and translational efficiency characteristic for the two mRNA classes was attributed to structural features of the mRNA(s) and to the reduced stability of the translation initiation complex formed at a 5'-terminal start codon at elevated temperature.     

Abundance of mRNAs encoding urea cycle enzymes in fetal and neonatal mouse liver

The relative abundances of mRNAs encoding the five urea cycle enzymes during development of mouse liver have been determined and compared with those of mRNAs encoding four other liver-specific proteins (phosphoenolpyruvate carboxykinase, tyrosine aminotransferase, alpha-fetoprotein, and albumin). Urea cycle enzyme mRNAs in fetal liver are expressed at 2-14% of the abundance in adult liver as early as 6 days before birth. Expression of the urea cycle enzyme mRNAs is not coordinate during the fetal and neonatal period. However, profiles of three urea cycle enzyme mRNAs are quite similar to that of alpha-fetoprotein mRNA, suggesting the possibility of a common response to regulatory signals during fetal development. With the exception of ornithine transcarbamylase mRNA, the urea cycle enzyme mRNAs have been shown previously to be inducible by cAMP and glucocorticoids. However, only argininosuccinate lyase mRNA exhibits any significant change in abundance at birth, resembling postnatal expression of tyrosine aminotransferase mRNA. The results indicate that the urea cycle enzyme mRNAs are potentially useful markers for elucidating various features of hepatocyte differentiation in mammals.     

Variation in the lack of polyadenylation of the rat milk protein mRNAs during the lactation cycle

Translationally active milk protein mRNAs were found as nonpolyadenylated mRNAs in the rat mammary gland during pregnancy, lactation and involution. Analyses of whey protein mRNA and casein mRNA with the corresponding cDNAs showed that the lack of polyadenylation of these mRNAs at different time points of the lactation cycle is not consistent with the hypothesis that polyadenylation may be incomplete in the mammary gland when large amounts of mRNA are synthesized. The fraction of whey protein mRNA and casein mRNA that lacked polyadenylation was inversely proportional to the concentration of each sequence in the tissue during pregnancy, lactation and involution. A model is proposed to explain the finding that in each animal the ratio of casein mRNA to whey protein mRNA was similar in polyadenylated RNA and in nonpolyadenylated RNA at all stages of the lactation cycle.