Dictyostelium discoideum mRNAs developmentally regulated during spore germination have short half-lives.

mRNA decay was studied during spore germination in Dictyoselium discoideum by the use of three previously isolated cDNA clones, pLK109, pLK229, and pRK270, which are specific for mRNAs developmentally regulated during spore germination. The half-life of a constitutive mRNA, pLK125, which is present throughout germination, growth, and development, as also determined. Nogalamycin, a DNA-intercalating compound, was used to inhibit RNA synthesis. Total RNA was isolated at intervals after addition of the drug, and the decay of mRNAs specific for the cDNA clones was determined by both Northern blot and RNA dot hybridization. If nogalamycin was added immediately after activation of dormant spores, neither pLK229 nor pLK109 mRNA decayed, but pLK125 mRNA did decay. Although pLK109 mRNA did not decay under these conditions, the RNA was smaller 1 h after activation than in dormant spores, indicating that it was processed normally. At 1 h after activation, pLK229-, pLK125-specific mRNAs decayed exponentially, with half-lives of 24, 39, and 165 min, respectively. Under the same conditions, decay of pLK109-specific mRNA was biphasic. Thirty-eight percent of the mRNA decayed with a half-life of 5.5 min, and the remainder decayed with a half-life of 115 min. It seems likely that nogalamycin inhibits the synthesis of an unstable component of the mRNA degradative pathway which is needed continuously for the decay of pLK109 mRNA. By extrapolating the curve representing the rapidly decaying component, a half-life of 18 min was calculated for pLK109-specific mRNA. The mRNAs developmentally regulated during spore germination have half-lives shorter than that of the constitutive messenger and shorter than the average half-life of 3 to 4 h previously determined for total Dicyostelium polyadenylated mRNA.     

Linking nuclear mRNP assembly and cytoplasmic destiny

From the very beginning, mRNAs have a complex existence. They are transcribed, capped, spliced, modified at the 3'end, exported from the nucleus, translated, and eventually degraded. These many events not only affect the overall survival and properties of an mRNA, but are also carefully co-ordinated and integrated with quality control mechanisms that function to ensure that only 'proper' mRNAs are translated at the correct developmental time and place. This does not mean that all mRNAs follow a single or uniform path from synthesis to death. Instead, there are diverse means by which the activities of specific mRNAs are regulated, and these controls often depend upon multiple events in the mRNA's life. mRNAs are not found naked in the cell, instead they are part of complex RNPs (ribonucleoproteins) that consist of many factors. These RNPs are highly dynamic structures that change during the lifetime of a given RNA; linking events such as synthesis and processing to the final fate of the mRNA. Here, we will discuss what is known of the assembly of RNPs in general, with specific reference to the myriad of connections between different nuclear events and the cytoplasmic activity of an mRNA. Due to space limitations this review is not comprehensive, instead we focus on specific examples to illustrate these emerging themes in gene expression.     

Gene expression in rat brain

191 randomly selected cDNA clones prepared from rat brain cytoplasmic poly (A)+ RNA were screened by Northern blot hybridization to rat brain, liver and kidney RNA to determine the tissue distribution, abundance and size of the corresponding brain mRNA. 18% hybridized to mRNAs each present equally in the three tissues, 26% to mRNAs differentially expressed in the tissues, and 30% to mRNAs present only in the brain. An additional 26% of the clones failed to detect mRNA in the three tissues at an abundance level of about 0.01%, but did contain rat cDNA as demonstrated by Southern blotting; this class probably represents rare mRNAs expressed in only some brain cells. Therefore, most mRNA expressed in brain is either specific to brain or otherwise displays regulation. Rarer mRNA species tend to be larger than the more abundant species, and tend to be brain specific; the rarest, specific mRNAs average 5000 nucleotides in length. Ten percent of the clones hybridize to multiple mRNAs, some of which are expressed from small multigenic families. From these data we estimate that there are probably at most 30,000 distinct mRNA species expressed in the rat brain, the majority of which are uniquely expressed in the brain.     

Direct mapping of rare messenger RNAs by means of oligomer-directed ribonuclease H cleavage

A method has been developed for characterizing rare messenger RNAs in the bulk population by using oligodeoxyribonucleotide: RNA hybrids as substrates for Escherichia coli ribonuclease H. Two 1.3-kb mRNAs in lymphocyte cytoplasm, interferon-gamma (0.002% of polyadenylated mRNA), and prothymosin-alpha, have been studied. Interferon-gamma mRNA was cut virtually completely into two fragments, each about 0.6 kb in length, by using an interferon-specific 24-mer to direct cleavage. Prothymosin-alpha mRNA in the same bulk population was unaffected by this treatment. When the 24-mer was replaced by a 12-mer, whose sequence was based on an incomplete cDNA clone for prothymosin-alpha, the products included two fragments of prothymosin-alpha mRNA. The sum of the fragment lengths equaled the length of the mRNA. Although the reaction directed by the smaller oligomer did not go to completion, the 12-mer, and hence the cDNA clone from which it was derived, could nevertheless be oriented with respect to prothymosin-alpha mRNA. With this technique, sequences in mRNA can be mapped without first isolating full-length cDNA clones.     

mRNA synthesis rates in vivo for androgen-inducible sequences in mouse kidney.

A method was developed for measuring in vivo rates of mRNA synthesis in mice by pulse-labeling with the RNA precursor [3H]orotate and then using hybridization to recover specific mRNAs. The efficiency of recovery is determined with synthetic RNAs as internal hybridization standards. The method is particularly applicable to the kidney since this organ shows a strong preferential uptake of the label. Rates of synthesis, expressed as a fraction of total RNA synthesis, were measured for the androgen-inducible mRNAs coding for beta-glucuronidase (GUS), ornithine decarboxylase (ODC), the protein coded by the RP-2 gene, and the so-called kidney androgen-regulated protein (KAP). Control mRNAs coded for beta-actin, phosphoenolpyruvate carboxykinase, and major urinary protein. Testosterone markedly increased the synthesis of the androgen-inducible mRNAs, but not the control mRNAs. Induction was not seen in mutant mice lacking functional androgen receptor protein. For GUS, ODC, and RP-2 mRNAs, the fold induction of synthesis was less than the fold induction of concentration, suggesting that mRNA stabilization also plays a part in the response to androgen. For GUS, ODC, and RP-2 mRNAs, but not KAP mRNA, induction of synthesis was rapidly reversed after testosterone removal. KAP mRNA was also exceptional in that its concentration was disproportionately high compared with its rate of synthesis, implying that it is a particularly stable mRNA.     

eIF4E is a central node of an RNA regulon that governs cellular proliferation

This study demonstrates that the eukaryotic translation initiation factor eIF4E is a critical node in an RNA regulon that impacts nearly every stage of cell cycle progression. Specifically, eIF4E coordinately promotes the messenger RNA (mRNA) export of several genes involved in the cell cycle. A common feature of these mRNAs is a structurally conserved, approximately 50-nucleotide element in the 3' untranslated region denoted as an eIF4E sensitivity element. This element is sufficient for localization of capped mRNAs to eIF4E nuclear bodies, formation of eIF4E-specific ribonucleoproteins in the nucleus, and eIF4E-dependent mRNA export. The roles of eIF4E in translation and mRNA export are distinct, as they rely on different mRNA elements. Furthermore, eIF4E-dependent mRNA export is independent of ongoing RNA or protein synthesis. Unlike the NXF1-mediated export of bulk mRNAs, eIF4E-dependent mRNA export is CRM1 dependent. Finally, the growth-suppressive promyelocytic leukemia protein (PML) inhibits this RNA regulon. These data provide novel perspectives into the proliferative and oncogenic properties of eIF4E.     

Stability of histone mRNAs is related to their location in polysomes

Synthesis of histone mRNAs is closely coupled to DNA synthesis. Following inhibition of DNA synthesis in L6 myoblasts with cytosine arabinoside, a coordinate and exaggerated rate of degradation of histone mRNAs occurs while other mRNAs, encoding ribosomal protein L32 and actin, are unaffected. Inhibition of protein synthesis by puromycin, emetine, or cycloheximide stabilizes histone mRNAs and results in their accumulation. When inhibition of DNA synthesis was followed immediately by inhibition of protein synthesis, the exaggerated rate of decay of the existing subspecies of histone H4 mRNAs was prevented and histone mRNA accumulated. If inhibition of protein synthesis was delayed longer than 3 minutes following inhibition of DNA synthesis, the ability to accumulate H4 mRNAs was lost. Furthermore, new protein synthesis was required to activate the mechanism which specifically destabilized histone mRNA. Puromycin was able to prevent the exaggerated rate of degradation of the various subspecies of H4 mRNA when added up to 15 min after inhibition of DNA synthesis, whereas emetine was effective only when added up to 5 min following inhibition of DNA synthesis. These data suggest that histone H4 mRNAs in polysomes are better targets than those released from polysomes for the specific mechanism which destabilizes histone mRNAs upon inhibition of DNA synthesis.